Journal of Virological Methods 205 (2014) 81–86
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Development of SYBR Green based real time PCR assay for detection of monodon baculovirus in Penaeus monodon D. Ramesh kumar, M. Sanjuktha, J.J.S. Rajan, R. Ananda Bharathi, T.C. Santiago, S.V. Alavandi, M. Poornima ∗ Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, 75, Santhome High Road, Raja Annamalai Puram, Chennai 600028, India
a b s t r a c t Article history: Received 26 December 2013 Received in revised form 26 April 2014 Accepted 7 May 2014 Available online 16 May 2014 Keywords: SYBR Green real time PCR Monodon baculovirus Penaeus monodon Specific pathogen free (SPF)
Shrimp farming is one of the most important aquaculture activities. Expansion and intensification of shrimp farming has been accompanied with the outbreak of diseases, which threaten the development and sustainability of the industry. Viral diseases are the major challenges faced by shrimp farming industries. The prevention/control of such diseases have become critical in determining the viability of the shrimp farming. The disease caused by monodon baculovirus (MBV) is the major limiting factor especially in shrimp hatchery. There are no therapeutic measures to control the viral diseases. So the detection of the disease is crucial in the prevention and spread of the disease. Hence, in this study, SYBR Green based real time polymerase chain reaction (PCR) assay was developed for the detection of MBV. The sensitivity of the real time PCR was determined using 10-fold dilutions of purified plasmid DNA with the concentration in the range of 101 –105 copies per reaction. The assay could detect as low as 12 copies indicating that the assay was sensitive and could be effectively used for the quantification of MBV. The real time PCR assay was found to be specific and did not show any amplification with P. monodon infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and hepatopancreatic parvo-like virus (HPV). The novelty of this assay is that it could be employed for diagnosis of low level MBV infection in broodstock using fecal matter of shrimp, a non-invasive diagnostic tool. © 2014 Published by Elsevier B.V.
1. Introduction The monodon baculovirus (MBV) also known as Penaeus monodon singly enveloped nuclear polyhedrosis virus (PmSNPV) was the first shrimp viral pathogen reported from Asia (Rajendran et al., 2012). It was first reported in laboratory reared adult P. monodon in Mexico in 1977, which was imported as post larvae (PL) from Taiwan. It was considered as the causative agent responsible for the massive shrimp production losses in Asia, Australia, Africa and Southern Europe (Lightner, 1993). It was reported to cause high mortalities in larva and early post larval stages of P. monodon (Lightner et al., 1983). It has diverse host range including cultured and captured shrimp and fresh water prawn. MBV infects the epithelial cells of the hepatopancreatic tubules and anterior midgut. The viral particles and occlusion bodies are released
∗ Corresponding author. Tel.: +91 44 24616948/24618817/24610565; fax: +91 44 24610311. E-mail addresses: poornima [email protected], [email protected] (M. Poornima). http://dx.doi.org/10.1016/j.jviromet.2014.05.006 0166-0934/© 2014 Published by Elsevier B.V.
to enter the intestinal tract through the hepatopancreatic lumen (Rajendran et al., 2012). The MBV occlusion bodies could be seen in high numbers of PLs and recede promptly when the PLs molt to juveniles. MBV is transmitted vertically or horizontally and spreads from the post larvae with latent infection (Fegan et al., 1991). MBV is a Nuclear Polyhedrosis Virus (NPV) of the family of Baculoviridae (Mari et al., 1993). MBV virions are rod shaped enveloped particles, measuring on average 265–324 nm in length and 42–77 nm in diameter (Lightner, 1996). The MBV genome is double stranded, circular DNA of 80–160 kb (Chen et al., 1989; Chang et al., 1993). In the squash preparations of hepatopancreas and shrimp fecal matter, the occlusion bodies measure less than 0.1 m to nearly 20 m (Lightner, 1996). It consists of crystalline matrices of protein particle called polyhedrin of 20–23 nm diameter and molecular weight of 58 kDa (Boonsanongchokying et al., 2006). Occlusion bodies are highly stable and maintain viability of viral particles under extreme external environmental conditions, thereby allowing virions to remain infectious for long durations (Entwistle et al., 1978; Rohrmann, 2008). The characteristic diagnostic feature of MBV infection is the presence of spherical occlusion bodies in hypertrophied nuclei of
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hepatopancreatic and anterior midgut epithelial cells. However, it could be observed only in advanced stages of infection. The early and rapid diagnosis of the disease with specificity and sensitivity is the need of the hour. Detection and avoidance of the viral pathogens using smart diagnostic tools such as real time PCR will enable the farmers to stock pathogen free seed. There are several techniques used for the detection of MBV including light microscopy (Lightner and Redman, 1998), in situ hybridization (Poulos et al., 1994), polymerase chain reaction (Surachetpong et al., 2005), colorimetric PCR based detection (Belcher and Young, 1998), immunohistochemistry (Boonsanongchokying et al., 2006), ELISA (Hsu et al., 2000), loop mediated isothermal amplification (LAMP) (Chaivisuthangkura et al., 2009) and real time PCR (Dongchun et al., 2009). Conventional PCR have been widely used for the detection of MBV infection. These assay target conserved region of the polyhedrin and DNA polymerase. In recent years, the SYBR Green based real-time PCR method had been used to detect and quantify shrimp and fish viruses such as white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic virus (IHHNV), Taura syndrome virus (TSV), yellow head virus (YHV) and infectious hematopoietic necrosis virus (IHNV) (Dhar et al., 2001; Mouillesseaux et al., 2003; Dhar et al., 2008). The SYBR Green-based real-time PCR have been described as an excellent alternative tool for the detection of viruses owing to easy identification of spurious or non-specific amplification, the ability to detect uncharacterized variants, and reduced time for analysis (Richards et al., 2004). The objectives of this study were (i) to develop a SYBR Green based realtime PCR assay for diagnosis of Indian MBV strains, (ii) to determine the sensitivity and specificity of SYBR Green PCR assay, and (iii) to determine the viral load in the clinical samples. 2. Materials and methods 2.1. Sample collection Brooders and post larvae of P. monodon were randomly collected from a commercial shrimp hatchery at Marakanam, Tamil Nadu (India) during the period February 2010 to January 2011. Shrimp samples were examined for MBV by wet mount and PCR investigation. 2.2. Detection of MBV by light microscopy The simplest MBV diagnostic method is the direct microscopic examination of squash preparation of the hepatopancreas of PL and brooders. For this, hepatopancreas from live shrimp were dissected and squash preparations prepared by staining with 0.05% aqueous solution of malachite green in 5.8% sodium chloride solution. Slides were microscopically examined to demonstrate polyhedra (occlusion bodies). MBV occlusions were distinguished from lipid droplets, nucleoli and other artifacts as described by Lightner (1996). 2.3. DNA extraction The DNA was extracted from hepatopancreas and fecal matter of shrimp as described by Van Engelenburg et al. (1993) with minor modifications. Approximately, 100 mg of hepatopancreas was homogenized and digested for 1 h at 90 ◦ C in 500 l of lysis buffer (10 mM Tris, 1 mM ethylenediamine tetra-acetic acid (EDTA), 100 mM NaCl) containing 0.5% sodium dodecyl sulphate and 0.1 mg proteinase K. The DNA was extracted with 500 l phenol:chloroform:isoamyl alcohol (25:24:1). The mixture was centrifuged 9000 × g (SIGMA 310 K) for 10 min at 4 ◦ C. After centrifugation, two volumes of 100% ethanol were added to the
aqueous phase and kept at −20 ◦ C for 1 h. The mixture was centrifuged at 9000 × g for 10 min at 4 ◦ C, and the DNA pellet obtained was washed with 70% cold ethanol, air-dried and re-suspended in 100 l of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4). 2.4. Detection of MBV by PCR MBV detection was performed with the PCR protocol and primers described by Belcher and Young (1998). The sequence of the oligonucleotide primers was (F: 5 -CGA TTC CAT ATC GGC CGA ATA-3 and R: 5 -TTG GCA TGC ACT CCC TGA GAT-3 ). The DNA extracted from the hepatopancreas of shrimp infected with MBV was used as the template. The thermocycling profile consisted of initial denaturation at 95 ◦ C for 5 min followed by 30 cycles of denaturation at 95 ◦ C for 45 s, annealing at 56 ◦ C for 40 s, extension at 72 ◦ C for 45 s and final extension at 72 ◦ C for 5 min. The PCR product were electrophoresed through 1.5% agarose gel and visualized under transilluminator (Bio Rad Laboratories, Hercules, CA, USA). 2.5. MBV real time PCR primer design The specific primers were designed from the MBV genome sequence retrieved from Genbank (Acc No. EU246944) using DNASTAR software. The primer sequences of MBVF and MBVR are (5 ATTTGAACGCACCAACAAGA-3 and 5 -GATTTTCAAGATCTGCACT CC-3 ) respectively. The predicted product size of the real time PCR with this primer pair is 115 bp. 2.6. PCR amplification and cloning PCR was performed with the above MBV specific real time PCR primer. The thermocycling profile was as follows: initial denaturation at 95 ◦ C for 5 min followed by 30 cycles of denaturation at 95 ◦ C for 45 s, annealing at 56 ◦ C for 40 s, extension at 72 ◦ C for 45 s and final extension at 72 ◦ C for 5 min. The amplified 115 bp PCR product was purified using HiYield Gel/PCR DNA purification kit (RBC Bioscience, Taiwan) according to the manufacturer’s instruction. The purified PCR product was cloned in pGEM-T Easy vector (Promega, USA) and transformed into E. coli DH5 ␣ competent cells. The recombinant clone was identified as the white colony on an Xgal and IPTG plate. The plasmid DNA was purified and used as the standard for the determination of the sensitivity of the real time PCR assay. 2.7. Standard curve analysis The recombinant plasmid DNA was isolated and purified using HiYield plasmid mini prep purification kit (RBC Bioscience, Taiwan) according to the manufacturer’s instruction. The recombinant plasmid DNA was quantified using spectrophotometer (Implen NanoPhotometer, Germany). The copy number of recombinant plasmid was determined using Thermo scientific copy number calculation tool (http://www.thermoscientificbio.com/ webtools/copynumber/). Thus, the dilution series of the plasmid standard of known copy number was used as the positive control and standards for quantitation in all real time PCR assays. 2.8. Specificity and sensitivity of real-time PCR The MBV real time PCR assay was performed with the optimized primer concentration. The reactions were carried out in a 48-well plate in a 10 l reaction volume containing 5.0 l of 2× SYBR Green Master Mix (PE Applied Biosystems), 0.5 l of each forward and reverse primer (0.4 M), 1.0 l template DNA and 3.0 l deionised water. The thermal cycling program of this assay consisted of initial denaturation at 95 ◦ C for 2 min, followed by 40
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Fig. 1. MBV occlusion bodies in hepatopancreas of infected P. monodon on staining with malachite green (a) highly positive, arrow indicates occlusion bodies and (b) low positive (no occlusion bodies). (Scale bars equal to 10 m).
cycles of denaturation at 95 ◦ C for 10 s; annealing, extension and fluorescent reading at 60 ◦ C for 1 min. After 40 cycles melting was performed from 60 to 95 ◦ C at 0.1 ◦ C/s with continuous measurement of fluorescence. Amplification and detection were performed with the Applied Biosystems, step one real time PCR system. The specificity of the real time-PCR assay was determined using DNA templates from tissues of P. monodon infected with WSSV, IHHNV and HPV viruses. The sensitivity of MBV real time PCR assay was determined using 10-fold dilutions of purified plasmid DNA with known viral copy numbers ranging from 101 to 105 copies per reaction. Real time PCR products were subjected to electrophoresis in 2% agarose gel containing ethidium bromide (0.5 g/ml) and the gel was visualized under ultraviolet transilluminator (Bio Rad Laboratories, Hercules, CA, USA) to confirm the SYBR Green assay. 3. Results 3.1. Detection of MBV by light microscopy The squash preparation of heavily infected PL showed the presence of MBV occlusion bodies upon malachite green staining under light microscopy (Fig. 1a). These shrimp are in the advanced stages of infection. The samples with mild infection did not reveal the presence of any occlusion bodies under light microscopy (Fig. 1b). 3.2. Detection of MBV by PCR DNA was extracted from PLs that were infected heavily, moderately and low level by MBV. These, DNA samples were used as templates for the detection of MBV by conventional PCR using the published primer sequence. Post larval samples with heavy and moderate infection could be detected by the amplification of specific band of 596 bp (Fig. 2). The post larval samples with mild infection could not be detected by this conventional PCR assay.
Fig. 2. Agarose gel electrophoresis of MBV diagnostic PCR, lane M: 100 bp marker, lane 1: low MBV positive sample, lane 2: moderate and lanes 3 and 4: high MBV positive samples.
3.3. Real time PCR amplification The thermal profile and the primer concentration for SYBR Green based real time PCR was optimized to increase the sensitivity and specificity. The optimal annealing temperature was found to be 60 ◦ C, and the optimal primer concentration was 0.4 M. The amplified SYBR Green real time PCR product was visualized by agarose gel electrophoresis showed the presence of 115 bp product (Fig. 3). 3.4. Melting and standard curve analysis Following amplification, a melting curve analysis was performed to verify the specific melting temperature (Tm ) of the PCR product. The measured melting temperature was found to be 71.0 ◦ C and matched with the expected PCR product melting temperature (Fig. 4). Standard curve for MBV specific real time PCR assay used for quantitation of viral load, generated from the Ct values obtained against the 10-fold serially diluted MBV DNA from 101 to 105 copies. A linear relationship between plasmid DNA and Ct value over a range from 101 to 105 copies was detected by SYBR Green PCR. The value of the linear regression (R2 ) calculated for the DNA copy number and Ct value was 0.994 (Fig. 5). 3.5. Specificity and sensitivity of SYBR Green based real-time PCR The DNA extracted from P. monodon infected with IHHNV, WSSV, HPV and SPF P. monodon did not show any amplification. These results clearly indicating that the selected primer pair for SYBR Green based real-time PCR was highly specific for the detection of MBV (Fig. 6). The sensitivity of this assay was performed with 10-fold serially diluted MBV plasmid DNA as a standard. The MBV infected clinical samples with high, moderate and mild level infection was tested to ascertain the sensitivity of this assay. The viral load in the MBV infected clinical samples were measured
Fig. 3. Agarose gel electrophoresis of amplified SYBR Green real time PCR product. Lane M: 100 bp marker, lane 1: 101 copy, lane 2: 102 , lane 3: 103 , lane 4: 104 , lane 5: 105 , lane 6: 106 , lane 7: negative control.
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Fig. 6. The specificity of MBV real time PCR assay using agarose gel electrophoresis of the amplified product. Lane M: 100 bp marker, lane 1: MBV DNA, lane 2: WSSV DNA, lane 3: IHHNV DNA, lane 4: HPV DNA, lane 5: SPF penaeid shrimp DNA, lane 6: deionized water.
by extrapolating the Ct value of the clinical samples into the standard curve of MBV. The viral load in these samples was found to be 12,239, 8622 and 78,150 copies l−1 (Fig. 7). It shows the considerable variation in viral load among these clinical samples. 4. Discussion
Fig. 4. The dissociation curve of MBV infected shrimp and negative control. The Tm values of MBV amplicons are indicated throughout their dissociation curve.
MBV has been reported to be controlled effectively by quarantine protocols. The rapid and accurate diagnosis of shrimp viral diseases is a better strategy for prevention and control of disease outbreaks in shrimp hatcheries. Lu et al. (1993) reported that the control and eradication of the disease through selection of virus free brood stock. There are several reports of continued high prevalence of this virus in shrimp hatcheries (Surachetpong et al., 2005). Therefore, the detection and avoidance of the virus in the broodstock would help in producing MBV-free post larvae. Quantitative real time PCR is a highly sensitive and specific assay for
Fig. 5. Standard curve shows a linear relationship between Ct values and the dilutions of the plasmid DNA of MBV.
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Fig. 7. Sensitivity of MBV real-time PCR assay using clinically infected samples: (a) amplification plot and (b) standard curves with copy number.
detection of shrimp viral pathogens. However, this assay was not available for the Indian strain of MBV. In the present study, a specific and sensitive SYBR Green based real time PCR assay was developed for the detection of Indian strain of MBV in affected P. monodon. This is also an efficient assay to quantify the viral copy number in infected P. monodon. SYBR Green is a fluorescent dye with a high affinity for double stranded DNA (Witter et al., 1997) and the SYBR Green based quantitative real time PCR is inexpensive, compared to TaqMan based assay (Mackay et al., 2002). SYBR Green real-time PCR methods have been applied successfully to various human and veterinary viruses (Mouillesseaux et al., 2003; Gibellini et al., 2006; Wilhelm et al., 2006; Martinez et al., 2008). Various conventional PCR methods have been developed for the detection of MBV from different geographical locations, including Thailand, Taiwan, Hawaii, Malaysia and Australia, with detection limit ranging from 100 to 800 copies of MBV DNA (Belcher and Young, 1998; Surachetpong et al., 2005). The detection range of real time PCR in the present study was 12–78,150 copies/l of MBV viral DNA in clinical samples. It was 10–80-fold more sensitive than traditional PCR. This wide dynamic range of detection level will be useful in measuring viral loads in animals with various level of infection. The larger amplicon size had a higher fluorescence derivative because more SYBR Green dye will bind to the larger amplicon compared to the smaller amplicon. This further increases the sensitivity of the assay (Mouillesseaux et al., 2003). Dhar et al. (2001) reported that the SYBR Green-based real-time PCR technique was 2000 times more sensitive than the conventional PCR technique in detecting IHHNV and WSSV. Conventional PCR and SYBR Green real time PCR assays have been compared and the real time PCR technique is found to be superior in terms of sensitivity (Dagher et al., 2004). The method with high sensitivity such as nested PCR is susceptible to carry over contamination due to exposure of high copy number amplicon to the laboratory environment during gel electrophoresis. This SYBR Green PCR does not require post PCR steps. Thus, it significantly reduces the PCR carry over contamination (Dhar et al., 2001). The high sensitivity of this assay was found to be coupled with high specificity. The SYBR Green dye can bind to any double stranded DNA. The specificity of the amplified product in SYBR
Green real time PCR assay is determined by the melting curve analysis. The precise verification of melting curve analysis was based on the specific temperature and primer dimer or other non-specific products (Ririe et al., 1997). Since the melting curve of amplicon is based on GC content, amplicon size and composition. So a melting curve peak with Tm within a variation of 2 ◦ C of the Tm of expected product suggests an amplification of a specific product. There was no primer dimer or non-specific products formation in this assay. The real-time PCR method amplifying a 135 bp DNA fragment with a TaqMan probe was reported to be very specific for MBV and did not cross react with HPV, WSSV, IHHNV and SPF shrimp DNA (Yan et al., 2009). The higher amplicon size of this assay increased the specificity due to higher Tm of the amplicon and higher amplicon fluorescence to noise ratio (Mouillesseaux et al., 2003). Screening of brood stock shrimp for MBV is one of the effective methods to control the vertical transmission of viral infection in the hatchery. The present study is the first attempt to develop a low cost assay with increased specificity and sensitivity. This assay could detect a wide dynamic range of MBV loads in shrimp with various levels of infection. Low levels of viral loads were found in the fecal matter of infected brooders. This could not be detected by either malachite green staining or conventional PCR. The novelty and sensitivity of this assay will allow detection of low viral load in fecal matter. Hence this can be used as a nonlethal/non-invasive diagnostic method to screen out infected shrimp brooders in hatcheries and achieve biosecurity with regard to MBV. Conflict of interest We have no conflict of interest. Acknowledgments The authors are thankful to the Department of Biotechnology, Govt of India, New Delhi, for funding this study (BT/PR9801/AGR/36/04/2007 dated 4 January 2008). Sincere thanks are also due to Dr. A.G. Ponniah, Director, Central Institute of Brackishwater Aquaculture (CIBA), Chennai for providing facilities.
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References Belcher, C.R., Young, P.R., 1998. Colourimetric PCR-based detection of monodon baculovirus in whole Penaeus monodon postlarvae. J. Virol. Methods 74, 21–29. Boonsanongchokying, C., Sang-oum, W., Sithigorngul, P., Sriurairatana, S., Flegel, T.W., 2006. Production of monoclonal antibodies to polyhedrin of Monodon Baculovirus (MBV) from shrimp. ScienceAsia 32, 371–376. Chaivisuthangkura, P., Srisuk, C., Rukpratanporn, S., Longyant, S., Sridulyakul, P., Sithigorngul, P., 2009. Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus by loop-mediated isothermal amplification. J. Virol. Methods 162, 188–193. Chang, P.S., Lo, C.F., Kou, G.H., Lu, C.C., Chen, S.N., 1993. Purification and amplification of DNA from Penaeus monodon-type baculovirus (MBV). J. Invertebr. Pathol. 62, 116–120. Chen, S.N., Chang, P.S., Kou, G.H., 1989. Observation on pathogenicity and epizootiology of Penaeus monodon Baculovirus (MBV) in cultured shrimp in Taiwan. Fish Pathol. 24, 189–195. Dagher, H., Donninger, H., Hatchinson, P., Ghildyal, R., Bardin, P., 2004. Rhinovirus detection: comparison of real-time and conventional PCR. J. Virol. Methods 117, 113–121. Dhar, A.K., Roux, M.R., Klimpel, K.R., 2001. Detection and quantification of infectious hematopoietic and haematopoietic necrosis virus (IHHNV) and white spot virus (WSV) of shrimp by real time quantitative PCR using SYBR Green chemistry. J. Clin. Microbiol. 39, 2835–2845. Dhar, A.K., Bowers, R.M., Licon, K.S., LaPatra, S.E., 2008. Detection and quantification of infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss) by SYBR Green real-time reverse transcriptase-polymerase chain reaction. J. Virol. Methods 147, 157–166. Dongchun, Y., Kathy, F.J.T., Donald, V.L., 2009. Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp. J. Invertebr. Pathol. 102, 97–100. Entwistle, P.F., Adams, P.H., Evans, H.F., 1978. Epizootiology of a nuclear polyhedrosis virus in European spruce sawfly (Gilpiniahercyniae): the rate of passage of infective virus through the gut of birds during cage tests. J. Invertebr. Pathol. 31 (3), 307–312. Fegan, D.F., Flegel, T.W., Sriurairatana, S., Waiakrutra, M., 1991. The occurrence, development and histopathology of monodon baculovirus in Penaeus monodon in Southern Thailand. Aquaculture 96, 205–217. Gibellini, D., Gardini, F., Vitone, F., Schiavone, P., Furlini, G., Re, M.C., 2006. Simultaneous detection of HCV and HIV-1 by SYBR Green real time multiplex RT-PCR technique in plasma samples. Mol. Cell. Probes 20, 223–229. Hsu, Y.L., Wang, K.H., Yang, Y.H., Tung, M.C., Hu, C.H., Lo, C.F., Wang, C.H., Hsu, T., 2000. Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies. Dis. Aquat. Organ. 40, 93–99. Lightner, D.V., 1993. Diseases of cultured Penaeid shrimp. In: McVey, J.P. (Ed.), CRC Handbook of Mariculture. Crustacean Aquaculture, vol. 1, second ed. CRC Press, Boca Raton, pp. 393–486. Lightner, D.V., 1996. A Handbook of Shrimp Pathology and Diagnostic Procedures for Disease of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, Louisiana, USA, pp. 304.
Lightner, D.V., Redman, R.M., Bell, T.A., 1983. Observations on the geographic distribution, pathogenesis and morphology of baculovirus from Penaeus monodon Fabricius. Aquaculture 32, 209–233. Lightner, D.V., Redman, R.M., 1998. Shrimp diseases and current diagnostic methods. Aquaculture 164, 201–220. Lu, C.C., Tang, F.J.K., Kou, G.H., Chen, S.N., 1993. Development of a Penaeus monodontype baculovirus (MBV) DNA probe by polymerase chain reaction and sequence analysis. J. Fish Dis. 16, 551–559. Mackay, I.M., Arden, K.E., Nitsche, A., 2002. Real-time PCR in virology. Nucleic Acids Res. 30, 1292–1305. Mari, J., Bonami, J.R., Poulos, B., Lightner, D., 1993. Preliminary characterization and partial cloning of the genome of a baculovirus from Penaeus monodon (PmSNPV = MBV). Dis. Aquat.Org. 16, 207–215. Martinez, E., Riera, P., Sitja, M., Fang, Y., Oliveira, S., Maldonado, J., 2008. Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time PCR and amplicon melting curve analysis using SYBR Green. Res. Vet. Sci. 85, 184–193. Mouillesseaux, K.P., Klimpel, K.R., Dhar, A.K., 2003. Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and yellow head virus of penaeid shrimp by increasing the amplicon size in SYBR Green real-time PCR. J. Virol. Methods 111, 121–127. Poulos, B.T., Mari, J., Bonami, J.R., Redman, R., Lightner, D.V., 1994. Use of nonradioactively labeled DNA probes for the detection of a baculovirus from Penaeus monodon by in situ hybridization on fixed tissue. J. Virol. Methods 49, 187–194. Rajendran, K.V., Makesh, M., Karunasagar, I., 2012. Monodon baculovirus of shrimp. Indian J. Virol. 23 (2), 149–160. Richards, G.P., Watson, M.A., Kingsley, D.H., 2004. A SYBR Green real-time RTPCR method to detect and quantitate Norwalk virus in stools. J. Virol. Methods 116, 63–70. Ririe, K.M., Rasmussen, R.P., Wittwer, C.T., 1997. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 270, 154–160. Rohrmann, G.F., 2008. Baculovirus Molecular Biology. National Library of Medicine, National Center for Biotechnology Information, Bethesda http://www.ncbi. nlm.gov/books/NBK1736/ Surachetpong, W., Poulos, B.T., Tang, K.F.J., Lightner, D.V., 2005. Improvement of PCR method for the detection of monodon baculovirus (MBV) in penaeid shrimp. Aquaculture 249, 69–75. Van Engelenburg, F.A.C., Maes, R.K., Van Oirschot, J.T., Rijsewijk, F.A., 1993. Development of a rapid and sensitive polymerase chain reaction assay for detection of bovine herpesvirus type 1 in bovine semen. J. Clin. Microbiol. 31, 3129–3135. Wilhelm, S., Zimmermann, P., Selbitz, H.J., Truyen, U., 2006. Real-time PCR protocol for the detection of porcine parvovirus in field samples. J. Virol. Methods 134, 257–260. Witter, C.T., Herrmann, M.G., Moss, A.A., Rasmussen, R.P., 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22, 130–138. Yan, D., Tang, K.F., Lightner, D.V., 2009. Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp. J. Invertebr. Pathol. 102, 97–100.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Development of SYBR Green based real time PCR assay for detection of monodon baculovirus in Penaeus monodon D. Ramesh kumar, M. Sanjuktha, J.J.S. Rajan, R. Ananda Bharathi, T.C. Santiago, S.V. Alavandi, M. Poornima ∗ Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, 75, Santhome High Road, Raja Annamalai Puram, Chennai 600028, India
a b s t r a c t Article history: Received 26 December 2013 Received in revised form 26 April 2014 Accepted 7 May 2014 Available online 16 May 2014 Keywords: SYBR Green real time PCR Monodon baculovirus Penaeus monodon Specific pathogen free (SPF)
Shrimp farming is one of the most important aquaculture activities. Expansion and intensification of shrimp farming has been accompanied with the outbreak of diseases, which threaten the development and sustainability of the industry. Viral diseases are the major challenges faced by shrimp farming industries. The prevention/control of such diseases have become critical in determining the viability of the shrimp farming. The disease caused by monodon baculovirus (MBV) is the major limiting factor especially in shrimp hatchery. There are no therapeutic measures to control the viral diseases. So the detection of the disease is crucial in the prevention and spread of the disease. Hence, in this study, SYBR Green based real time polymerase chain reaction (PCR) assay was developed for the detection of MBV. The sensitivity of the real time PCR was determined using 10-fold dilutions of purified plasmid DNA with the concentration in the range of 101 –105 copies per reaction. The assay could detect as low as 12 copies indicating that the assay was sensitive and could be effectively used for the quantification of MBV. The real time PCR assay was found to be specific and did not show any amplification with P. monodon infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and hepatopancreatic parvo-like virus (HPV). The novelty of this assay is that it could be employed for diagnosis of low level MBV infection in broodstock using fecal matter of shrimp, a non-invasive diagnostic tool. © 2014 Published by Elsevier B.V.
1. Introduction The monodon baculovirus (MBV) also known as Penaeus monodon singly enveloped nuclear polyhedrosis virus (PmSNPV) was the first shrimp viral pathogen reported from Asia (Rajendran et al., 2012). It was first reported in laboratory reared adult P. monodon in Mexico in 1977, which was imported as post larvae (PL) from Taiwan. It was considered as the causative agent responsible for the massive shrimp production losses in Asia, Australia, Africa and Southern Europe (Lightner, 1993). It was reported to cause high mortalities in larva and early post larval stages of P. monodon (Lightner et al., 1983). It has diverse host range including cultured and captured shrimp and fresh water prawn. MBV infects the epithelial cells of the hepatopancreatic tubules and anterior midgut. The viral particles and occlusion bodies are released
∗ Corresponding author. Tel.: +91 44 24616948/24618817/24610565; fax: +91 44 24610311. E-mail addresses: poornima [email protected], [email protected] (M. Poornima). http://dx.doi.org/10.1016/j.jviromet.2014.05.006 0166-0934/© 2014 Published by Elsevier B.V.
to enter the intestinal tract through the hepatopancreatic lumen (Rajendran et al., 2012). The MBV occlusion bodies could be seen in high numbers of PLs and recede promptly when the PLs molt to juveniles. MBV is transmitted vertically or horizontally and spreads from the post larvae with latent infection (Fegan et al., 1991). MBV is a Nuclear Polyhedrosis Virus (NPV) of the family of Baculoviridae (Mari et al., 1993). MBV virions are rod shaped enveloped particles, measuring on average 265–324 nm in length and 42–77 nm in diameter (Lightner, 1996). The MBV genome is double stranded, circular DNA of 80–160 kb (Chen et al., 1989; Chang et al., 1993). In the squash preparations of hepatopancreas and shrimp fecal matter, the occlusion bodies measure less than 0.1 m to nearly 20 m (Lightner, 1996). It consists of crystalline matrices of protein particle called polyhedrin of 20–23 nm diameter and molecular weight of 58 kDa (Boonsanongchokying et al., 2006). Occlusion bodies are highly stable and maintain viability of viral particles under extreme external environmental conditions, thereby allowing virions to remain infectious for long durations (Entwistle et al., 1978; Rohrmann, 2008). The characteristic diagnostic feature of MBV infection is the presence of spherical occlusion bodies in hypertrophied nuclei of
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hepatopancreatic and anterior midgut epithelial cells. However, it could be observed only in advanced stages of infection. The early and rapid diagnosis of the disease with specificity and sensitivity is the need of the hour. Detection and avoidance of the viral pathogens using smart diagnostic tools such as real time PCR will enable the farmers to stock pathogen free seed. There are several techniques used for the detection of MBV including light microscopy (Lightner and Redman, 1998), in situ hybridization (Poulos et al., 1994), polymerase chain reaction (Surachetpong et al., 2005), colorimetric PCR based detection (Belcher and Young, 1998), immunohistochemistry (Boonsanongchokying et al., 2006), ELISA (Hsu et al., 2000), loop mediated isothermal amplification (LAMP) (Chaivisuthangkura et al., 2009) and real time PCR (Dongchun et al., 2009). Conventional PCR have been widely used for the detection of MBV infection. These assay target conserved region of the polyhedrin and DNA polymerase. In recent years, the SYBR Green based real-time PCR method had been used to detect and quantify shrimp and fish viruses such as white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic virus (IHHNV), Taura syndrome virus (TSV), yellow head virus (YHV) and infectious hematopoietic necrosis virus (IHNV) (Dhar et al., 2001; Mouillesseaux et al., 2003; Dhar et al., 2008). The SYBR Green-based real-time PCR have been described as an excellent alternative tool for the detection of viruses owing to easy identification of spurious or non-specific amplification, the ability to detect uncharacterized variants, and reduced time for analysis (Richards et al., 2004). The objectives of this study were (i) to develop a SYBR Green based realtime PCR assay for diagnosis of Indian MBV strains, (ii) to determine the sensitivity and specificity of SYBR Green PCR assay, and (iii) to determine the viral load in the clinical samples. 2. Materials and methods 2.1. Sample collection Brooders and post larvae of P. monodon were randomly collected from a commercial shrimp hatchery at Marakanam, Tamil Nadu (India) during the period February 2010 to January 2011. Shrimp samples were examined for MBV by wet mount and PCR investigation. 2.2. Detection of MBV by light microscopy The simplest MBV diagnostic method is the direct microscopic examination of squash preparation of the hepatopancreas of PL and brooders. For this, hepatopancreas from live shrimp were dissected and squash preparations prepared by staining with 0.05% aqueous solution of malachite green in 5.8% sodium chloride solution. Slides were microscopically examined to demonstrate polyhedra (occlusion bodies). MBV occlusions were distinguished from lipid droplets, nucleoli and other artifacts as described by Lightner (1996). 2.3. DNA extraction The DNA was extracted from hepatopancreas and fecal matter of shrimp as described by Van Engelenburg et al. (1993) with minor modifications. Approximately, 100 mg of hepatopancreas was homogenized and digested for 1 h at 90 ◦ C in 500 l of lysis buffer (10 mM Tris, 1 mM ethylenediamine tetra-acetic acid (EDTA), 100 mM NaCl) containing 0.5% sodium dodecyl sulphate and 0.1 mg proteinase K. The DNA was extracted with 500 l phenol:chloroform:isoamyl alcohol (25:24:1). The mixture was centrifuged 9000 × g (SIGMA 310 K) for 10 min at 4 ◦ C. After centrifugation, two volumes of 100% ethanol were added to the
aqueous phase and kept at −20 ◦ C for 1 h. The mixture was centrifuged at 9000 × g for 10 min at 4 ◦ C, and the DNA pellet obtained was washed with 70% cold ethanol, air-dried and re-suspended in 100 l of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4). 2.4. Detection of MBV by PCR MBV detection was performed with the PCR protocol and primers described by Belcher and Young (1998). The sequence of the oligonucleotide primers was (F: 5 -CGA TTC CAT ATC GGC CGA ATA-3 and R: 5 -TTG GCA TGC ACT CCC TGA GAT-3 ). The DNA extracted from the hepatopancreas of shrimp infected with MBV was used as the template. The thermocycling profile consisted of initial denaturation at 95 ◦ C for 5 min followed by 30 cycles of denaturation at 95 ◦ C for 45 s, annealing at 56 ◦ C for 40 s, extension at 72 ◦ C for 45 s and final extension at 72 ◦ C for 5 min. The PCR product were electrophoresed through 1.5% agarose gel and visualized under transilluminator (Bio Rad Laboratories, Hercules, CA, USA). 2.5. MBV real time PCR primer design The specific primers were designed from the MBV genome sequence retrieved from Genbank (Acc No. EU246944) using DNASTAR software. The primer sequences of MBVF and MBVR are (5 ATTTGAACGCACCAACAAGA-3 and 5 -GATTTTCAAGATCTGCACT CC-3 ) respectively. The predicted product size of the real time PCR with this primer pair is 115 bp. 2.6. PCR amplification and cloning PCR was performed with the above MBV specific real time PCR primer. The thermocycling profile was as follows: initial denaturation at 95 ◦ C for 5 min followed by 30 cycles of denaturation at 95 ◦ C for 45 s, annealing at 56 ◦ C for 40 s, extension at 72 ◦ C for 45 s and final extension at 72 ◦ C for 5 min. The amplified 115 bp PCR product was purified using HiYield Gel/PCR DNA purification kit (RBC Bioscience, Taiwan) according to the manufacturer’s instruction. The purified PCR product was cloned in pGEM-T Easy vector (Promega, USA) and transformed into E. coli DH5 ␣ competent cells. The recombinant clone was identified as the white colony on an Xgal and IPTG plate. The plasmid DNA was purified and used as the standard for the determination of the sensitivity of the real time PCR assay. 2.7. Standard curve analysis The recombinant plasmid DNA was isolated and purified using HiYield plasmid mini prep purification kit (RBC Bioscience, Taiwan) according to the manufacturer’s instruction. The recombinant plasmid DNA was quantified using spectrophotometer (Implen NanoPhotometer, Germany). The copy number of recombinant plasmid was determined using Thermo scientific copy number calculation tool (http://www.thermoscientificbio.com/ webtools/copynumber/). Thus, the dilution series of the plasmid standard of known copy number was used as the positive control and standards for quantitation in all real time PCR assays. 2.8. Specificity and sensitivity of real-time PCR The MBV real time PCR assay was performed with the optimized primer concentration. The reactions were carried out in a 48-well plate in a 10 l reaction volume containing 5.0 l of 2× SYBR Green Master Mix (PE Applied Biosystems), 0.5 l of each forward and reverse primer (0.4 M), 1.0 l template DNA and 3.0 l deionised water. The thermal cycling program of this assay consisted of initial denaturation at 95 ◦ C for 2 min, followed by 40
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Fig. 1. MBV occlusion bodies in hepatopancreas of infected P. monodon on staining with malachite green (a) highly positive, arrow indicates occlusion bodies and (b) low positive (no occlusion bodies). (Scale bars equal to 10 m).
cycles of denaturation at 95 ◦ C for 10 s; annealing, extension and fluorescent reading at 60 ◦ C for 1 min. After 40 cycles melting was performed from 60 to 95 ◦ C at 0.1 ◦ C/s with continuous measurement of fluorescence. Amplification and detection were performed with the Applied Biosystems, step one real time PCR system. The specificity of the real time-PCR assay was determined using DNA templates from tissues of P. monodon infected with WSSV, IHHNV and HPV viruses. The sensitivity of MBV real time PCR assay was determined using 10-fold dilutions of purified plasmid DNA with known viral copy numbers ranging from 101 to 105 copies per reaction. Real time PCR products were subjected to electrophoresis in 2% agarose gel containing ethidium bromide (0.5 g/ml) and the gel was visualized under ultraviolet transilluminator (Bio Rad Laboratories, Hercules, CA, USA) to confirm the SYBR Green assay. 3. Results 3.1. Detection of MBV by light microscopy The squash preparation of heavily infected PL showed the presence of MBV occlusion bodies upon malachite green staining under light microscopy (Fig. 1a). These shrimp are in the advanced stages of infection. The samples with mild infection did not reveal the presence of any occlusion bodies under light microscopy (Fig. 1b). 3.2. Detection of MBV by PCR DNA was extracted from PLs that were infected heavily, moderately and low level by MBV. These, DNA samples were used as templates for the detection of MBV by conventional PCR using the published primer sequence. Post larval samples with heavy and moderate infection could be detected by the amplification of specific band of 596 bp (Fig. 2). The post larval samples with mild infection could not be detected by this conventional PCR assay.
Fig. 2. Agarose gel electrophoresis of MBV diagnostic PCR, lane M: 100 bp marker, lane 1: low MBV positive sample, lane 2: moderate and lanes 3 and 4: high MBV positive samples.
3.3. Real time PCR amplification The thermal profile and the primer concentration for SYBR Green based real time PCR was optimized to increase the sensitivity and specificity. The optimal annealing temperature was found to be 60 ◦ C, and the optimal primer concentration was 0.4 M. The amplified SYBR Green real time PCR product was visualized by agarose gel electrophoresis showed the presence of 115 bp product (Fig. 3). 3.4. Melting and standard curve analysis Following amplification, a melting curve analysis was performed to verify the specific melting temperature (Tm ) of the PCR product. The measured melting temperature was found to be 71.0 ◦ C and matched with the expected PCR product melting temperature (Fig. 4). Standard curve for MBV specific real time PCR assay used for quantitation of viral load, generated from the Ct values obtained against the 10-fold serially diluted MBV DNA from 101 to 105 copies. A linear relationship between plasmid DNA and Ct value over a range from 101 to 105 copies was detected by SYBR Green PCR. The value of the linear regression (R2 ) calculated for the DNA copy number and Ct value was 0.994 (Fig. 5). 3.5. Specificity and sensitivity of SYBR Green based real-time PCR The DNA extracted from P. monodon infected with IHHNV, WSSV, HPV and SPF P. monodon did not show any amplification. These results clearly indicating that the selected primer pair for SYBR Green based real-time PCR was highly specific for the detection of MBV (Fig. 6). The sensitivity of this assay was performed with 10-fold serially diluted MBV plasmid DNA as a standard. The MBV infected clinical samples with high, moderate and mild level infection was tested to ascertain the sensitivity of this assay. The viral load in the MBV infected clinical samples were measured
Fig. 3. Agarose gel electrophoresis of amplified SYBR Green real time PCR product. Lane M: 100 bp marker, lane 1: 101 copy, lane 2: 102 , lane 3: 103 , lane 4: 104 , lane 5: 105 , lane 6: 106 , lane 7: negative control.
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Fig. 6. The specificity of MBV real time PCR assay using agarose gel electrophoresis of the amplified product. Lane M: 100 bp marker, lane 1: MBV DNA, lane 2: WSSV DNA, lane 3: IHHNV DNA, lane 4: HPV DNA, lane 5: SPF penaeid shrimp DNA, lane 6: deionized water.
by extrapolating the Ct value of the clinical samples into the standard curve of MBV. The viral load in these samples was found to be 12,239, 8622 and 78,150 copies l−1 (Fig. 7). It shows the considerable variation in viral load among these clinical samples. 4. Discussion
Fig. 4. The dissociation curve of MBV infected shrimp and negative control. The Tm values of MBV amplicons are indicated throughout their dissociation curve.
MBV has been reported to be controlled effectively by quarantine protocols. The rapid and accurate diagnosis of shrimp viral diseases is a better strategy for prevention and control of disease outbreaks in shrimp hatcheries. Lu et al. (1993) reported that the control and eradication of the disease through selection of virus free brood stock. There are several reports of continued high prevalence of this virus in shrimp hatcheries (Surachetpong et al., 2005). Therefore, the detection and avoidance of the virus in the broodstock would help in producing MBV-free post larvae. Quantitative real time PCR is a highly sensitive and specific assay for
Fig. 5. Standard curve shows a linear relationship between Ct values and the dilutions of the plasmid DNA of MBV.
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Fig. 7. Sensitivity of MBV real-time PCR assay using clinically infected samples: (a) amplification plot and (b) standard curves with copy number.
detection of shrimp viral pathogens. However, this assay was not available for the Indian strain of MBV. In the present study, a specific and sensitive SYBR Green based real time PCR assay was developed for the detection of Indian strain of MBV in affected P. monodon. This is also an efficient assay to quantify the viral copy number in infected P. monodon. SYBR Green is a fluorescent dye with a high affinity for double stranded DNA (Witter et al., 1997) and the SYBR Green based quantitative real time PCR is inexpensive, compared to TaqMan based assay (Mackay et al., 2002). SYBR Green real-time PCR methods have been applied successfully to various human and veterinary viruses (Mouillesseaux et al., 2003; Gibellini et al., 2006; Wilhelm et al., 2006; Martinez et al., 2008). Various conventional PCR methods have been developed for the detection of MBV from different geographical locations, including Thailand, Taiwan, Hawaii, Malaysia and Australia, with detection limit ranging from 100 to 800 copies of MBV DNA (Belcher and Young, 1998; Surachetpong et al., 2005). The detection range of real time PCR in the present study was 12–78,150 copies/l of MBV viral DNA in clinical samples. It was 10–80-fold more sensitive than traditional PCR. This wide dynamic range of detection level will be useful in measuring viral loads in animals with various level of infection. The larger amplicon size had a higher fluorescence derivative because more SYBR Green dye will bind to the larger amplicon compared to the smaller amplicon. This further increases the sensitivity of the assay (Mouillesseaux et al., 2003). Dhar et al. (2001) reported that the SYBR Green-based real-time PCR technique was 2000 times more sensitive than the conventional PCR technique in detecting IHHNV and WSSV. Conventional PCR and SYBR Green real time PCR assays have been compared and the real time PCR technique is found to be superior in terms of sensitivity (Dagher et al., 2004). The method with high sensitivity such as nested PCR is susceptible to carry over contamination due to exposure of high copy number amplicon to the laboratory environment during gel electrophoresis. This SYBR Green PCR does not require post PCR steps. Thus, it significantly reduces the PCR carry over contamination (Dhar et al., 2001). The high sensitivity of this assay was found to be coupled with high specificity. The SYBR Green dye can bind to any double stranded DNA. The specificity of the amplified product in SYBR
Green real time PCR assay is determined by the melting curve analysis. The precise verification of melting curve analysis was based on the specific temperature and primer dimer or other non-specific products (Ririe et al., 1997). Since the melting curve of amplicon is based on GC content, amplicon size and composition. So a melting curve peak with Tm within a variation of 2 ◦ C of the Tm of expected product suggests an amplification of a specific product. There was no primer dimer or non-specific products formation in this assay. The real-time PCR method amplifying a 135 bp DNA fragment with a TaqMan probe was reported to be very specific for MBV and did not cross react with HPV, WSSV, IHHNV and SPF shrimp DNA (Yan et al., 2009). The higher amplicon size of this assay increased the specificity due to higher Tm of the amplicon and higher amplicon fluorescence to noise ratio (Mouillesseaux et al., 2003). Screening of brood stock shrimp for MBV is one of the effective methods to control the vertical transmission of viral infection in the hatchery. The present study is the first attempt to develop a low cost assay with increased specificity and sensitivity. This assay could detect a wide dynamic range of MBV loads in shrimp with various levels of infection. Low levels of viral loads were found in the fecal matter of infected brooders. This could not be detected by either malachite green staining or conventional PCR. The novelty and sensitivity of this assay will allow detection of low viral load in fecal matter. Hence this can be used as a nonlethal/non-invasive diagnostic method to screen out infected shrimp brooders in hatcheries and achieve biosecurity with regard to MBV. Conflict of interest We have no conflict of interest. Acknowledgments The authors are thankful to the Department of Biotechnology, Govt of India, New Delhi, for funding this study (BT/PR9801/AGR/36/04/2007 dated 4 January 2008). Sincere thanks are also due to Dr. A.G. Ponniah, Director, Central Institute of Brackishwater Aquaculture (CIBA), Chennai for providing facilities.
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References Belcher, C.R., Young, P.R., 1998. Colourimetric PCR-based detection of monodon baculovirus in whole Penaeus monodon postlarvae. J. Virol. Methods 74, 21–29. Boonsanongchokying, C., Sang-oum, W., Sithigorngul, P., Sriurairatana, S., Flegel, T.W., 2006. Production of monoclonal antibodies to polyhedrin of Monodon Baculovirus (MBV) from shrimp. ScienceAsia 32, 371–376. Chaivisuthangkura, P., Srisuk, C., Rukpratanporn, S., Longyant, S., Sridulyakul, P., Sithigorngul, P., 2009. Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus by loop-mediated isothermal amplification. J. Virol. Methods 162, 188–193. Chang, P.S., Lo, C.F., Kou, G.H., Lu, C.C., Chen, S.N., 1993. Purification and amplification of DNA from Penaeus monodon-type baculovirus (MBV). J. Invertebr. Pathol. 62, 116–120. Chen, S.N., Chang, P.S., Kou, G.H., 1989. Observation on pathogenicity and epizootiology of Penaeus monodon Baculovirus (MBV) in cultured shrimp in Taiwan. Fish Pathol. 24, 189–195. Dagher, H., Donninger, H., Hatchinson, P., Ghildyal, R., Bardin, P., 2004. Rhinovirus detection: comparison of real-time and conventional PCR. J. Virol. Methods 117, 113–121. Dhar, A.K., Roux, M.R., Klimpel, K.R., 2001. Detection and quantification of infectious hematopoietic and haematopoietic necrosis virus (IHHNV) and white spot virus (WSV) of shrimp by real time quantitative PCR using SYBR Green chemistry. J. Clin. Microbiol. 39, 2835–2845. Dhar, A.K., Bowers, R.M., Licon, K.S., LaPatra, S.E., 2008. Detection and quantification of infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss) by SYBR Green real-time reverse transcriptase-polymerase chain reaction. J. Virol. Methods 147, 157–166. Dongchun, Y., Kathy, F.J.T., Donald, V.L., 2009. Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp. J. Invertebr. Pathol. 102, 97–100. Entwistle, P.F., Adams, P.H., Evans, H.F., 1978. Epizootiology of a nuclear polyhedrosis virus in European spruce sawfly (Gilpiniahercyniae): the rate of passage of infective virus through the gut of birds during cage tests. J. Invertebr. Pathol. 31 (3), 307–312. Fegan, D.F., Flegel, T.W., Sriurairatana, S., Waiakrutra, M., 1991. The occurrence, development and histopathology of monodon baculovirus in Penaeus monodon in Southern Thailand. Aquaculture 96, 205–217. Gibellini, D., Gardini, F., Vitone, F., Schiavone, P., Furlini, G., Re, M.C., 2006. Simultaneous detection of HCV and HIV-1 by SYBR Green real time multiplex RT-PCR technique in plasma samples. Mol. Cell. Probes 20, 223–229. Hsu, Y.L., Wang, K.H., Yang, Y.H., Tung, M.C., Hu, C.H., Lo, C.F., Wang, C.H., Hsu, T., 2000. Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies. Dis. Aquat. Organ. 40, 93–99. Lightner, D.V., 1993. Diseases of cultured Penaeid shrimp. In: McVey, J.P. (Ed.), CRC Handbook of Mariculture. Crustacean Aquaculture, vol. 1, second ed. CRC Press, Boca Raton, pp. 393–486. Lightner, D.V., 1996. A Handbook of Shrimp Pathology and Diagnostic Procedures for Disease of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, Louisiana, USA, pp. 304.
Lightner, D.V., Redman, R.M., Bell, T.A., 1983. Observations on the geographic distribution, pathogenesis and morphology of baculovirus from Penaeus monodon Fabricius. Aquaculture 32, 209–233. Lightner, D.V., Redman, R.M., 1998. Shrimp diseases and current diagnostic methods. Aquaculture 164, 201–220. Lu, C.C., Tang, F.J.K., Kou, G.H., Chen, S.N., 1993. Development of a Penaeus monodontype baculovirus (MBV) DNA probe by polymerase chain reaction and sequence analysis. J. Fish Dis. 16, 551–559. Mackay, I.M., Arden, K.E., Nitsche, A., 2002. Real-time PCR in virology. Nucleic Acids Res. 30, 1292–1305. Mari, J., Bonami, J.R., Poulos, B., Lightner, D., 1993. Preliminary characterization and partial cloning of the genome of a baculovirus from Penaeus monodon (PmSNPV = MBV). Dis. Aquat.Org. 16, 207–215. Martinez, E., Riera, P., Sitja, M., Fang, Y., Oliveira, S., Maldonado, J., 2008. Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time PCR and amplicon melting curve analysis using SYBR Green. Res. Vet. Sci. 85, 184–193. Mouillesseaux, K.P., Klimpel, K.R., Dhar, A.K., 2003. Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and yellow head virus of penaeid shrimp by increasing the amplicon size in SYBR Green real-time PCR. J. Virol. Methods 111, 121–127. Poulos, B.T., Mari, J., Bonami, J.R., Redman, R., Lightner, D.V., 1994. Use of nonradioactively labeled DNA probes for the detection of a baculovirus from Penaeus monodon by in situ hybridization on fixed tissue. J. Virol. Methods 49, 187–194. Rajendran, K.V., Makesh, M., Karunasagar, I., 2012. Monodon baculovirus of shrimp. Indian J. Virol. 23 (2), 149–160. Richards, G.P., Watson, M.A., Kingsley, D.H., 2004. A SYBR Green real-time RTPCR method to detect and quantitate Norwalk virus in stools. J. Virol. Methods 116, 63–70. Ririe, K.M., Rasmussen, R.P., Wittwer, C.T., 1997. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 270, 154–160. Rohrmann, G.F., 2008. Baculovirus Molecular Biology. National Library of Medicine, National Center for Biotechnology Information, Bethesda http://www.ncbi. nlm.gov/books/NBK1736/ Surachetpong, W., Poulos, B.T., Tang, K.F.J., Lightner, D.V., 2005. Improvement of PCR method for the detection of monodon baculovirus (MBV) in penaeid shrimp. Aquaculture 249, 69–75. Van Engelenburg, F.A.C., Maes, R.K., Van Oirschot, J.T., Rijsewijk, F.A., 1993. Development of a rapid and sensitive polymerase chain reaction assay for detection of bovine herpesvirus type 1 in bovine semen. J. Clin. Microbiol. 31, 3129–3135. Wilhelm, S., Zimmermann, P., Selbitz, H.J., Truyen, U., 2006. Real-time PCR protocol for the detection of porcine parvovirus in field samples. J. Virol. Methods 134, 257–260. Witter, C.T., Herrmann, M.G., Moss, A.A., Rasmussen, R.P., 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22, 130–138. Yan, D., Tang, K.F., Lightner, D.V., 2009. Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp. J. Invertebr. Pathol. 102, 97–100.
