Indian Journal of Medical Microbiology, (2014) 32(2): 124-129
1
Original Article
Detection of Enterovirus 71 gene from clinical specimens by reverse‑transcription loop‑mediated isothermal amplification D Wang, X Wang, Y Geng, *C An
Abstract Purpose: The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop‑mediated isothermal amplification (LAMP) technique. Materials and Methods: A reverse‑transcription loop‑mediated isothermal amplification (RT‑LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT‑LAMP was tested, and the clinical specimens was assayed by the RT‑LAMP comparing with conventional reverse‑transcription polymerase chain reaction (RT‑PCR) and real‑time PCR. Results: A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT‑LAMP. The detection rate for EV71 was 56.89% by RT‑LAMP, 41.38% by real‑time PCR and 34.48% by RT‑PCR. The minimum detection limit of RT‑LAMP was 0.01 PFU, both of RT‑PCR and real‑time PCR was 0.1PFU. Non‑cross‑reactive amplification with other enteroviruses was detected in the survey reports. Conclusions: The effectiveness of RT‑LAMP is higher than RT‑PCR and real‑time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource‑poor countries or in developing countries. Key words: Clinical specimens, Enterovirus 71, hand foot and mouth disease, reverse transcription loop‑mediated isothermal amplification
Introduction Hand foot and mouth disease (HFMD) is a severe infective disease among young children. Several new epidemics of HFMD have broken out around the world[1,2] and a tendency of outbreak seems to rise significantly in China mainland in the recent years.[3,4] The data from Ministry of Health of China have indicated that HFMD is a quite common infectious disease of class C since 2008 (Official website of Chinese Centre for Disease Control of the People’s Republic of China). HFMD can be caused *Corresponding author (email: ) Department of Obstetrics and Gynecology (DW), Shengjing Affiliated Hospital of China Medical University, Shenyang 110004, Department of Medical Microbiology and Parasitology (DW, YG, CA), College of Basic Medical Sciences, China Medical University, Department of Endocrinology and Metabolism (XW), 1st Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Center for Disease Control and Prevention (YG), Shenyang 110005, China Received: 04‑03‑2013 Accepted: 27‑10‑2013 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.129779
by many species and different types of human enterovirues, such as human enterovirus 71 (EV71), coxsackievirus A16, A4, A5 (CVA16, CVA4, CVA5), etc., However, the EV71 has been recently identified as the primary and major pathogen of HFMD among young children, and could result in severe neuphrotic syndrome, even fatal.[5,6] Until now, the diagnosis of HFMD is meanly dependent on the clinical symptoms, detection of virus‑specific antibodies or virus culture and identification. But, the serodiagnosis is easy to cross‑response with other pathogen and the process of virus identification is time‑consuming. Reverse‑transcription polymerase chain reaction (RT‑PCR) and real‑time PCR have been recently reported to detect EV71 with high sensitivities and specificity.[7,8] However, these methods are limited in clinical applications because of the expensive equipment and complicated operation process. Therefore it is essential to develop a sensitive, specific and rapid approach to diagnose HFMD for an early treatment. Materials and Methods Samples A total of 116 specimens (from 116 patients), including 88 stool samples and 28 throat swabs were collected from clinically suspected HFMD patients in Liaoning province of China during February to October, 2009. The patients were selected following the guidelines for HFMD diagnosis from the Chinese Centre for Disease Control (China CDC) and the patients suffered from some typical symptoms of the disease, such as febricity, maculopapule and vesicular
April-June 2014
Wang, et al.: RT‑LAMP in diagnosis of HFMD
on the skin of hands and feet or palms and soles. All the patients are aged 1‑6 years; the throat swabs and stool samples were collected on the day of admission. All samples were collected between 2 and 5 days after the onset of illness. Samples were stored in 3‑5 ml of preservation solution (Hanks solution containing 10 μg/ml gentamicin and 0.25 μg/ml amphotericin B) at −70°C until the genomic viral RNA was extracted. The study was conducted in accordance with the amended declaration of Helsinki and was approved by the Provincial CDC Review Board. The clinical samples used in this study were appropriately coded to be anonymous, and guidelines were followed for use of clinical material and access to diagnostic results. RNA extraction Total RNA was isolated from 100 μl viral culture supernatant or 0.2 g of stool sample, which was suspended in 1.0 ml phosphate buffered saline (PBS) buffer, or 100 μl aliquot swab specimens from each patient, using RNeasy mini kit (QIAGEN Co., Ltd, Shanghai, China) following the instructions of manufacture. Total RNA were eluted in a final 60 μl of RNase‑free water and stored at −80°C. RT‑PCR The primers of RT‑PCP for EV71 were 5′‑ GCAGCCC AAAAGAACTTCAC‑3′ and 5′‑ ATTTCAGCAGCTTGG AGTGC‑3′. Conventional RT‑PCR was carried out as described by the one‑step RT‑PCR detection kit (Promega Beijing Biotech Co. Ltd, Beijing, China). All the protocols refer to “Guide for Control and Prevention of HFMD” issued by the Ministry of Public Health of China (China Ministry of Health, 2010). The 50 μl of mixture containing extracted RNA 5 μl, 10 × buffer 5 μl, dNTP 2 μl, 5 × enhancer 10 μl, Quant RTase 0.5 μl, Hotmaster Taq 2.5 μl, Rnasin 0.5 μl and EV71‑S and EV71‑A primer each 1 μl (20 pmol). The reaction was performed for 30 min at 50°C, 2 s at 45°C, 30 s at 70°C with 32 cycles of amplification, extensions at 72°C for 10 min. The product of amplification was checked by 1.5% agarose gel electrophoresis and EB staining. Real‑time PCR The real‑time PCR were carried out in the Light Cycler 480 (Roche, real time thermal cycler), according to the instruction of EV71 fluorescence PCR detection kit (Da An Gene Co., Ltd. of Sun Yat‑sen University, Guang Zhou, China). Briefly, total 20 µl mixture containing template RNA 5 μl, Taq DNA polymerases 1 μl, reverse transcriptase 1 μl, forward primer 0.2 µM and 0.2 µM reverse primer. The reaction was performed for 20 min at 42°C, 2 min at 93°C, following 40 cycles amplification of 95°C 30s, 55°C 45 s and 72°C, 30 s for denaturation, annealing and extension steps, respectively.
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Design of reverse‑transcription loop‑mediated isothermal amplification primers DNA Star was employed to analyse the gene nucleotide sequence of EV71 VP1 from GenBank (GQ121432). Three sets of universal primers, including inner primers (FIP, BIP), outer primers (F3, B3) and loop primers (Loop F, Loop B) targeting highly conserved immunogenic regions of VP1 were selected with the primer design software (LAMP Primer Explorer 4, Eiken Chemical Co. Ltd). The specificity of the primers was analysed online BLAST [Table 1]. The primers were synthesised by Sangon Biotech Co. Ltd., Shanghai, China. RT‑LAMP assay Total 25 μl amplification system consists of RNA template 2.5 μl, Bst DNA polymerase (8 U/μL, NEB Inc.) 1 μl, Bst DNA polymerase buffer (10×) 2.5 μl, AMV inverse transcriptase (10 U/μl, Promega Beijing Biotech Co.Ltd. Beijing, China) 1 μl, dNTP (10 mmol/μl) 1 μl, Betaine (5 mol/L, Sigma) 5 μl, MgSO4 (100 nmol/μl) 1.5 μl, RNase Inhibitor 0.5 μl, six primers corresponding to VP1 gene 1 μl each (5 pmol each of outer primers F3 and B3, 40 pmol each of inner primers FIP and BIP, 20 pmol each of loop primers LB and LF, respectively) and H2O 4 μl. The reaction mixture was incubated at 64°C for 60 min in thermal cycler. The products were analysed by agarose gel and capillary electrophoresis. Specificity of RT‑LAMP assay The specificity of the assay was evaluated by cross‑reactivity tests with RNA extracted from EV71 PV1, Coxsackie virus A16, Rotavirus, Norovirus, Sapovirus and Table 1: The sequences and genomic position of primers Primer Genome Length of Sequence (5′‑3′) list position oligonucleotides (bp) F3 386-404 19 TGCGCTTTGA TGCAGAGTT B3 571-590 20 GGTGACATGA ATGGCACTGA FIP 459-480, 42 GGCTCCAGGTG (F2, 4052424 GCACAAACATA‑ F1c) CACTTTTGTTG CGTGCACAC BIP 493-514, 42 TCTAGGGAATC (B1c, 539-558 CCTTGCATGGC‑ B2) AGGGTCTGACA GCTTAACAA LF 428-444 17 TGGGACAACT TCCCCGG LB 515-532 18 AAACCGCCAC TAACCCCT
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Astrovirus. All of the virus positive samples were confirmed by RT–PCR and kindly given as a present by Center for Disease Prevention and Control of Liaoning province of China.
limit of RT‑PCR and real‑time PCR was about 0.1PFU [Figure 3B and C].
Sensitivity of RT‑LAMP assay
RT‑LAMP, RT‑PCR and real‑time PCR were performed to analyse EV71 for 88 stool samples and 28 throat swabs [Figure 4]. Comparative analysis of positive detection rates was carried out with RT‑LAMP and compared with RT‑PCR or real‑time PCR, respectively. The results showed that the positive detection rate of RT‑LAMP for EV71 gene was significantly higher than RT‑PCR and real‑time PCR (c2 = 6.20, P ﹤ 0.05). The agreement analysis between different assays showed that Kappa = 0.188, P = 0.036 between RT‑LAMP and real‑time PCR assays, Kappa = 0.438, P = 0.000 between RT‑LAMP and RT‑PCR assays.
The EV71 PV1 was cultured in vero cells and the concentration of virus was determined by plaque assay. The sensitivity and the lowest detection limit of RT‑LAMP assay was tested with various concentrations of virus RNA, which ranged from 105 to 0.1 PFU/ml in a serial 10‑fold dilution (1.0 × 10‑1-1.0 × 10‑6). The sensitivity of RT‑LAMP assay was simultaneously compared with real‑time PCR and RT‑PCR. Statistical analysis
RT‑LAMP assay from clinical samples
Statistically significant of this study was evaluated by using Chi‑square test. The agreement analysis between different assays was measured. A significant difference was considered at the P value of less than 0.05.
The concordance and discordance of three assay methods for 36 cell culture positive samples, 28 from stool samples and 8 from swab samples, as shown in Table 2.
RESULTS
Discussion
Specificity of RT‑LAMP for EV71 detection
HFMD caused by EV71 is an emerging worldwide epidemic infection most commonly occurring in young children, especially pre‑school‑aged children.[1‑6] EV71 can cause severe neurological disease with complex transmission routes, rapid progression and high mortality. There is no vaccine or specific anti‑viral drugs against EV71 infection. Laboratory detection methods include viral isolation from cell cultures, serum antibody assay, nucleic acid detection by RT‑PCR. The time‑consuming cell culture analysis is complicated. Serological measurement cannot refrain from the effect of enterovirus cross‑reaction, and thus neither method is suitable for emergency diagnosis at the earlier stage of HFMD. Even though RT‑PCR is
The products of amplified were analysed by1.5% agarose gel electrophoresis and capillary electrophoresis. The specific nucleic acid of EV71 can be observed as a series of disparate long strand DNA. The ladder‑like mixture of stem‑loop DNAs was consistent with expected. The RT‑LAMP of EV71‑specific assay did not show any cross reactivity with Coxsackievirus A16, Rotavirus, Norovirus, Sapovirus or stellavirus [Figure 1]. Similar results also can be proved by capillary electrophoresis [Figure 2]. Sensitivity of RT‑LAMP for EV71 detection The typical amplified bands were observed for 10‑1–10‑5 serial dilution of specimen with signals gradually weaker. The minimum limit detection by RT‑LAMP assay was 0.01PFU (Figure 3A, Real time monitoring of RT‑LAMP see Supplemental Figures 1 and 2). The minimum
Figure 1: Specificity of RT‑LAMP for Human Enterovirus 71 The specific product of EV71 nucleic acid specimen can be observed as a series of disparate length macromolecular, long strand DNA in the 1.5% agarose gel electrophoresis. The ladder‑like mixture of stem‑loop DNAs was consistent with theoretical results. The RT‑LAMP primers for EV71 did not show any specific bands or cross reactivity of Coxsackievirus A16, Rotavirus, Norovirus, Sapovirus or stellavirusis.
Figure 2: Specificity detection of Human Enterovirus 71 gene by RT‑LAMP with Capillary electrophoresis. Capillary electrophoresis was used to analyse the amplification products performed by RT‑LAMP. Nucleic acid of EV71 showed (1) a series of disparate length macromolecular. The ladder‑like mixture of stem‑loop DNAs was consistent with theoretical consequence. RT‑LAMP primers for EV71 did not show any specific bands or cross reactivity of Coxsackievirus A16 (2), Rotavirus (3), Norovirus (4), Sapovirus (5) and negative control (6). M: 50 bp Marker.
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April-June 2014
Wang, et al.: RT‑LAMP in diagnosis of HFMD
a
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b
c
Figure 3: Sensitivity of three methods for detection EV71 (A) Sensitivity of RT‑LAMP for detection EV71, the detection limit is 0.01PFU, (B) Sensitivity of RT‑PCR for detection EV71, the detection limit is 0.1PFU, (C) Sensitivity of real‑time PCR for detection EV71, the detection limit is 0.1PFU
Figure 4: Comparison of detection rates with three assay methods from clinical samples Total of 116 clinical samples were collected from suspected HFMD infant patients. The assay results exhibited a higher detection rate by RT‑LAMP (56.9%, 66/116) than real‑time PCR (43.1%, 50/116) and RT‑PCR 34.5%, 40/116), (P 0.05
respectively, in cell culture positive samples, the negative cases may be infected by the CA16 or other human enterovirus. We also noticed there were several publications on the evaluation of the LAMP assay for the detection of EV71 in the region of China, but all of the cases were from south of China, our cases were from north‑east of China. And we used different sequences in VP1 to attempt getting more sensitive and specific result compared with other researches. Moreover, in our research we compared the samples of stool and throat swab by the three assays and proved RT‑LAMP is effective in detecting both stool and throat swab samples.
www.ijmm.org
April-June 2014
Wang, et al.: RT‑LAMP in diagnosis of HFMD
In conclusion, the detecting rate of RT‑LAMP is higher, the protocol is easy to operate and time saving, which can be finished in single condition within 60 min, and no expensive instrument was needed. It can be applied for rapid diagnosis of the disease, especially in poor areas or developing countries. Acknowledgements The authors thank all of the members of our laboratories for their helpful assistance.
References 1. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 2010;10:778‑90. 2. Ryu WS, Kang B, Hong J, Hwang S, Kim J, Cheon DS. Clinical and etiological characteristics of enterovirus 71‑related diseases during a recent 2‑year period in Korea. J Clin Microbiol 2010;48:2490‑4. 3. Yang F, Ren L, Xiong Z, Li J, Xiao Y, Zhao R, et al. Enterovirus 71 outbreak in the People’s Republic of China in 2008. J Clin Microbiol 2009;47:2351‑2. 4. Hu, XY, Zheng X, Wen ZF. Investigation of epidemiology of hand‑foot‑mouth disease in 2346 patients. Chin J Nosocomiol 2012;22:2333‑5. 5. Ni H, Yi B, Yin J, Fang T, He T, Du Y, et al. Epidemiological and etiological characteristics of hand, foot, and mouth disease in Ningbo, China, 2008‑2011. J Clin Virol 2012;54:342‑8.
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6. Deng AP, Zhang YH, Sun LM. Survey on the recessive infection of pathogen to hand‑foot‑mouth disease among healthy adults and children in Guangdong province. Zhozhonghua Liu Xing Bing Xue Za Zhi 2012;33:189‑91. 7. Chen TC, Chen GW, Hsiung CA, Yang JY, Shih SR, Lai YK, et al. Combining multiplex reverse transcription‑PCR and a diagnostic microarray to detect and differentiate enterovirus 71 and coxsackievirus A16. J Clin Microbiol 2006;44:2212‑9. 8. Chiang PS, Huang ML, Luo ST, Lin TY, Tsao KC, Lee MS. Comparing molecular methods for early detection and serotyping of enteroviruses in throat swabs of pediatric patients. PLoS One 2012;7:e48269. 9. Oberste MS, Maher K, Kilpatrick DR, Pallansch MA. Molecular evolution of the human enteroviruses: Correlation of serotype with VP1 sequence and application to picornavirus classification. J Virol 1999;73:1941‑8. 10. Shih SR, Li YS, Chiou CC, Suen PC, Lin TY, Chang LY, et al. Expression of capsid proteinVP1for use as antigen for the diagnosis of enterovirus71 infection. J Med Virol 2000;61:228‑34. How to cite this article: Wang D, Wang X, Geng Y, An C. Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification. Indian J Med Microbiol 2014;32:124-9. Source of Support: This work was supported by the National Natural Science Foundation of China (NSFC 81200653) and Natural Science Funds of Liaoning province, China (2011225020 and 20111108). Conflict of Interest: We declare that we have no potential conflicts of interest relevant to this article.
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1
Original Article
Detection of Enterovirus 71 gene from clinical specimens by reverse‑transcription loop‑mediated isothermal amplification D Wang, X Wang, Y Geng, *C An
Abstract Purpose: The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop‑mediated isothermal amplification (LAMP) technique. Materials and Methods: A reverse‑transcription loop‑mediated isothermal amplification (RT‑LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT‑LAMP was tested, and the clinical specimens was assayed by the RT‑LAMP comparing with conventional reverse‑transcription polymerase chain reaction (RT‑PCR) and real‑time PCR. Results: A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT‑LAMP. The detection rate for EV71 was 56.89% by RT‑LAMP, 41.38% by real‑time PCR and 34.48% by RT‑PCR. The minimum detection limit of RT‑LAMP was 0.01 PFU, both of RT‑PCR and real‑time PCR was 0.1PFU. Non‑cross‑reactive amplification with other enteroviruses was detected in the survey reports. Conclusions: The effectiveness of RT‑LAMP is higher than RT‑PCR and real‑time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource‑poor countries or in developing countries. Key words: Clinical specimens, Enterovirus 71, hand foot and mouth disease, reverse transcription loop‑mediated isothermal amplification
Introduction Hand foot and mouth disease (HFMD) is a severe infective disease among young children. Several new epidemics of HFMD have broken out around the world[1,2] and a tendency of outbreak seems to rise significantly in China mainland in the recent years.[3,4] The data from Ministry of Health of China have indicated that HFMD is a quite common infectious disease of class C since 2008 (Official website of Chinese Centre for Disease Control of the People’s Republic of China). HFMD can be caused *Corresponding author (email: ) Department of Obstetrics and Gynecology (DW), Shengjing Affiliated Hospital of China Medical University, Shenyang 110004, Department of Medical Microbiology and Parasitology (DW, YG, CA), College of Basic Medical Sciences, China Medical University, Department of Endocrinology and Metabolism (XW), 1st Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Center for Disease Control and Prevention (YG), Shenyang 110005, China Received: 04‑03‑2013 Accepted: 27‑10‑2013 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.129779
by many species and different types of human enterovirues, such as human enterovirus 71 (EV71), coxsackievirus A16, A4, A5 (CVA16, CVA4, CVA5), etc., However, the EV71 has been recently identified as the primary and major pathogen of HFMD among young children, and could result in severe neuphrotic syndrome, even fatal.[5,6] Until now, the diagnosis of HFMD is meanly dependent on the clinical symptoms, detection of virus‑specific antibodies or virus culture and identification. But, the serodiagnosis is easy to cross‑response with other pathogen and the process of virus identification is time‑consuming. Reverse‑transcription polymerase chain reaction (RT‑PCR) and real‑time PCR have been recently reported to detect EV71 with high sensitivities and specificity.[7,8] However, these methods are limited in clinical applications because of the expensive equipment and complicated operation process. Therefore it is essential to develop a sensitive, specific and rapid approach to diagnose HFMD for an early treatment. Materials and Methods Samples A total of 116 specimens (from 116 patients), including 88 stool samples and 28 throat swabs were collected from clinically suspected HFMD patients in Liaoning province of China during February to October, 2009. The patients were selected following the guidelines for HFMD diagnosis from the Chinese Centre for Disease Control (China CDC) and the patients suffered from some typical symptoms of the disease, such as febricity, maculopapule and vesicular
April-June 2014
Wang, et al.: RT‑LAMP in diagnosis of HFMD
on the skin of hands and feet or palms and soles. All the patients are aged 1‑6 years; the throat swabs and stool samples were collected on the day of admission. All samples were collected between 2 and 5 days after the onset of illness. Samples were stored in 3‑5 ml of preservation solution (Hanks solution containing 10 μg/ml gentamicin and 0.25 μg/ml amphotericin B) at −70°C until the genomic viral RNA was extracted. The study was conducted in accordance with the amended declaration of Helsinki and was approved by the Provincial CDC Review Board. The clinical samples used in this study were appropriately coded to be anonymous, and guidelines were followed for use of clinical material and access to diagnostic results. RNA extraction Total RNA was isolated from 100 μl viral culture supernatant or 0.2 g of stool sample, which was suspended in 1.0 ml phosphate buffered saline (PBS) buffer, or 100 μl aliquot swab specimens from each patient, using RNeasy mini kit (QIAGEN Co., Ltd, Shanghai, China) following the instructions of manufacture. Total RNA were eluted in a final 60 μl of RNase‑free water and stored at −80°C. RT‑PCR The primers of RT‑PCP for EV71 were 5′‑ GCAGCCC AAAAGAACTTCAC‑3′ and 5′‑ ATTTCAGCAGCTTGG AGTGC‑3′. Conventional RT‑PCR was carried out as described by the one‑step RT‑PCR detection kit (Promega Beijing Biotech Co. Ltd, Beijing, China). All the protocols refer to “Guide for Control and Prevention of HFMD” issued by the Ministry of Public Health of China (China Ministry of Health, 2010). The 50 μl of mixture containing extracted RNA 5 μl, 10 × buffer 5 μl, dNTP 2 μl, 5 × enhancer 10 μl, Quant RTase 0.5 μl, Hotmaster Taq 2.5 μl, Rnasin 0.5 μl and EV71‑S and EV71‑A primer each 1 μl (20 pmol). The reaction was performed for 30 min at 50°C, 2 s at 45°C, 30 s at 70°C with 32 cycles of amplification, extensions at 72°C for 10 min. The product of amplification was checked by 1.5% agarose gel electrophoresis and EB staining. Real‑time PCR The real‑time PCR were carried out in the Light Cycler 480 (Roche, real time thermal cycler), according to the instruction of EV71 fluorescence PCR detection kit (Da An Gene Co., Ltd. of Sun Yat‑sen University, Guang Zhou, China). Briefly, total 20 µl mixture containing template RNA 5 μl, Taq DNA polymerases 1 μl, reverse transcriptase 1 μl, forward primer 0.2 µM and 0.2 µM reverse primer. The reaction was performed for 20 min at 42°C, 2 min at 93°C, following 40 cycles amplification of 95°C 30s, 55°C 45 s and 72°C, 30 s for denaturation, annealing and extension steps, respectively.
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Design of reverse‑transcription loop‑mediated isothermal amplification primers DNA Star was employed to analyse the gene nucleotide sequence of EV71 VP1 from GenBank (GQ121432). Three sets of universal primers, including inner primers (FIP, BIP), outer primers (F3, B3) and loop primers (Loop F, Loop B) targeting highly conserved immunogenic regions of VP1 were selected with the primer design software (LAMP Primer Explorer 4, Eiken Chemical Co. Ltd). The specificity of the primers was analysed online BLAST [Table 1]. The primers were synthesised by Sangon Biotech Co. Ltd., Shanghai, China. RT‑LAMP assay Total 25 μl amplification system consists of RNA template 2.5 μl, Bst DNA polymerase (8 U/μL, NEB Inc.) 1 μl, Bst DNA polymerase buffer (10×) 2.5 μl, AMV inverse transcriptase (10 U/μl, Promega Beijing Biotech Co.Ltd. Beijing, China) 1 μl, dNTP (10 mmol/μl) 1 μl, Betaine (5 mol/L, Sigma) 5 μl, MgSO4 (100 nmol/μl) 1.5 μl, RNase Inhibitor 0.5 μl, six primers corresponding to VP1 gene 1 μl each (5 pmol each of outer primers F3 and B3, 40 pmol each of inner primers FIP and BIP, 20 pmol each of loop primers LB and LF, respectively) and H2O 4 μl. The reaction mixture was incubated at 64°C for 60 min in thermal cycler. The products were analysed by agarose gel and capillary electrophoresis. Specificity of RT‑LAMP assay The specificity of the assay was evaluated by cross‑reactivity tests with RNA extracted from EV71 PV1, Coxsackie virus A16, Rotavirus, Norovirus, Sapovirus and Table 1: The sequences and genomic position of primers Primer Genome Length of Sequence (5′‑3′) list position oligonucleotides (bp) F3 386-404 19 TGCGCTTTGA TGCAGAGTT B3 571-590 20 GGTGACATGA ATGGCACTGA FIP 459-480, 42 GGCTCCAGGTG (F2, 4052424 GCACAAACATA‑ F1c) CACTTTTGTTG CGTGCACAC BIP 493-514, 42 TCTAGGGAATC (B1c, 539-558 CCTTGCATGGC‑ B2) AGGGTCTGACA GCTTAACAA LF 428-444 17 TGGGACAACT TCCCCGG LB 515-532 18 AAACCGCCAC TAACCCCT
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Astrovirus. All of the virus positive samples were confirmed by RT–PCR and kindly given as a present by Center for Disease Prevention and Control of Liaoning province of China.
limit of RT‑PCR and real‑time PCR was about 0.1PFU [Figure 3B and C].
Sensitivity of RT‑LAMP assay
RT‑LAMP, RT‑PCR and real‑time PCR were performed to analyse EV71 for 88 stool samples and 28 throat swabs [Figure 4]. Comparative analysis of positive detection rates was carried out with RT‑LAMP and compared with RT‑PCR or real‑time PCR, respectively. The results showed that the positive detection rate of RT‑LAMP for EV71 gene was significantly higher than RT‑PCR and real‑time PCR (c2 = 6.20, P ﹤ 0.05). The agreement analysis between different assays showed that Kappa = 0.188, P = 0.036 between RT‑LAMP and real‑time PCR assays, Kappa = 0.438, P = 0.000 between RT‑LAMP and RT‑PCR assays.
The EV71 PV1 was cultured in vero cells and the concentration of virus was determined by plaque assay. The sensitivity and the lowest detection limit of RT‑LAMP assay was tested with various concentrations of virus RNA, which ranged from 105 to 0.1 PFU/ml in a serial 10‑fold dilution (1.0 × 10‑1-1.0 × 10‑6). The sensitivity of RT‑LAMP assay was simultaneously compared with real‑time PCR and RT‑PCR. Statistical analysis
RT‑LAMP assay from clinical samples
Statistically significant of this study was evaluated by using Chi‑square test. The agreement analysis between different assays was measured. A significant difference was considered at the P value of less than 0.05.
The concordance and discordance of three assay methods for 36 cell culture positive samples, 28 from stool samples and 8 from swab samples, as shown in Table 2.
RESULTS
Discussion
Specificity of RT‑LAMP for EV71 detection
HFMD caused by EV71 is an emerging worldwide epidemic infection most commonly occurring in young children, especially pre‑school‑aged children.[1‑6] EV71 can cause severe neurological disease with complex transmission routes, rapid progression and high mortality. There is no vaccine or specific anti‑viral drugs against EV71 infection. Laboratory detection methods include viral isolation from cell cultures, serum antibody assay, nucleic acid detection by RT‑PCR. The time‑consuming cell culture analysis is complicated. Serological measurement cannot refrain from the effect of enterovirus cross‑reaction, and thus neither method is suitable for emergency diagnosis at the earlier stage of HFMD. Even though RT‑PCR is
The products of amplified were analysed by1.5% agarose gel electrophoresis and capillary electrophoresis. The specific nucleic acid of EV71 can be observed as a series of disparate long strand DNA. The ladder‑like mixture of stem‑loop DNAs was consistent with expected. The RT‑LAMP of EV71‑specific assay did not show any cross reactivity with Coxsackievirus A16, Rotavirus, Norovirus, Sapovirus or stellavirus [Figure 1]. Similar results also can be proved by capillary electrophoresis [Figure 2]. Sensitivity of RT‑LAMP for EV71 detection The typical amplified bands were observed for 10‑1–10‑5 serial dilution of specimen with signals gradually weaker. The minimum limit detection by RT‑LAMP assay was 0.01PFU (Figure 3A, Real time monitoring of RT‑LAMP see Supplemental Figures 1 and 2). The minimum
Figure 1: Specificity of RT‑LAMP for Human Enterovirus 71 The specific product of EV71 nucleic acid specimen can be observed as a series of disparate length macromolecular, long strand DNA in the 1.5% agarose gel electrophoresis. The ladder‑like mixture of stem‑loop DNAs was consistent with theoretical results. The RT‑LAMP primers for EV71 did not show any specific bands or cross reactivity of Coxsackievirus A16, Rotavirus, Norovirus, Sapovirus or stellavirusis.
Figure 2: Specificity detection of Human Enterovirus 71 gene by RT‑LAMP with Capillary electrophoresis. Capillary electrophoresis was used to analyse the amplification products performed by RT‑LAMP. Nucleic acid of EV71 showed (1) a series of disparate length macromolecular. The ladder‑like mixture of stem‑loop DNAs was consistent with theoretical consequence. RT‑LAMP primers for EV71 did not show any specific bands or cross reactivity of Coxsackievirus A16 (2), Rotavirus (3), Norovirus (4), Sapovirus (5) and negative control (6). M: 50 bp Marker.
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April-June 2014
Wang, et al.: RT‑LAMP in diagnosis of HFMD
a
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b
c
Figure 3: Sensitivity of three methods for detection EV71 (A) Sensitivity of RT‑LAMP for detection EV71, the detection limit is 0.01PFU, (B) Sensitivity of RT‑PCR for detection EV71, the detection limit is 0.1PFU, (C) Sensitivity of real‑time PCR for detection EV71, the detection limit is 0.1PFU
Figure 4: Comparison of detection rates with three assay methods from clinical samples Total of 116 clinical samples were collected from suspected HFMD infant patients. The assay results exhibited a higher detection rate by RT‑LAMP (56.9%, 66/116) than real‑time PCR (43.1%, 50/116) and RT‑PCR 34.5%, 40/116), (P 0.05
respectively, in cell culture positive samples, the negative cases may be infected by the CA16 or other human enterovirus. We also noticed there were several publications on the evaluation of the LAMP assay for the detection of EV71 in the region of China, but all of the cases were from south of China, our cases were from north‑east of China. And we used different sequences in VP1 to attempt getting more sensitive and specific result compared with other researches. Moreover, in our research we compared the samples of stool and throat swab by the three assays and proved RT‑LAMP is effective in detecting both stool and throat swab samples.
www.ijmm.org
April-June 2014
Wang, et al.: RT‑LAMP in diagnosis of HFMD
In conclusion, the detecting rate of RT‑LAMP is higher, the protocol is easy to operate and time saving, which can be finished in single condition within 60 min, and no expensive instrument was needed. It can be applied for rapid diagnosis of the disease, especially in poor areas or developing countries. Acknowledgements The authors thank all of the members of our laboratories for their helpful assistance.
References 1. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 2010;10:778‑90. 2. Ryu WS, Kang B, Hong J, Hwang S, Kim J, Cheon DS. Clinical and etiological characteristics of enterovirus 71‑related diseases during a recent 2‑year period in Korea. J Clin Microbiol 2010;48:2490‑4. 3. Yang F, Ren L, Xiong Z, Li J, Xiao Y, Zhao R, et al. Enterovirus 71 outbreak in the People’s Republic of China in 2008. J Clin Microbiol 2009;47:2351‑2. 4. Hu, XY, Zheng X, Wen ZF. Investigation of epidemiology of hand‑foot‑mouth disease in 2346 patients. Chin J Nosocomiol 2012;22:2333‑5. 5. Ni H, Yi B, Yin J, Fang T, He T, Du Y, et al. Epidemiological and etiological characteristics of hand, foot, and mouth disease in Ningbo, China, 2008‑2011. J Clin Virol 2012;54:342‑8.
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6. Deng AP, Zhang YH, Sun LM. Survey on the recessive infection of pathogen to hand‑foot‑mouth disease among healthy adults and children in Guangdong province. Zhozhonghua Liu Xing Bing Xue Za Zhi 2012;33:189‑91. 7. Chen TC, Chen GW, Hsiung CA, Yang JY, Shih SR, Lai YK, et al. Combining multiplex reverse transcription‑PCR and a diagnostic microarray to detect and differentiate enterovirus 71 and coxsackievirus A16. J Clin Microbiol 2006;44:2212‑9. 8. Chiang PS, Huang ML, Luo ST, Lin TY, Tsao KC, Lee MS. Comparing molecular methods for early detection and serotyping of enteroviruses in throat swabs of pediatric patients. PLoS One 2012;7:e48269. 9. Oberste MS, Maher K, Kilpatrick DR, Pallansch MA. Molecular evolution of the human enteroviruses: Correlation of serotype with VP1 sequence and application to picornavirus classification. J Virol 1999;73:1941‑8. 10. Shih SR, Li YS, Chiou CC, Suen PC, Lin TY, Chang LY, et al. Expression of capsid proteinVP1for use as antigen for the diagnosis of enterovirus71 infection. J Med Virol 2000;61:228‑34. How to cite this article: Wang D, Wang X, Geng Y, An C. Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification. Indian J Med Microbiol 2014;32:124-9. Source of Support: This work was supported by the National Natural Science Foundation of China (NSFC 81200653) and Natural Science Funds of Liaoning province, China (2011225020 and 20111108). Conflict of Interest: We declare that we have no potential conflicts of interest relevant to this article.
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