Original Paper
Intervirology 1992;34:184-191
N. Jothikumar P Khanna S. Kamatchiammal R. Paul Murugan
Rapid Detection of Waterborne Viruses Using the Polymerase Chain Reaction and a Gene Probe
National Environmental Engineering Research Institute, Nagpur, India
Introduction Waterborne viruses, especially the entero viruses, are the most common causative agents of childhood infection. The WHO Scientific Group [1] has concluded that the presence of even a few enteric viruses in a large volume of drinking water poses a threat to public health. In the absence of a reliable, reproducible and rapid method for detecting viruses in water and subsequent corrective measures, in less devel oped countries the mortality and morbidity due to waterborne viruses are high. The standard methods [2] for detecting viruses by inoculation in cell culture require 6-10 days, thereby defeat
Received: July 7,1992 Accepted: February 7,1993
ing the very purpose of analysis with respect to preventive measures. Furthermore, some viruses are not amenable to culture while others are expensive to culture. Alternative detection techniques have been reported for viruses but require high titres in the range of 10,000 viral particles. In addition, the gene probes detect around 1 pg of viral RN A /D N A equivalent to 10,000 infectious units [3]. Thus, there is an urgent need for rapid and sensitive techniques for detecting lower viral titres to enable preven tive measures in water supply practices. With the advent of recombinant DNA tech nology, it is now possible to detect waterborne viruses [3-7] without the need for tissue culture.
Prof. P. Khanna Director National Environmental Engineering Research Institute Nagpur 440 020 (India)
©1992 S. Karger AG, Basel 0300-5526/92/ 0344-0184S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
Key Words Waterborne viruses Poliovirus 1 Virus concentration Detection Gene amplification Gene probe
Summary We describe a membrane-filter-based urea-arginine phosphate buffer method for concentrating waterborne viruses from large volumes of water to microlitre volumes, and their subsequent detection by the polymerase chain reaction (PCR). The detec tion step involves the extraction of RNA, synthesis of com plementary DNA, amplification by PCR of target DNA with specific primers, and confirmation through nucleic acid hybridi zation with a radiolabelled oligonucleotide probe. The PCR technique detected the presence of enteroviruses in spiked as well as in contaminated water samples. The technique is sensi tive and detects as few as 120 waterborne viral particles. PCR is simple, rapid, sensitive, specific and adaptable for water quality surveillance in less developed countries.
Table 1. Bacteriological quali fy 0f water and sewage samples
Most probable number per 100 ml Sampling points
TC
FC
Hand pump water 4.9x 10 1.7x 10 Open-well water (Open dug) 2 .2x10s l.lx lO 5 Sewage l.l x I0X 7 .0 x l0 7
EC
FS
1.3x10 4.9 xIO4 7.0x107
2.3x10 5.4x103 7 .9 x l0 J
TC = Total Coliforms; FC = faecal conforms: EC = E. coli; FS = faecal streptococci.
Materials and Methods Enteroviruses Poliovirus l was chosen as a model virus in ihis study for optimizing the experimental protocol, because it is present in large numbers in contaminated water particu larly in less developed countries. It also has the advan tage ofbeing easy to quantitate through plaqueassay. In addition, the complete nucleotide sequences of the genomes of the different poliovirus serotypes are avail able in the published literature [I7], A poliovirus I sus
pension was thawed and frozen three times repeatedly to release virions from infected cells, and viral stocks of known titre (12 x l05/m l) were diluted and enumerated by the agar overlay plaque assay technique as described by Jothikumar et al. [I8). Coxsackievirus B5 was propa gated in HeLa cell monolayer culture. Adsorption o f Viruses from Water Samples onto Membrane Filter For the initial experiments, 5-litre water samples were conditioned to pH 3.5 with I N HCI, and AICI, was added to a final molarity o f0.0005 M. I ml of poliovirus l suspension of different log dilutions as added to the sample and mixed for I0 min with a magnetic stirrer. Water samples were then passed through a millipore HA-type cellulose nitrate membrane filter of 0.45 pm pore size for virus adsorption. The remainder of the viral inoculum was enumerated on cell lines [18], and plaque counts were expressed as PFU /l. Both hand pump and well water sources located within 30 m of an open sewerage system in Madras, India, were examined for bacteriological quality using standard methods [2], and analysis of the data (table I) reveals that the water sources were highly contaminated by groundwater pol lution. Water samples of 50 I each from hand pump water and open well water were concentrated by twostep concentration and inoculated into cell culture to raise the titre, because no amplification could be achieved directly except from the sewage sample. A sewage sample of 500 ml was blended to desorb viruses from organic matter and centrifuged at 5,000 g for 20 min to remove solids. Further concentration of superna tant by membrane-filter-based urea-arginine phosphate buffer (U-APB) was carried out as in water samples, followed by nucleic acid extraction and PCR amplifica tion.
185
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
The polymerase chain reaction (PCR) has the advantage of amplifying even one genome copy exponentially to several million in a few hours without culturing the viruses [8-10], Due to the high sensitivity [ll] PCR detection of viruses in drinking water has great potential. Several researchers have reported the use of PCR for detecting enterovirus in clinical sam ples [12-I4], rotavirus [8, 15], and hepatitis E virus [16]. Notwithstanding these advances, no literature citations are available on the use of PCR for detecting waterborne viruses in envi ronmental samples, mainly due to the presence of low viral titres in drinking-water samples. We describe here a method for concentrat ing viruses from a large volume of water to a microlitre quantity, and a highly specific and sensitive technique for detection through nu cleic acid amplification with PCR, followed by confirmation with a radiolabelled oligonucleo tide probe.
Table 2. Recovery of poliovi rus I from water samples during the concentration procedure
Observation number
1 2 3 4 5
Input PFU/I
265 195 182 360 218
M ean±SD
Recovery. % second-step concentration1
Third-step concentration2
80.70 84.09 82.40 83.30 82.56
64.15 71.79 65.93 71.11 70.19
82.6± 1.3
68.6±3.4
1 After membrane filtration adsorption, viruses were eluted with U-APB. reconcentrated with MgCE and dissolved in 4 ml of Mcllvaines buffer, followed by enumeration. 2 4 ml Mcllvaines buffer were filtered and eluted with U-APB, recon centrated with MgCl2 and dissolved in 400 pi of Mcllvaines buffer follo wed by enumeration.
Third-Step Concentration o f Viruses to a Microlitre Volume Viruses concentrated in 4 ml of Mcllvaine’s buffer were passed through a swinnex filter containing a 0.45iim membrane filter, using a syringe. Adsorbed viruses were eluted with 5 ml of U-APB and the eluate was precipitated by the addition of 50 pi of I M M gC k The precipitate was pelleted by centrifugation at 7,000 rpm for 30 min. The pellet was dissolved in 400-600 pi of Mcllvaine’s buffer and transferred to 2-mI siliconised Eppendorf centrifuge tubes. In order to determine the percentage recovery of virus in water at each stage of the concentration proce dure, poliovirus I (182-360 PFU/ml) was added to 11of water and concentrated as described previously. The percentage recovery was recorded by enumerating on cell lines throughout the concentration procedure to
186
assess the sensitivity of the method in terms of the virus concentration in the original sample. The proposed U-APB method gives 82.6 and 68.6% recovery of polio virus I at the second and third steps of the concentration procedure respectively (table 2). Extraction o f RNA Virus titres > 120 PFU per 400-600 pi volume were treated with SDS and proteinase K to a final concentra tion of 0.5% and 5 mg/ml, respectively, followed by extraction with a phenol: chloroform isoamyl alcohol (PCI) mixture. The procedure is summarised in figure 1. Samples with viral titres < 120 PFU per 400-600 pi were inoculated into cell cultures to raise the titre value, and the cytopathic effect was observed after overnight infection. Culture bottles were frozen and thawed three times. Approximately 100 pi of cell lysate were boiled for 2 min and ice-chilled immediately. Twenty microli tres were used forcDN A synthesis in a 500-pl microfuge tube with a final reaction volume of 50 pi. Primers Enterovirus primers and probe were chosen to am plify a part of the 5' terminal non-coding part and a region of the single-stranded enteroviral genome encod ing non-structural proteins which is highly conserved among human picomaviruses [12], The downstream primer 3' ACC GAC GAA TAC CAC TGTTA located between 584-603 and the upstream primer 5' CC’TCCG GCC CCT GAA TGC GGC TAAT, located between
Jothikum ar/Khanna/Kam atchiam m al/ Murugan
Detection of Waterborne Viruses with PC R
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
EluIion o f Virus with U-APB Adsorbed viruses were eluted with 50 ml of U-APB (1.5 Murea:0.02 M arginine:0.008 M phosphate) at pH 9.0. The buffer was constituted by adding 4.5 g freshly dissolved urea, 2 ml 0.2 M sodium dihydrogen phos phate dihydrate and 2 ml of 0.5 M L-arginine to 42 ml of double-distilled water. The eluate was further reconcen trated by the addition of 0.5 ml of I M M gCk Samples were mixed in a beaker with a magnetic stirrer. The resultant precipitate was recovered through centrifuga tion at 5,000 gfor 30 min and the pellet was resuspended in 4 ml of Mcllvaine's buffer (pH 5.0).
Water sample 1 «•- HCI + AICI3 Adjust pH to 3.5 and 0.0005 M AICI3
4
Pass through membrane filter (0.45 pm. 142 mm diameter) 4 Discard filtrate Elute with 50 ml U-APB
4
Add 0.5 ml 1 M MgCI2 to eluate
4
Stir for 2 min
4 Centrifuge (5,000 g, 30 min) 4 - * Discard supernatant Dissolve pellet In 4 ml Mcllvaines buffer (pH 5.0)
4
Pass through smaller diameter membrane filter (0.45 pm. 13 mm diameter) 4 -■» Discard filtrate Elute with 5 ml U-APB
4
Add 50 pi 1 M MgCI2 to eluate
4
Centrifuge (7,000 g, 30 min) 4 - » Discard supernatant Dissolve pellet in 400-600 pi Mcllvaines buffer (pH 5.0)
4
Treat with proteinase/SDS
4
Incubate at 37 "C
4
Add equal volume of PCI
4
Vortex and centrifuge at 15.000 g, 10 min
4 4
“ l
Organic phase
Aqueous phase
I
Re-extract with equal volume of 10 mMTris HCI. 100 m M NaCI, 1 mM EDTA
I Centrifuge 15,000 g, 10 min
I
Aqueous phase
4
Mix two aqueous phases
4
Add ammonium acetate and 2.5 volumes of cold ethanol ( 100%)
4
Overnight precipitation at -20 'C
4
Centrifuge (15,000 g. 30 min)
4 Precipitate dissolved in 20 pi of Tris EDTA buffer
4
cDNA synthesis
4
4“ Agarose-gel ethidium bromide staining
4
Southern hybridization
“1 Slot blot analysis
187
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
Fig-1- Procedure for concentra tion, amplification and detection of viruses from contaminated water samples.
PCR amplification |
cDNA Synthesis cDNA synthesis was carried out in a final volume of 50 pi of 1x reverse transcriptase (RTase) buffer (5 x RTase buffer: 250 mMTris HC1 (pH 7.6), 300 mM KCI, 50 mMMgCI2, 5 mM DTT, 250 ul/100 ml vanadyl ribonucleoside complex), 4 pi containing 10 u M of each dNTP, 2 ul of 25 ng/pl downstream primer, 20 ul of test sample and 10 pi containing 5 units of AMV transcrip tase. The reaction mixture was incubated at 37 °C for 75 min, the reaction stopped by boiling or heat denaturing at 96°C for 2 min, and the reaction tube quick-chilled on ice. PCR Amplification was performed on the reverse-tran scription cDNA product (20 ul in a final volume of 100 pi containing 10 pi of 10 x PCR buffer [I x PCR buffer: 50 mM KCI, 10 mMTris HC1 (pH 8.3 at 25°C), 1.5 mM MgCI2, 100 pg/ml gelatin], 2 units of Taq polymerase, 2 pi each of upstream and downstream primers, 200 mM of 2 pi dNTP concentration of each nucleotide, and 10 pi ofl0% bovine serum albumin. The reaction mixture was finally overlaid with 100 pi of light mineral oil to prevent evaporation. The amplification reactions were per formed through 40 cycles of alternating temperature with each cycle consisting of denaturing at 94°C for 2 min, annealing at 50 °C for 2 min, and extension at 72°C for 3 min. After 40 cycles, the DNA was extended for an additional 10 min at 72 °C. Southern Transfer and Hybridization 10 pi of amplified PCR product were electrophoresed on a 2% agarose gel, photographed under UV light, and transferred to nitrocellulose membrane for hybridization according to the procedure of Maniatis et al. [19], and then fixed by baking at 80"C for 2 h. After prehybridization (6 x SSC, 1 x Denhardt’s solution and 0.3% SDS) at 60°C for 4 h the filter was hybridized overnight with fresh buffer containing 50 pg/ml salmon sperm DNA and I06 c, p, m/m l of y 32P ATP end-la belled (polynucleotide kinase) probe at 60°C. After six washing steps (three times in 2 x SSC, 0,1% SDS at room temperature for 10 min and thrice in 0.1 x SSC,0.1%SDS
188
at 50“C for 10 min) the filter was air dried and exposed to X-ray film.
Results The procedure for concentrating viruses from contaminated water samples, cDNA syn thesis, PCR amplification and detection is shown in figure 1. Primers selected from highly conserved regions of the genome could amplify both poliovirus 1 and Coxsackievirus B5, as depicted in figure 2. None of the negative con trols in the experiments resulted in false posi tives, and no spurious bands representing non specific amplification were observed. No am plification was observed with rotavirus and bacteriophage when the same procedure with the same set of primers was used. Carry-over DNA leading to false positives was prevented by exercising strict precautions including the control experiments in which the reverse tran scriptase of RNA was excluded, giving no sig nal after amplification, indicating absence of contamination in all the experiments. In all the PCR-positive samples, a distinct 154-bp PCR product was visible on the agarose gel when stained with ethidium bromide. The identify of the 154-bp band was further con firmed by Southern hybridization analysis with a 32P-labelled probe. To assess the sensitivity of the PCR technique, log dilutions of poliovirus (1,200, 600, 300, and 120 PFU viral titre) were added to water samples. The effectiveness of PCR for detecting virus in spiked water sam ples with only 20 ul cDNA used for PCR ampli fication corresponding to titre values of 480, 240,120 and 48 PFU/1, respectively is shown in figure 3. The sensitivity of the proposed detec tion scheme was 120 PFU viral titre, and no amplification was observed in lane E for < 120 PFU viral titre. Identical results for the sensitiv ity of the PCR technique were obtained when duplicate samples were tested.
Jothikum ar/Khanna/Kam atchiam m al/ Muruean
Detection of Waterborne Viruses with PCR
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
450-474 served as flanking sequences for PCR. The probe was synthesized antisense to genomic viral RNA from the middle part of the amplified sequence and was located between 548-568, 3' ATG AAA CCC ACA GGC ACA AAG, The downstream primer served as a cDNA primer and the size of the amplified PCR pro duct was 154 base pairs (bp) which included a 109-base intervening sequence between the two primers plus the incorporated 20 and 25 primers.
A
B
C
D
A B O D E
A B C
D E
b b
Despite the > 10,000-fold concentration of viral particles from the contaminated water samples, the titre was not adequate for direct amplification of cDNA. Accordingly, fortitres < 120 PFU, the viruses were allowed to infect MAI04 cells overnight, followed by PCR am plification. The results are shown in figure 4. Two water samples of 50 1 each (hand pump water and open well water), showed the typical
Fig. 3. Evaluation of U-APB method for concen trating viruses from spiked water samples to a microlitre volume, a Gel electrophoresis of PCR products. Lanes B, C, D and E show the results of poliovirus 1 at concentrations of 1.200, 600, 300 and 120 PFU, respec tively, spiked in 5 I samples. Only 20 |il of 50 ul cDNA were used for PCR amplification corresponding to titre values of 480, 240,120 and 48 PFU, respectively. Lane A = Hae\Y\ (pX 174 restriction fragment pattern. The 154-bp PCR product is indicated by the arrow, b Auto radiogram of the Southern blot of the same gel hy bridized with radiolabelled oligonucleotide probe.
PCR product of a 154-bp band (lanes C and D). Sewage samples contained sufficient titre to concentrate through the successive steps fol lowed by phenol-chloroform extraction of viral nucleic acid, and amplification of cDNA in lane E shows the typical 154-bp band. The
189
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
Fig. 2. Evaluation of enterovirus primer for cDNA synthesis followed by PCR amplification of poliovirus I (lane B) and Coxsackievirus B5 (lane C). Lane A = Hae\ 11 (pX 174 restriction fragment pattern; lane D = negative control (no RN A), a Gel electrophoresis of PCR product; the arrow indicates the 154-bp product, b Autoradiogram of Southern blot hybridization of PCR products shown in the above gel.
D E F
A B O D E
F
154 b p -»
b Fig. 4. Evaluation of PCR for waterborne viruses, a Gel electrophoresis of PCR products. Lane A =
Intervirology 1992;34:184-191
N. Jothikumar P Khanna S. Kamatchiammal R. Paul Murugan
Rapid Detection of Waterborne Viruses Using the Polymerase Chain Reaction and a Gene Probe
National Environmental Engineering Research Institute, Nagpur, India
Introduction Waterborne viruses, especially the entero viruses, are the most common causative agents of childhood infection. The WHO Scientific Group [1] has concluded that the presence of even a few enteric viruses in a large volume of drinking water poses a threat to public health. In the absence of a reliable, reproducible and rapid method for detecting viruses in water and subsequent corrective measures, in less devel oped countries the mortality and morbidity due to waterborne viruses are high. The standard methods [2] for detecting viruses by inoculation in cell culture require 6-10 days, thereby defeat
Received: July 7,1992 Accepted: February 7,1993
ing the very purpose of analysis with respect to preventive measures. Furthermore, some viruses are not amenable to culture while others are expensive to culture. Alternative detection techniques have been reported for viruses but require high titres in the range of 10,000 viral particles. In addition, the gene probes detect around 1 pg of viral RN A /D N A equivalent to 10,000 infectious units [3]. Thus, there is an urgent need for rapid and sensitive techniques for detecting lower viral titres to enable preven tive measures in water supply practices. With the advent of recombinant DNA tech nology, it is now possible to detect waterborne viruses [3-7] without the need for tissue culture.
Prof. P. Khanna Director National Environmental Engineering Research Institute Nagpur 440 020 (India)
©1992 S. Karger AG, Basel 0300-5526/92/ 0344-0184S2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
Key Words Waterborne viruses Poliovirus 1 Virus concentration Detection Gene amplification Gene probe
Summary We describe a membrane-filter-based urea-arginine phosphate buffer method for concentrating waterborne viruses from large volumes of water to microlitre volumes, and their subsequent detection by the polymerase chain reaction (PCR). The detec tion step involves the extraction of RNA, synthesis of com plementary DNA, amplification by PCR of target DNA with specific primers, and confirmation through nucleic acid hybridi zation with a radiolabelled oligonucleotide probe. The PCR technique detected the presence of enteroviruses in spiked as well as in contaminated water samples. The technique is sensi tive and detects as few as 120 waterborne viral particles. PCR is simple, rapid, sensitive, specific and adaptable for water quality surveillance in less developed countries.
Table 1. Bacteriological quali fy 0f water and sewage samples
Most probable number per 100 ml Sampling points
TC
FC
Hand pump water 4.9x 10 1.7x 10 Open-well water (Open dug) 2 .2x10s l.lx lO 5 Sewage l.l x I0X 7 .0 x l0 7
EC
FS
1.3x10 4.9 xIO4 7.0x107
2.3x10 5.4x103 7 .9 x l0 J
TC = Total Coliforms; FC = faecal conforms: EC = E. coli; FS = faecal streptococci.
Materials and Methods Enteroviruses Poliovirus l was chosen as a model virus in ihis study for optimizing the experimental protocol, because it is present in large numbers in contaminated water particu larly in less developed countries. It also has the advan tage ofbeing easy to quantitate through plaqueassay. In addition, the complete nucleotide sequences of the genomes of the different poliovirus serotypes are avail able in the published literature [I7], A poliovirus I sus
pension was thawed and frozen three times repeatedly to release virions from infected cells, and viral stocks of known titre (12 x l05/m l) were diluted and enumerated by the agar overlay plaque assay technique as described by Jothikumar et al. [I8). Coxsackievirus B5 was propa gated in HeLa cell monolayer culture. Adsorption o f Viruses from Water Samples onto Membrane Filter For the initial experiments, 5-litre water samples were conditioned to pH 3.5 with I N HCI, and AICI, was added to a final molarity o f0.0005 M. I ml of poliovirus l suspension of different log dilutions as added to the sample and mixed for I0 min with a magnetic stirrer. Water samples were then passed through a millipore HA-type cellulose nitrate membrane filter of 0.45 pm pore size for virus adsorption. The remainder of the viral inoculum was enumerated on cell lines [18], and plaque counts were expressed as PFU /l. Both hand pump and well water sources located within 30 m of an open sewerage system in Madras, India, were examined for bacteriological quality using standard methods [2], and analysis of the data (table I) reveals that the water sources were highly contaminated by groundwater pol lution. Water samples of 50 I each from hand pump water and open well water were concentrated by twostep concentration and inoculated into cell culture to raise the titre, because no amplification could be achieved directly except from the sewage sample. A sewage sample of 500 ml was blended to desorb viruses from organic matter and centrifuged at 5,000 g for 20 min to remove solids. Further concentration of superna tant by membrane-filter-based urea-arginine phosphate buffer (U-APB) was carried out as in water samples, followed by nucleic acid extraction and PCR amplifica tion.
185
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
The polymerase chain reaction (PCR) has the advantage of amplifying even one genome copy exponentially to several million in a few hours without culturing the viruses [8-10], Due to the high sensitivity [ll] PCR detection of viruses in drinking water has great potential. Several researchers have reported the use of PCR for detecting enterovirus in clinical sam ples [12-I4], rotavirus [8, 15], and hepatitis E virus [16]. Notwithstanding these advances, no literature citations are available on the use of PCR for detecting waterborne viruses in envi ronmental samples, mainly due to the presence of low viral titres in drinking-water samples. We describe here a method for concentrat ing viruses from a large volume of water to a microlitre quantity, and a highly specific and sensitive technique for detection through nu cleic acid amplification with PCR, followed by confirmation with a radiolabelled oligonucleo tide probe.
Table 2. Recovery of poliovi rus I from water samples during the concentration procedure
Observation number
1 2 3 4 5
Input PFU/I
265 195 182 360 218
M ean±SD
Recovery. % second-step concentration1
Third-step concentration2
80.70 84.09 82.40 83.30 82.56
64.15 71.79 65.93 71.11 70.19
82.6± 1.3
68.6±3.4
1 After membrane filtration adsorption, viruses were eluted with U-APB. reconcentrated with MgCE and dissolved in 4 ml of Mcllvaines buffer, followed by enumeration. 2 4 ml Mcllvaines buffer were filtered and eluted with U-APB, recon centrated with MgCl2 and dissolved in 400 pi of Mcllvaines buffer follo wed by enumeration.
Third-Step Concentration o f Viruses to a Microlitre Volume Viruses concentrated in 4 ml of Mcllvaine’s buffer were passed through a swinnex filter containing a 0.45iim membrane filter, using a syringe. Adsorbed viruses were eluted with 5 ml of U-APB and the eluate was precipitated by the addition of 50 pi of I M M gC k The precipitate was pelleted by centrifugation at 7,000 rpm for 30 min. The pellet was dissolved in 400-600 pi of Mcllvaine’s buffer and transferred to 2-mI siliconised Eppendorf centrifuge tubes. In order to determine the percentage recovery of virus in water at each stage of the concentration proce dure, poliovirus I (182-360 PFU/ml) was added to 11of water and concentrated as described previously. The percentage recovery was recorded by enumerating on cell lines throughout the concentration procedure to
186
assess the sensitivity of the method in terms of the virus concentration in the original sample. The proposed U-APB method gives 82.6 and 68.6% recovery of polio virus I at the second and third steps of the concentration procedure respectively (table 2). Extraction o f RNA Virus titres > 120 PFU per 400-600 pi volume were treated with SDS and proteinase K to a final concentra tion of 0.5% and 5 mg/ml, respectively, followed by extraction with a phenol: chloroform isoamyl alcohol (PCI) mixture. The procedure is summarised in figure 1. Samples with viral titres < 120 PFU per 400-600 pi were inoculated into cell cultures to raise the titre value, and the cytopathic effect was observed after overnight infection. Culture bottles were frozen and thawed three times. Approximately 100 pi of cell lysate were boiled for 2 min and ice-chilled immediately. Twenty microli tres were used forcDN A synthesis in a 500-pl microfuge tube with a final reaction volume of 50 pi. Primers Enterovirus primers and probe were chosen to am plify a part of the 5' terminal non-coding part and a region of the single-stranded enteroviral genome encod ing non-structural proteins which is highly conserved among human picomaviruses [12], The downstream primer 3' ACC GAC GAA TAC CAC TGTTA located between 584-603 and the upstream primer 5' CC’TCCG GCC CCT GAA TGC GGC TAAT, located between
Jothikum ar/Khanna/Kam atchiam m al/ Murugan
Detection of Waterborne Viruses with PC R
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
EluIion o f Virus with U-APB Adsorbed viruses were eluted with 50 ml of U-APB (1.5 Murea:0.02 M arginine:0.008 M phosphate) at pH 9.0. The buffer was constituted by adding 4.5 g freshly dissolved urea, 2 ml 0.2 M sodium dihydrogen phos phate dihydrate and 2 ml of 0.5 M L-arginine to 42 ml of double-distilled water. The eluate was further reconcen trated by the addition of 0.5 ml of I M M gCk Samples were mixed in a beaker with a magnetic stirrer. The resultant precipitate was recovered through centrifuga tion at 5,000 gfor 30 min and the pellet was resuspended in 4 ml of Mcllvaine's buffer (pH 5.0).
Water sample 1 «•- HCI + AICI3 Adjust pH to 3.5 and 0.0005 M AICI3
4
Pass through membrane filter (0.45 pm. 142 mm diameter) 4 Discard filtrate Elute with 50 ml U-APB
4
Add 0.5 ml 1 M MgCI2 to eluate
4
Stir for 2 min
4 Centrifuge (5,000 g, 30 min) 4 - * Discard supernatant Dissolve pellet In 4 ml Mcllvaines buffer (pH 5.0)
4
Pass through smaller diameter membrane filter (0.45 pm. 13 mm diameter) 4 -■» Discard filtrate Elute with 5 ml U-APB
4
Add 50 pi 1 M MgCI2 to eluate
4
Centrifuge (7,000 g, 30 min) 4 - » Discard supernatant Dissolve pellet in 400-600 pi Mcllvaines buffer (pH 5.0)
4
Treat with proteinase/SDS
4
Incubate at 37 "C
4
Add equal volume of PCI
4
Vortex and centrifuge at 15.000 g, 10 min
4 4
“ l
Organic phase
Aqueous phase
I
Re-extract with equal volume of 10 mMTris HCI. 100 m M NaCI, 1 mM EDTA
I Centrifuge 15,000 g, 10 min
I
Aqueous phase
4
Mix two aqueous phases
4
Add ammonium acetate and 2.5 volumes of cold ethanol ( 100%)
4
Overnight precipitation at -20 'C
4
Centrifuge (15,000 g. 30 min)
4 Precipitate dissolved in 20 pi of Tris EDTA buffer
4
cDNA synthesis
4
4“ Agarose-gel ethidium bromide staining
4
Southern hybridization
“1 Slot blot analysis
187
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
Fig-1- Procedure for concentra tion, amplification and detection of viruses from contaminated water samples.
PCR amplification |
cDNA Synthesis cDNA synthesis was carried out in a final volume of 50 pi of 1x reverse transcriptase (RTase) buffer (5 x RTase buffer: 250 mMTris HC1 (pH 7.6), 300 mM KCI, 50 mMMgCI2, 5 mM DTT, 250 ul/100 ml vanadyl ribonucleoside complex), 4 pi containing 10 u M of each dNTP, 2 ul of 25 ng/pl downstream primer, 20 ul of test sample and 10 pi containing 5 units of AMV transcrip tase. The reaction mixture was incubated at 37 °C for 75 min, the reaction stopped by boiling or heat denaturing at 96°C for 2 min, and the reaction tube quick-chilled on ice. PCR Amplification was performed on the reverse-tran scription cDNA product (20 ul in a final volume of 100 pi containing 10 pi of 10 x PCR buffer [I x PCR buffer: 50 mM KCI, 10 mMTris HC1 (pH 8.3 at 25°C), 1.5 mM MgCI2, 100 pg/ml gelatin], 2 units of Taq polymerase, 2 pi each of upstream and downstream primers, 200 mM of 2 pi dNTP concentration of each nucleotide, and 10 pi ofl0% bovine serum albumin. The reaction mixture was finally overlaid with 100 pi of light mineral oil to prevent evaporation. The amplification reactions were per formed through 40 cycles of alternating temperature with each cycle consisting of denaturing at 94°C for 2 min, annealing at 50 °C for 2 min, and extension at 72°C for 3 min. After 40 cycles, the DNA was extended for an additional 10 min at 72 °C. Southern Transfer and Hybridization 10 pi of amplified PCR product were electrophoresed on a 2% agarose gel, photographed under UV light, and transferred to nitrocellulose membrane for hybridization according to the procedure of Maniatis et al. [19], and then fixed by baking at 80"C for 2 h. After prehybridization (6 x SSC, 1 x Denhardt’s solution and 0.3% SDS) at 60°C for 4 h the filter was hybridized overnight with fresh buffer containing 50 pg/ml salmon sperm DNA and I06 c, p, m/m l of y 32P ATP end-la belled (polynucleotide kinase) probe at 60°C. After six washing steps (three times in 2 x SSC, 0,1% SDS at room temperature for 10 min and thrice in 0.1 x SSC,0.1%SDS
188
at 50“C for 10 min) the filter was air dried and exposed to X-ray film.
Results The procedure for concentrating viruses from contaminated water samples, cDNA syn thesis, PCR amplification and detection is shown in figure 1. Primers selected from highly conserved regions of the genome could amplify both poliovirus 1 and Coxsackievirus B5, as depicted in figure 2. None of the negative con trols in the experiments resulted in false posi tives, and no spurious bands representing non specific amplification were observed. No am plification was observed with rotavirus and bacteriophage when the same procedure with the same set of primers was used. Carry-over DNA leading to false positives was prevented by exercising strict precautions including the control experiments in which the reverse tran scriptase of RNA was excluded, giving no sig nal after amplification, indicating absence of contamination in all the experiments. In all the PCR-positive samples, a distinct 154-bp PCR product was visible on the agarose gel when stained with ethidium bromide. The identify of the 154-bp band was further con firmed by Southern hybridization analysis with a 32P-labelled probe. To assess the sensitivity of the PCR technique, log dilutions of poliovirus (1,200, 600, 300, and 120 PFU viral titre) were added to water samples. The effectiveness of PCR for detecting virus in spiked water sam ples with only 20 ul cDNA used for PCR ampli fication corresponding to titre values of 480, 240,120 and 48 PFU/1, respectively is shown in figure 3. The sensitivity of the proposed detec tion scheme was 120 PFU viral titre, and no amplification was observed in lane E for < 120 PFU viral titre. Identical results for the sensitiv ity of the PCR technique were obtained when duplicate samples were tested.
Jothikum ar/Khanna/Kam atchiam m al/ Muruean
Detection of Waterborne Viruses with PCR
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
450-474 served as flanking sequences for PCR. The probe was synthesized antisense to genomic viral RNA from the middle part of the amplified sequence and was located between 548-568, 3' ATG AAA CCC ACA GGC ACA AAG, The downstream primer served as a cDNA primer and the size of the amplified PCR pro duct was 154 base pairs (bp) which included a 109-base intervening sequence between the two primers plus the incorporated 20 and 25 primers.
A
B
C
D
A B O D E
A B C
D E
b b
Despite the > 10,000-fold concentration of viral particles from the contaminated water samples, the titre was not adequate for direct amplification of cDNA. Accordingly, fortitres < 120 PFU, the viruses were allowed to infect MAI04 cells overnight, followed by PCR am plification. The results are shown in figure 4. Two water samples of 50 1 each (hand pump water and open well water), showed the typical
Fig. 3. Evaluation of U-APB method for concen trating viruses from spiked water samples to a microlitre volume, a Gel electrophoresis of PCR products. Lanes B, C, D and E show the results of poliovirus 1 at concentrations of 1.200, 600, 300 and 120 PFU, respec tively, spiked in 5 I samples. Only 20 |il of 50 ul cDNA were used for PCR amplification corresponding to titre values of 480, 240,120 and 48 PFU, respectively. Lane A = Hae\Y\ (pX 174 restriction fragment pattern. The 154-bp PCR product is indicated by the arrow, b Auto radiogram of the Southern blot of the same gel hy bridized with radiolabelled oligonucleotide probe.
PCR product of a 154-bp band (lanes C and D). Sewage samples contained sufficient titre to concentrate through the successive steps fol lowed by phenol-chloroform extraction of viral nucleic acid, and amplification of cDNA in lane E shows the typical 154-bp band. The
189
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:39:02 AM
Fig. 2. Evaluation of enterovirus primer for cDNA synthesis followed by PCR amplification of poliovirus I (lane B) and Coxsackievirus B5 (lane C). Lane A = Hae\ 11 (pX 174 restriction fragment pattern; lane D = negative control (no RN A), a Gel electrophoresis of PCR product; the arrow indicates the 154-bp product, b Autoradiogram of Southern blot hybridization of PCR products shown in the above gel.
D E F
A B O D E
F
154 b p -»
b Fig. 4. Evaluation of PCR for waterborne viruses, a Gel electrophoresis of PCR products. Lane A =
