- Email: [email protected]
Application of polymerase chain reaction to detect rna coliphage Qß in environmental water samples
e>
Pergamon
Wat. ScL Tech. Vol. 31. No. 5-6. pp. 383-390.1995.
0273-1223(95)00297-9
CopyrigbtC 1995 IAWQ Printed in Great Britain. All rigbts reserved. 0273-1223195 $9'50 + 00()()
APPLICATION OF POLYMERASE CHAIN REACTION TO DETECT RNA COLIPHAGE Q~ IN ENVIRONMENTAL WATER SAMPLES Limsawat Sunun, Naoyuki Kamiko, Kazuo Yamamoto and Shinichiro Ohgaki Department of Urban Engineering. Faculty ofEngineering, University of Tokyo. 7-3-1 Hongo, Bunkyo, Tokyo 113. Japan
ABSTRACT An RT·PCR technique for direct detection of enteric viruses in environmental water samples was developed by using RNA coliphage Oil as a model virus. RNA coliphage Oil was prepared from the lysate broth of single-plaque-isolated stock and extracted by direct heating. The sensitivity of the PCR amplification was improved by optimizing the parameters to be evaluated for efficient PCR inclUding KCI in buffer components, Taq polymerase, annealing temperature, and number of cycles. The higher KCI concentration (up to 90 mM) in reaction incorporating with lower Taq polymerase concentration (0.5 unit/40 111 total reaction) caused quite low. sensitivity and inconsistency of RT-PCR. The KCI concentration (30 to 90 mM) implied no significant effect to the yields of PCR products when higher Taq polymerase (1.0 to 2.5 units/40 iii total reaction) was included. Increasing the number of cycle from 25 to 35 cycles was able to increase the sensitivity. The increasing of annealing temperature reduced amplifying background products seen on gel electrophoreses. The maximum sensitivity is 0.3 PFU/reaction using condition of 35-cycle amplification with Taq polymerase 2.5 units and 55°C annealing temperature. This RT-PCR technique will be used as a protocol for further study for enteroviruses. The success of detection of indigenous RNA coliphages (group III) in night soil sample using the developed RT-PCR methods means that the developed method has great potential as a direct detection method of viruses in environmental sample. The inconsistency of the detection limit between plaque assay and RT-PCR assay may be caused by the effects of inhibitors in night soil sample. Further study on the relationship between plaque assay and PCR assay is required. KEYWORDS
Virol detection, polymerase chain reaction, coliphage, enterovirus
INTRODUCflON The polymerase chain reaction process has been developed as a useful technique for identification of clinically important pathogens and also applied to detect microorganisms in environmental surveillance (1,2,3,4,5). Since the PCR technique is an enzymatic amplification assay that will increase amount of target nucleic acid in a given sample for detecting the low level of microorganisms in environmental samples and it does not involve the culturing of cell. Therefore it suggests easier, sensitive, and specific approaches for the monitoring of viruses in environmental samples. To apply the PCR for direct detection of microorganisms in environmental samples, which often contained low level of microorganisms together with high level of inhibitors which can interfere wilh the activity of 383
384
L. SUNUN et al.
enzyme such as organic matters, metal ions, and other substances (2,4,6), requires adjustment of sample and optimization of reaction for efficient amplification of specific targets. Several methods other than proteinase K digestion and the phenol-chloroform extraction have been developed for the rapid methods for extraction and purification of sample prior to PCR, such as the use of glass powder suspension and heating for extraction methods (7,8). And for optimizing of PCR, the reaction mixture and the thermocyciing profiles are likely to be optimized in addition to the selected primers (9,10,11,12). Changing the buffering capacity of the PCR reaction. eg.by increasing the concentration of the Tris-HCI up to 50 mM(pH 8.9), sometimes increase the yield of the amplified DNA products (13). In this study the RNA coliphage Qll was used as model virus for developing RT·PCR amplification for direct detection of enteric viruses in water sample by modifying the PCR technique reported in previous studies (8,14). The direct heating (8) was used for the rapid extraction viral RNA in sample for amplifying in RT-PCR. The f11"st objective of this study is to improve the sensitivity of PCR method to obtain the theoritical detection limit at least as 1 PFU by optimizing the KO concentration in buffer component, Taq polymerase concentration, annealing temperature and number of cycles. Especially, the effect of KCI incorporating with Taq polymerase is discussed for enhancing the sensitivity of method. And the second objective is to apply the developed technique for direct detection of RNA coliphage Q II in night soil sample.
MATERIALS AND METHODS RNA coliphage Qll sample; The RNA coliphage Qll sample was prepared from the lysate broth of single_ plaque-isolated stock. The plaque assay method used was the double-agar-Iayer method with E.coli K12,F+, N').. as host organism (15). Extraction Method : The direct heating method (8) was used for extracting RNA of Qll coliphage in sample. The 100 ILl sample in phage broth medium was added in 0.5 J.Il Eppendorf tube and heated 10 minutes at 90·C then quick cooled down to 4·C and chilled on ice. Primers: The primers for detecting coliphage RNA QIl are the published sequences for amplifying the 3' terminal region on the replicase gene of Qll RNA genome and specific to F-specific RNA coliphage (group III) (8). The sequences of the primers are 5'CCA TCG ATC AGC TTA TCT GT-3' (position 3971-3990) for sense primer and 5'ATT CAC AAT TAG GCG CC-\T-3' (position 4142-4160) for antisense primer. Reverse Transcription: Three ILl of direct heating extracted RNA sample were added into the RT mixture tube. The RT mixtures combined in one 0.5 ILl Eppendorf tube include 1 ILl of RNasin (40 units), 2.5 ILl of 5x reverse transcription buffer [ 250 mM Tris-HCI(pH 8.3), 15 mM MgC12. 350 mM KCI, 50 mM DTT), 4 111 of dNTPs (10 mM. each), 1 ILl antisense primer (10 pmoVILI), and 1 ILl avian myeloblastosis virus reverse transcriptase (5 units! J.Il). Then the mixture is overlaid with 30 ILl of mineral oil. The thermal profile of RT step was incubation of90 minutes at 37·C and heating at 95·C for 5 minutes, then quick cooling down to 4·C. Polymerase chain reaction: To each of the reverse transcription mixture the following reagents were directly added to obtain 40 ILl final volume; 8 ILl of sterile MilIiQ H20, 4 J.Il of lOx PCR buffer [ *( vary to adjust final concentration to be 50 - 90 mM in reaction) KCI, 100 roM Tris-HCI (pH 8.3), 15 mM MgCI2, 0.1 % Gelatin], 6.5 ILl of dNTPs (2 roM, each), 4 ILl of antisense primer (10 pmoVI1I), 4 ILl of sense Primer (10 pmoVILI). and 1 ILl Taq polymerase. The reaction tubes are incubated in a Perkin Elmer Cetus DNA Thermal Cycler according to the following protocol : 3 min at 95·C, then 25 or 35 cycles of 95·C for 30 sec, 50· or SS·C for 30 sec, and for 30 sec, then fmally 7 min at and quick cool down to 4·C and chill on ice. Analysis of PCR product: The 10 ILl of reaction mixture is electrophoresed on a 4% Nusieve-Agarose 3:1 composite gel made in TAE buffer. The gel is then stained with ethidium bromide and photographed by polaroid film using UV transillumination. The photograph is scanned using scanner and measured the integrated brightness area of the amplified PCR product using computer software "Image 1.45".
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Application of polymerase chain reaction to detect RNA coliphage Qll
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Figure 1 : Effects of KCI incorporating with Taq polymerase for 25 and 35 cycle-amplification PCR.
386
L. SUNUN el al.
RESULTS Changing buffer condition and testing sensitivity : The effects of KO incorporating with Taq polymerase in PCR were studied in order to increase the sensitivity of RT-PCR. The experiments Were conducted by varying final KO concentration from 50 to 90 mM with Taq polymerase 0.5, 1.0, and 2.0 units for 25-cycle amplification and with Taq polymerase 1.0 unit for 35-cycle amplification. The results are shown in Figure 1. In Figure 1, the experiments using Taq polymerase 0.5 unit per reaction at the initial coliphage Q~ titer 4.1x106 and 7.5xl(P PFU showed tendency of linear decrease of the amplified PCR products when Ka concentration increased from 50 to 90 mM. The lower KO concentration gave higher PCR products and the maximum) ields were obtained at the concentration around 50 mM. These results agreed with the recommendation criteria that KO up to 75 mM caused significantly inhibiting the activity of Taq DNA polymerase (16). However, when the experiment using Taq polymerase 1.0 and 2.0 units were perfonned at the various initial coliphage O~ titer 5.7x106, 7.5 x103, and 7.5 x102 PFU, the results showed less effect of KCI and no significant difference of the amplified PCR products obtained at different KCl concentration.
The higher KO (up to 90 mM) in reaction incorporating with lower Taq polymerase concentration (0.5 unit) gave quite low sensitivity RT-PCR. In the case of higher concentration Taq polymerase (1.0 and 2.0 units), the KCl concentration in range of 50-90 mM implied no significant effect to the yields of peR products which were detected by Ethidium Bromide staining. Also the 35-cycle amplification with Taq polymerase 1.0 unit at the initial coliphage OP titer 2.3x1OS PFU showed no significant difference in yields of PCR products at different KCI concentrations (50-90 roM). Testing sensitivity ofRT·PCR method: Serial dilutions of coliphage QP stock were made in phage broth and 100 III of each dilution was heated at 90·C for 10 minutes. Then 3 III of each dilution was used fo; amplification in RT-PCR. The results are shown in Table 1. "Sensitivity" means the detection limit of amount of coliphage OP in unit PFU/reaction. Table 1: The sensitivities of the RT-PCR methods performed by changing Taq polymerase incorporating with KO concentration and 50·C annealing temperature. KO c:one.in reaction (mM)
(0.5,25)
8.1"10 6 • 4.2"10 6 4.2"103
90
80
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70
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Application of polymerase chain reaction to detect RNA coliphage Q(3
387
The column (0.5,25) in Table 1 sh~ws the res~l~s .obtained from the experiment using Taq polymerase 0.5 units. The effect of KCI concentratIon on senstttvlty of PCR can be seen from the concentration of KCI 90 mM. Consequently, this condition showed low sensitivity and reproductivity. In cases of KCI concentration in the range 30-70 mM. the range of sensitivities are 4.2 x 104 to 4.2x10 3 PFU/reaction which equal to 1.4xl07 to 1.4xI06 PFUlml, respectively.
In the experiments using Taq polymerase 1.0 and 2.0 units with 25-cycle amplification, the sensitivities are 4.2x103 PFU/reaction and 4.2x10 3 to 4.2x102 PFU/reaction, respectively. This case, the sensitivities are not affected by KCl concentration (30-90 mM). These results mean consistency of sensitivity with effect of KCI concentration. The experiment of 35-cycle amplification with Taq polymerase 1.0 unit (column (1.0,35) in Table 1), all sensitivities are 4.2x10 2 PFU/reaction. When this result was compared to the experiment using Taq polymerase 1.0 unit with 25-cycle amplification (column (1.0,25) in Table 1). It is showed that increasing the number of cycles from 25 to 35 cycles can increase sensitivity from 4.2xl03 to 4.2x102 PFU/reaction. However, when number of cycles were increased, the non-specific or background products were observed on gel electrophoresis. To solve this problem, the annealing temperature was increased from 50'C to 55'C. The increasing of annealing temperature has been suggested for improving the problem of smear of PCR products seen on the agarose gel (17). And to improve the sensitivity, the concentration of Taq polymerase was increased to 2.5 units with 35-cycle PCR amplification. Then this condition was tested for determining the sensitivity of the primers. The results are shown in Table 2. Table 2 : Testing sensitivity of F-specitic RNA coliphage QII primers with the condition of SS'C annealing temperature and Taq polymerase 2.5 units for 3S-cycle PCR amplification. KCI conc.(mM)
Sensitivity (2.5,35)'
50
0.3 PFU
(1.06x10 2 PFU/ml)
70
0.3 PFU
(l.06x 102 PFU/ml)
• (unit of Taq polymerase, number of cycles) 1 2 3 4 5 6 7 8 9 10
Annealing temperature SO'C (Taq polymerase 1.0 unit, KCI 50 mM)
1 2 3 4 5 6 7 8 9 10
Annealing temperature SST (Taq polymerase 2.5 units, KCI 50 mM)
Figure 2: Comparison of 35-cycie PCR amplification between tbe conditions of annealing temperature 50'C and 55·C. Lane (1) DNA marker 0X174, HaeIII, (2) Negative control, (3) 3.0 x 104 PFU, (4) 3.0 x 103 PFU, (5) 3.0 x 102 PFU, (6) 3.0 x 10 1 PFU, (7) 3.0 x 10° PFU, (8) 3.0 x 10-1 PFU, (9) 3.0 x 10-2 PFU, (10) 3.0 x 10-3 PFU. MSI H-5f6·.u,
388
L SUNlIN et al.
These results show that the detection limit or the sensitivity can be 0.3 PAl/reaction. The increasing of annealing temperature reduced amplifying background products and gave clearer image of amplified products on gel electrophoreses (see Figure 2). This developed RT-PCR technique ( 35-cycle amplification with Taq polymerase 25 units, 55"C annealing temperature, and 50 mM KO) was tested for detection of F-specific RNA coliphage (group III) in night soil sample. Testing detection of F·spedOc RNA coliphages ( group Ill) In Night soli sample: The above mentioned PCR condition which gave the maximum sensitivity (0.3 PAl/reaction) in the experiment for testing the sensitivity was used for detection of F-specific RNA coliphages (group UI) in night soil sample. The night soil sample which was mixed with sludge of onsite small treatment plant was collected from Urayasu Night Soil Treatment Plant, Cbiba Prefecture, Japan. The supernatant after centrifuged at 2O,000)(g for 30 minutes was filtered by 0.45 I!m membrane filter. The pH of filtrate was about 8.2. This filtrate was divided for determining plaque assay and extracting for RT-PCR. The plaque assay method used was the double-agar-Iayer method with E.coli K12,F+,AI>" as host organism. The extracted and diluted samples were kept at -20·C until RT-PCR was performed. The extraction methods used are as follows: 1) phenol-chlorofonn method (8), 2) direct heating at 9O"C for 10 min, 3) treating with RNasin (40 units/lOO Ill. sample) and then heating at 9O"C for 10 min, and 4) treating with Proteinase K then heating at 9O"C for 10 minutes: Fifty III of buffer solution (10 111M Tris-HO(pH 7.5), 100 mM NaC!, 1 mM EDTA) was added into 500 III sample and incubated with Proteinase K, 200 Ilglml at 55"C for 60 minutes. Then the mixture was heated at 90"C for 10 minutes quick cooled down to 4·C and chilled on ice. ' The results are summarized in Table 3 and shown in Figure 2. Table 3 : Testing detection of F.speclfic RNA coliphages ( group Ill) In night soil sample. Extraction method
No dilution
Phenol-chloroform method Direct heatiDl]; at 9O"C for 10 min. RNasin treatin/! and heatin/! at 9O·C for 10 min. Proteinase K treatinl! and heatinl! at 9O"C, for 10 min.
+
-
Diluted sample 10- 1fold
10-2 fold
10-3 fold
NO
NO
NO
+ + +
+ + +
-
-
NO
+ : positive, -- : negative, NO: not done. The concentration of F-specific RNA coliphages in night soil sample determined by plaque assay method is 1.8 x 103 PFU/ml. The results obtained from RT-PCR method showed positive bands of F-sr:cific RNA coliphages (group m) from samples which were extracted by Phenol-chloroform method, 10- and 10-2 fold dilution of direct heating samples without RNasin treating, with RNasin treating, and with proteinase K treating. In addition, direct heated samples without further dilution showed negative results (no visible band of amplified products). This indicates the presence of inhibitors in night soil sample which can obstruct the RT-PCR.
Application of polymerase cbain reaction to detect RNA coliphage Oil
389
This inhibitors effect could be reduced by dilution in the case of direct heating extraction methods, while the inhibitors were removed during Phenol-chloroform method. Based on the results of plaque assay, the 10-2 fold dilution of heat extracted samples, which showed positive results by RT-PCR assay in Table 3, contain 1.8 x 101 PFUlml. This value (1.8 x 101 PFU/ml ) is lower than the detection limit or sensitivity (10 2 PFU/ml which is equivalent to 0.3 PFU/reaction as mentioned above in Table 2). This inconsistency between plaque assay and PCR assay may be caused by various effect of inhibitors in night soil sample on both methods. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
•
194 bp
Figure 3
Testing detection of F-specific RNA coliphages (group III) in night soil sample. Lane (I) DNA marker 0XI74.HaeIIl, (2) Positive control 1()4 PFU, (3) and (4) Phenol-chloroform extracted sample, (5) Direct heating, (6), (7), and (8) lO- t , 10-2, 10-3 dilution, respectively, (9) RNasin treating and heating, (10), (11), and (12) 10- 1, 10-2, 10-3 dilution, respectively, (13) and (14) Proteinase K treating and heating, (15) Negative control.
CONCLUSION The RT-PCR method for direct detection of enteric viruses in environmental samples was developed by using RNA coliphage QJ3 as the model virus with direct heating (90·C, 10 min) as viral extraction method. This developed method was applied for detection of F-specific RNA coliphages (group III) in night soil sample. The RT-PCR method was improved by optimizing the concentrationn of KCI, Taq polymerase, annealing temperature, and number of cycle. Optimization concentration of KCI and Taq polymerase has illustrated that the high KCI concentration (up to 90 mM) in reaction incorporating with low Taq polymerase concentration (0.5 unit/40 III total reaction) causes low sensitivity and reproductivity of PCR. The KCI concentration (30 to 90 mM) implied no significant effect to the yields of PCR products when higher Taq polymerase (1.0 to 2.5 units/40 !.ll total reaction) was included. Increasing the number of cycle from 25 to 35 cycles can increase the sensitivity. The increasing of annealing temperature reduced amount of amplifying background products seen on gel electrophoreses. The maximum sensitivity is 0.3 PFU/reaction which is obtained by using the condition of 35-cycle PCR amplification with Taq polymerase 2.5 units and 55·C annealing temperature. This RT-PCR technique will be able to be used as a protocol for further study for enteroviruses.
L. SUNUN et al.
390
The success of detection of indigenous RNA coliphages (group III) in night soil sample using the developed RT-PCR methods means that the developed method has great potential as a direct detection method of viruses in environmental sample. The inconsistency of the detection limit between plaque assay and RT-PCR assay may be caused by the effects of inhibitors in night soil sample. Further study on the relationship between plaque assay and peR assay is required. REFERENCES 1. Vandervelde, C., Verstraete, M., Beers, D. V. (1990). Fast multiplex polymerase chain reaction On boiled clinical samples for rapid viral diagnosis. J. o/Virological Methods. 30,215-228. 2. Tsai, Y.-L., Palmer, C. J., Sangermano, L. R. (1993). Detection of Escherichia coli in sewage and sludge by polymerase chain reaction. Appl. Environ. Microbiol. 59,353-357. 3. Almar, R. L, Metcalf, T. G., Neil, F. H., Estes, M. K. (1993). Detection of enteric viruses in oysters by using the polymerase chain reaction. Appl. Environ. Microbiol. 59, 631-635. 4. Kopecka, H., Dubrou, S., Prevot, J., Marchal, J., Lopez-pila, J. M. (1993). Detection of naturaally occurring enteroviruses in waters by reverse transcriptase, polymerase chain reaction, and hybridization. Appl. Environ. Microbiol. 59, 1213-1219. 5. Tsai, Y.-L., Sobsey, M. D., Sangermano, L. R., Palmer, C. 1. (1993). Simple method of concentrating enteroviruses and Hepatitis A virus from sewage and ocean water for rapid detection by reverse transcriptase-polymerase chain reaction. Appl. Environ. Microbiol. 59, 3488-3491. 6. Abbaszadegan, M., Huber, M. S., Gerba, C. P. (1993). Detection of enteroviruses in groundwater with the polymerase chain reaction. Appl. Environ. Microbiol. 59, 1318-1324. 7. Yamada, 0., Matsumoto, T., Nakashima, M., Hagari, S., Kamhora, T., Ueyama, H., Kishi, Y., Uemura H., Kurimura, T. (1990). A new method for extracting DNA or RNA for polymerase chain reaction. 0/ Virological Methods. 27, 203-210. 8. Dantheeravanich, S. (1992). Application of polymerase chain reaction for health-related viral indicators detection in wastewater. Doctoral dissertation, The University of Tokyo, Tokyo, Japan. 9. Innis, M. A, Gelfand, D. H. (1990). Optimization ofPCRs, p.3-12. in Innis, M. A, Gelfand, D. H., Sninsky, J. J., White, T. J. (ed.), peR Protocols: A Guide to Methods and Applications. Academic Press, Inc., U.S.A. 10. Saiki, R. K. (1989). The design and optimization of the PCR, p.7-16. in Erlich, H. A. (ed.), PCR Technology: Principles and Application for DNA amplification. Stockton Press, U.S.A. 11. Bej, A. K., Mahbubani, M. H., Atlas, R. M. (1991). Amplification of nucleic acid by polymerase chain reaction (PCR) and other methods and their applications. Critical Reviews in Biochemistry and Molecular Biology. 26, 301-334. 12. Steffan, R. J., Atlas, R. M. (1991). Polymerase chain reaction: Applications in Environmental Microbiology. Annu. Rev. Microbiol. 45, 137-161. 13. Bej, A. K., DiCesare, J. L., Haff, L., Atlas, R. M. (1991). Detection of E.coli and Shigella sPp. in water by using polymerase chaain reaction (PCR) and gene probes for uid. Appl. Environ. Microbiol. 57, 1013-1017. 14. Rotbart, H. A (1990). PCR amplification of enteroviruses, p.372-377. In Innis, M. A. (ed.), PCR Protocols: A Guide to MetllOds and Applications. Academic Press, Inc., U.S.A. 15. Adams, M. (1959). Bacteriophages. Interscience Pub\. Inc., New York. 16. Innis, M. A, Myambo, K. B., Gelfand, D. H., Brow, M. D. (1988). DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. 85, 9436-9440. 17. Williams, J. F. (1989). Optimization strategies for the polymerase chain reaction. Biotec1lltiques. 7, 762-769.
J.
Pergamon
Wat. ScL Tech. Vol. 31. No. 5-6. pp. 383-390.1995.
0273-1223(95)00297-9
CopyrigbtC 1995 IAWQ Printed in Great Britain. All rigbts reserved. 0273-1223195 $9'50 + 00()()
APPLICATION OF POLYMERASE CHAIN REACTION TO DETECT RNA COLIPHAGE Q~ IN ENVIRONMENTAL WATER SAMPLES Limsawat Sunun, Naoyuki Kamiko, Kazuo Yamamoto and Shinichiro Ohgaki Department of Urban Engineering. Faculty ofEngineering, University of Tokyo. 7-3-1 Hongo, Bunkyo, Tokyo 113. Japan
ABSTRACT An RT·PCR technique for direct detection of enteric viruses in environmental water samples was developed by using RNA coliphage Oil as a model virus. RNA coliphage Oil was prepared from the lysate broth of single-plaque-isolated stock and extracted by direct heating. The sensitivity of the PCR amplification was improved by optimizing the parameters to be evaluated for efficient PCR inclUding KCI in buffer components, Taq polymerase, annealing temperature, and number of cycles. The higher KCI concentration (up to 90 mM) in reaction incorporating with lower Taq polymerase concentration (0.5 unit/40 111 total reaction) caused quite low. sensitivity and inconsistency of RT-PCR. The KCI concentration (30 to 90 mM) implied no significant effect to the yields of PCR products when higher Taq polymerase (1.0 to 2.5 units/40 iii total reaction) was included. Increasing the number of cycle from 25 to 35 cycles was able to increase the sensitivity. The increasing of annealing temperature reduced amplifying background products seen on gel electrophoreses. The maximum sensitivity is 0.3 PFU/reaction using condition of 35-cycle amplification with Taq polymerase 2.5 units and 55°C annealing temperature. This RT-PCR technique will be used as a protocol for further study for enteroviruses. The success of detection of indigenous RNA coliphages (group III) in night soil sample using the developed RT-PCR methods means that the developed method has great potential as a direct detection method of viruses in environmental sample. The inconsistency of the detection limit between plaque assay and RT-PCR assay may be caused by the effects of inhibitors in night soil sample. Further study on the relationship between plaque assay and PCR assay is required. KEYWORDS
Virol detection, polymerase chain reaction, coliphage, enterovirus
INTRODUCflON The polymerase chain reaction process has been developed as a useful technique for identification of clinically important pathogens and also applied to detect microorganisms in environmental surveillance (1,2,3,4,5). Since the PCR technique is an enzymatic amplification assay that will increase amount of target nucleic acid in a given sample for detecting the low level of microorganisms in environmental samples and it does not involve the culturing of cell. Therefore it suggests easier, sensitive, and specific approaches for the monitoring of viruses in environmental samples. To apply the PCR for direct detection of microorganisms in environmental samples, which often contained low level of microorganisms together with high level of inhibitors which can interfere wilh the activity of 383
384
L. SUNUN et al.
enzyme such as organic matters, metal ions, and other substances (2,4,6), requires adjustment of sample and optimization of reaction for efficient amplification of specific targets. Several methods other than proteinase K digestion and the phenol-chloroform extraction have been developed for the rapid methods for extraction and purification of sample prior to PCR, such as the use of glass powder suspension and heating for extraction methods (7,8). And for optimizing of PCR, the reaction mixture and the thermocyciing profiles are likely to be optimized in addition to the selected primers (9,10,11,12). Changing the buffering capacity of the PCR reaction. eg.by increasing the concentration of the Tris-HCI up to 50 mM(pH 8.9), sometimes increase the yield of the amplified DNA products (13). In this study the RNA coliphage Qll was used as model virus for developing RT·PCR amplification for direct detection of enteric viruses in water sample by modifying the PCR technique reported in previous studies (8,14). The direct heating (8) was used for the rapid extraction viral RNA in sample for amplifying in RT-PCR. The f11"st objective of this study is to improve the sensitivity of PCR method to obtain the theoritical detection limit at least as 1 PFU by optimizing the KO concentration in buffer component, Taq polymerase concentration, annealing temperature and number of cycles. Especially, the effect of KCI incorporating with Taq polymerase is discussed for enhancing the sensitivity of method. And the second objective is to apply the developed technique for direct detection of RNA coliphage Q II in night soil sample.
MATERIALS AND METHODS RNA coliphage Qll sample; The RNA coliphage Qll sample was prepared from the lysate broth of single_ plaque-isolated stock. The plaque assay method used was the double-agar-Iayer method with E.coli K12,F+, N').. as host organism (15). Extraction Method : The direct heating method (8) was used for extracting RNA of Qll coliphage in sample. The 100 ILl sample in phage broth medium was added in 0.5 J.Il Eppendorf tube and heated 10 minutes at 90·C then quick cooled down to 4·C and chilled on ice. Primers: The primers for detecting coliphage RNA QIl are the published sequences for amplifying the 3' terminal region on the replicase gene of Qll RNA genome and specific to F-specific RNA coliphage (group III) (8). The sequences of the primers are 5'CCA TCG ATC AGC TTA TCT GT-3' (position 3971-3990) for sense primer and 5'ATT CAC AAT TAG GCG CC-\T-3' (position 4142-4160) for antisense primer. Reverse Transcription: Three ILl of direct heating extracted RNA sample were added into the RT mixture tube. The RT mixtures combined in one 0.5 ILl Eppendorf tube include 1 ILl of RNasin (40 units), 2.5 ILl of 5x reverse transcription buffer [ 250 mM Tris-HCI(pH 8.3), 15 mM MgC12. 350 mM KCI, 50 mM DTT), 4 111 of dNTPs (10 mM. each), 1 ILl antisense primer (10 pmoVILI), and 1 ILl avian myeloblastosis virus reverse transcriptase (5 units! J.Il). Then the mixture is overlaid with 30 ILl of mineral oil. The thermal profile of RT step was incubation of90 minutes at 37·C and heating at 95·C for 5 minutes, then quick cooling down to 4·C. Polymerase chain reaction: To each of the reverse transcription mixture the following reagents were directly added to obtain 40 ILl final volume; 8 ILl of sterile MilIiQ H20, 4 J.Il of lOx PCR buffer [ *( vary to adjust final concentration to be 50 - 90 mM in reaction) KCI, 100 roM Tris-HCI (pH 8.3), 15 mM MgCI2, 0.1 % Gelatin], 6.5 ILl of dNTPs (2 roM, each), 4 ILl of antisense primer (10 pmoVI1I), 4 ILl of sense Primer (10 pmoVILI). and 1 ILl Taq polymerase. The reaction tubes are incubated in a Perkin Elmer Cetus DNA Thermal Cycler according to the following protocol : 3 min at 95·C, then 25 or 35 cycles of 95·C for 30 sec, 50· or SS·C for 30 sec, and for 30 sec, then fmally 7 min at and quick cool down to 4·C and chill on ice. Analysis of PCR product: The 10 ILl of reaction mixture is electrophoresed on a 4% Nusieve-Agarose 3:1 composite gel made in TAE buffer. The gel is then stained with ethidium bromide and photographed by polaroid film using UV transillumination. The photograph is scanned using scanner and measured the integrated brightness area of the amplified PCR product using computer software "Image 1.45".
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Application of polymerase chain reaction to detect RNA coliphage Qll
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Figure 1 : Effects of KCI incorporating with Taq polymerase for 25 and 35 cycle-amplification PCR.
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L. SUNUN el al.
RESULTS Changing buffer condition and testing sensitivity : The effects of KO incorporating with Taq polymerase in PCR were studied in order to increase the sensitivity of RT-PCR. The experiments Were conducted by varying final KO concentration from 50 to 90 mM with Taq polymerase 0.5, 1.0, and 2.0 units for 25-cycle amplification and with Taq polymerase 1.0 unit for 35-cycle amplification. The results are shown in Figure 1. In Figure 1, the experiments using Taq polymerase 0.5 unit per reaction at the initial coliphage Q~ titer 4.1x106 and 7.5xl(P PFU showed tendency of linear decrease of the amplified PCR products when Ka concentration increased from 50 to 90 mM. The lower KO concentration gave higher PCR products and the maximum) ields were obtained at the concentration around 50 mM. These results agreed with the recommendation criteria that KO up to 75 mM caused significantly inhibiting the activity of Taq DNA polymerase (16). However, when the experiment using Taq polymerase 1.0 and 2.0 units were perfonned at the various initial coliphage O~ titer 5.7x106, 7.5 x103, and 7.5 x102 PFU, the results showed less effect of KCI and no significant difference of the amplified PCR products obtained at different KCl concentration.
The higher KO (up to 90 mM) in reaction incorporating with lower Taq polymerase concentration (0.5 unit) gave quite low sensitivity RT-PCR. In the case of higher concentration Taq polymerase (1.0 and 2.0 units), the KCl concentration in range of 50-90 mM implied no significant effect to the yields of peR products which were detected by Ethidium Bromide staining. Also the 35-cycle amplification with Taq polymerase 1.0 unit at the initial coliphage OP titer 2.3x1OS PFU showed no significant difference in yields of PCR products at different KCI concentrations (50-90 roM). Testing sensitivity ofRT·PCR method: Serial dilutions of coliphage QP stock were made in phage broth and 100 III of each dilution was heated at 90·C for 10 minutes. Then 3 III of each dilution was used fo; amplification in RT-PCR. The results are shown in Table 1. "Sensitivity" means the detection limit of amount of coliphage OP in unit PFU/reaction. Table 1: The sensitivities of the RT-PCR methods performed by changing Taq polymerase incorporating with KO concentration and 50·C annealing temperature. KO c:one.in reaction (mM)
(0.5,25)
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80
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70
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Application of polymerase chain reaction to detect RNA coliphage Q(3
387
The column (0.5,25) in Table 1 sh~ws the res~l~s .obtained from the experiment using Taq polymerase 0.5 units. The effect of KCI concentratIon on senstttvlty of PCR can be seen from the concentration of KCI 90 mM. Consequently, this condition showed low sensitivity and reproductivity. In cases of KCI concentration in the range 30-70 mM. the range of sensitivities are 4.2 x 104 to 4.2x10 3 PFU/reaction which equal to 1.4xl07 to 1.4xI06 PFUlml, respectively.
In the experiments using Taq polymerase 1.0 and 2.0 units with 25-cycle amplification, the sensitivities are 4.2x103 PFU/reaction and 4.2x10 3 to 4.2x102 PFU/reaction, respectively. This case, the sensitivities are not affected by KCl concentration (30-90 mM). These results mean consistency of sensitivity with effect of KCI concentration. The experiment of 35-cycle amplification with Taq polymerase 1.0 unit (column (1.0,35) in Table 1), all sensitivities are 4.2x10 2 PFU/reaction. When this result was compared to the experiment using Taq polymerase 1.0 unit with 25-cycle amplification (column (1.0,25) in Table 1). It is showed that increasing the number of cycles from 25 to 35 cycles can increase sensitivity from 4.2xl03 to 4.2x102 PFU/reaction. However, when number of cycles were increased, the non-specific or background products were observed on gel electrophoresis. To solve this problem, the annealing temperature was increased from 50'C to 55'C. The increasing of annealing temperature has been suggested for improving the problem of smear of PCR products seen on the agarose gel (17). And to improve the sensitivity, the concentration of Taq polymerase was increased to 2.5 units with 35-cycle PCR amplification. Then this condition was tested for determining the sensitivity of the primers. The results are shown in Table 2. Table 2 : Testing sensitivity of F-specitic RNA coliphage QII primers with the condition of SS'C annealing temperature and Taq polymerase 2.5 units for 3S-cycle PCR amplification. KCI conc.(mM)
Sensitivity (2.5,35)'
50
0.3 PFU
(1.06x10 2 PFU/ml)
70
0.3 PFU
(l.06x 102 PFU/ml)
• (unit of Taq polymerase, number of cycles) 1 2 3 4 5 6 7 8 9 10
Annealing temperature SO'C (Taq polymerase 1.0 unit, KCI 50 mM)
1 2 3 4 5 6 7 8 9 10
Annealing temperature SST (Taq polymerase 2.5 units, KCI 50 mM)
Figure 2: Comparison of 35-cycie PCR amplification between tbe conditions of annealing temperature 50'C and 55·C. Lane (1) DNA marker 0X174, HaeIII, (2) Negative control, (3) 3.0 x 104 PFU, (4) 3.0 x 103 PFU, (5) 3.0 x 102 PFU, (6) 3.0 x 10 1 PFU, (7) 3.0 x 10° PFU, (8) 3.0 x 10-1 PFU, (9) 3.0 x 10-2 PFU, (10) 3.0 x 10-3 PFU. MSI H-5f6·.u,
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These results show that the detection limit or the sensitivity can be 0.3 PAl/reaction. The increasing of annealing temperature reduced amplifying background products and gave clearer image of amplified products on gel electrophoreses (see Figure 2). This developed RT-PCR technique ( 35-cycle amplification with Taq polymerase 25 units, 55"C annealing temperature, and 50 mM KO) was tested for detection of F-specific RNA coliphage (group III) in night soil sample. Testing detection of F·spedOc RNA coliphages ( group Ill) In Night soli sample: The above mentioned PCR condition which gave the maximum sensitivity (0.3 PAl/reaction) in the experiment for testing the sensitivity was used for detection of F-specific RNA coliphages (group UI) in night soil sample. The night soil sample which was mixed with sludge of onsite small treatment plant was collected from Urayasu Night Soil Treatment Plant, Cbiba Prefecture, Japan. The supernatant after centrifuged at 2O,000)(g for 30 minutes was filtered by 0.45 I!m membrane filter. The pH of filtrate was about 8.2. This filtrate was divided for determining plaque assay and extracting for RT-PCR. The plaque assay method used was the double-agar-Iayer method with E.coli K12,F+,AI>" as host organism. The extracted and diluted samples were kept at -20·C until RT-PCR was performed. The extraction methods used are as follows: 1) phenol-chlorofonn method (8), 2) direct heating at 9O"C for 10 min, 3) treating with RNasin (40 units/lOO Ill. sample) and then heating at 9O"C for 10 min, and 4) treating with Proteinase K then heating at 9O"C for 10 minutes: Fifty III of buffer solution (10 111M Tris-HO(pH 7.5), 100 mM NaC!, 1 mM EDTA) was added into 500 III sample and incubated with Proteinase K, 200 Ilglml at 55"C for 60 minutes. Then the mixture was heated at 90"C for 10 minutes quick cooled down to 4·C and chilled on ice. ' The results are summarized in Table 3 and shown in Figure 2. Table 3 : Testing detection of F.speclfic RNA coliphages ( group Ill) In night soil sample. Extraction method
No dilution
Phenol-chloroform method Direct heatiDl]; at 9O"C for 10 min. RNasin treatin/! and heatin/! at 9O·C for 10 min. Proteinase K treatinl! and heatinl! at 9O"C, for 10 min.
+
-
Diluted sample 10- 1fold
10-2 fold
10-3 fold
NO
NO
NO
+ + +
+ + +
-
-
NO
+ : positive, -- : negative, NO: not done. The concentration of F-specific RNA coliphages in night soil sample determined by plaque assay method is 1.8 x 103 PFU/ml. The results obtained from RT-PCR method showed positive bands of F-sr:cific RNA coliphages (group m) from samples which were extracted by Phenol-chloroform method, 10- and 10-2 fold dilution of direct heating samples without RNasin treating, with RNasin treating, and with proteinase K treating. In addition, direct heated samples without further dilution showed negative results (no visible band of amplified products). This indicates the presence of inhibitors in night soil sample which can obstruct the RT-PCR.
Application of polymerase cbain reaction to detect RNA coliphage Oil
389
This inhibitors effect could be reduced by dilution in the case of direct heating extraction methods, while the inhibitors were removed during Phenol-chloroform method. Based on the results of plaque assay, the 10-2 fold dilution of heat extracted samples, which showed positive results by RT-PCR assay in Table 3, contain 1.8 x 101 PFUlml. This value (1.8 x 101 PFU/ml ) is lower than the detection limit or sensitivity (10 2 PFU/ml which is equivalent to 0.3 PFU/reaction as mentioned above in Table 2). This inconsistency between plaque assay and PCR assay may be caused by various effect of inhibitors in night soil sample on both methods. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
•
194 bp
Figure 3
Testing detection of F-specific RNA coliphages (group III) in night soil sample. Lane (I) DNA marker 0XI74.HaeIIl, (2) Positive control 1()4 PFU, (3) and (4) Phenol-chloroform extracted sample, (5) Direct heating, (6), (7), and (8) lO- t , 10-2, 10-3 dilution, respectively, (9) RNasin treating and heating, (10), (11), and (12) 10- 1, 10-2, 10-3 dilution, respectively, (13) and (14) Proteinase K treating and heating, (15) Negative control.
CONCLUSION The RT-PCR method for direct detection of enteric viruses in environmental samples was developed by using RNA coliphage QJ3 as the model virus with direct heating (90·C, 10 min) as viral extraction method. This developed method was applied for detection of F-specific RNA coliphages (group III) in night soil sample. The RT-PCR method was improved by optimizing the concentrationn of KCI, Taq polymerase, annealing temperature, and number of cycle. Optimization concentration of KCI and Taq polymerase has illustrated that the high KCI concentration (up to 90 mM) in reaction incorporating with low Taq polymerase concentration (0.5 unit/40 III total reaction) causes low sensitivity and reproductivity of PCR. The KCI concentration (30 to 90 mM) implied no significant effect to the yields of PCR products when higher Taq polymerase (1.0 to 2.5 units/40 !.ll total reaction) was included. Increasing the number of cycle from 25 to 35 cycles can increase the sensitivity. The increasing of annealing temperature reduced amount of amplifying background products seen on gel electrophoreses. The maximum sensitivity is 0.3 PFU/reaction which is obtained by using the condition of 35-cycle PCR amplification with Taq polymerase 2.5 units and 55·C annealing temperature. This RT-PCR technique will be able to be used as a protocol for further study for enteroviruses.
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The success of detection of indigenous RNA coliphages (group III) in night soil sample using the developed RT-PCR methods means that the developed method has great potential as a direct detection method of viruses in environmental sample. The inconsistency of the detection limit between plaque assay and RT-PCR assay may be caused by the effects of inhibitors in night soil sample. Further study on the relationship between plaque assay and peR assay is required. REFERENCES 1. Vandervelde, C., Verstraete, M., Beers, D. V. (1990). Fast multiplex polymerase chain reaction On boiled clinical samples for rapid viral diagnosis. J. o/Virological Methods. 30,215-228. 2. Tsai, Y.-L., Palmer, C. J., Sangermano, L. R. (1993). Detection of Escherichia coli in sewage and sludge by polymerase chain reaction. Appl. Environ. Microbiol. 59,353-357. 3. Almar, R. L, Metcalf, T. G., Neil, F. H., Estes, M. K. (1993). Detection of enteric viruses in oysters by using the polymerase chain reaction. Appl. Environ. Microbiol. 59, 631-635. 4. Kopecka, H., Dubrou, S., Prevot, J., Marchal, J., Lopez-pila, J. M. (1993). Detection of naturaally occurring enteroviruses in waters by reverse transcriptase, polymerase chain reaction, and hybridization. Appl. Environ. Microbiol. 59, 1213-1219. 5. Tsai, Y.-L., Sobsey, M. D., Sangermano, L. R., Palmer, C. 1. (1993). Simple method of concentrating enteroviruses and Hepatitis A virus from sewage and ocean water for rapid detection by reverse transcriptase-polymerase chain reaction. Appl. Environ. Microbiol. 59, 3488-3491. 6. Abbaszadegan, M., Huber, M. S., Gerba, C. P. (1993). Detection of enteroviruses in groundwater with the polymerase chain reaction. Appl. Environ. Microbiol. 59, 1318-1324. 7. Yamada, 0., Matsumoto, T., Nakashima, M., Hagari, S., Kamhora, T., Ueyama, H., Kishi, Y., Uemura H., Kurimura, T. (1990). A new method for extracting DNA or RNA for polymerase chain reaction. 0/ Virological Methods. 27, 203-210. 8. Dantheeravanich, S. (1992). Application of polymerase chain reaction for health-related viral indicators detection in wastewater. Doctoral dissertation, The University of Tokyo, Tokyo, Japan. 9. Innis, M. A, Gelfand, D. H. (1990). Optimization ofPCRs, p.3-12. in Innis, M. A, Gelfand, D. H., Sninsky, J. J., White, T. J. (ed.), peR Protocols: A Guide to Methods and Applications. Academic Press, Inc., U.S.A. 10. Saiki, R. K. (1989). The design and optimization of the PCR, p.7-16. in Erlich, H. A. (ed.), PCR Technology: Principles and Application for DNA amplification. Stockton Press, U.S.A. 11. Bej, A. K., Mahbubani, M. H., Atlas, R. M. (1991). Amplification of nucleic acid by polymerase chain reaction (PCR) and other methods and their applications. Critical Reviews in Biochemistry and Molecular Biology. 26, 301-334. 12. Steffan, R. J., Atlas, R. M. (1991). Polymerase chain reaction: Applications in Environmental Microbiology. Annu. Rev. Microbiol. 45, 137-161. 13. Bej, A. K., DiCesare, J. L., Haff, L., Atlas, R. M. (1991). Detection of E.coli and Shigella sPp. in water by using polymerase chaain reaction (PCR) and gene probes for uid. Appl. Environ. Microbiol. 57, 1013-1017. 14. Rotbart, H. A (1990). PCR amplification of enteroviruses, p.372-377. In Innis, M. A. (ed.), PCR Protocols: A Guide to MetllOds and Applications. Academic Press, Inc., U.S.A. 15. Adams, M. (1959). Bacteriophages. Interscience Pub\. Inc., New York. 16. Innis, M. A, Myambo, K. B., Gelfand, D. H., Brow, M. D. (1988). DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. 85, 9436-9440. 17. Williams, J. F. (1989). Optimization strategies for the polymerase chain reaction. Biotec1lltiques. 7, 762-769.
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