Optimized RT-PCR with universal primers detection of enteroviruses from water samples Introduction Because enteroviruses have been associated with outbreaks of waterborne diseases, their presence in these waters is of important concem for public health Enteroviruses have been found in various waters including natural water, raw sewage and treated effluents, and even in treated drinking water. However, the conventional methods have disadvantages of being inaccurate, time consuming and expensive. In order to establish a rapid and sensitive method to simultaneously detect enterovirus from different water samples, the detection method of RT-PCR with universal primers combined the modified adsorption elution was described in this study. The operational conditions of detection method were optimized By tested source water, secondary effluent and sewage sample with seeded virus, detection limits of RT-PCR system was determined Results Discussion Analysis of RT-PCR amplification Virus concentration and RT-PCR analysis The universal primers tested by RT-PCR on RNA templates prepared The water sample seeded with PV1 was filtered through cellulose mixed- from Poliovirus type1, Poliovirus type2, Poliovirus type3 and ester filter with different nominal pore size, respectively. The water sample Coxsackievirus B3. As expected, four limpid specific straps all appeared at seeded with PV1 was filtered through 0.22um filters with different 400bp for fours kinds of enteroviruses on the electrophoregrams materials The result showed that the concentrated effect of enterovirus respectively(Fig. 1). Analysis results verified that the universal primers with cellulose mixed-ester filter with nominal pore size of 0. 22um was showed specificities only for their corresponding target genes. Direct superior than others. The isolation of the enteroviruses from water sample sequencing was conducted to further confirm the identity depends on virus adsorption to the filter by multi-action including electrostatic attraction, hydrogen bond and hydrophobic bond Fig 1 RT-PCR amplification of four virus sample Fig. 3 different nominal pore size Fig. 4 Different materials of filters M: DNA marker: 1: CVB3: 2. PV1: 3: PV2 M: DNA marker M: DNA marker: 1: NC membrane 4: PV3: 5: negative control 1: concentrated with 0. 22um filter: 2: Nylon membrane 2: concentrated with 0.8um filter; 3: Cellulose mixed-ester membrane(not Optimization of operational conditions of RT-PCR 3: concentrated with 5um filter. adjust Mg2+ and pH in solution) 4: Cellulose mixed-ester membrane AMV reverse transcriptase was adopted for use with sewage sample 5: negative control Annealing temperature seems to be no obvious effect to the results of detection From 0 to 4.5mM Mg2+ ions concentration were tested in PCR Detection limits of RT-PCR with universal primers reaction system with same other components, the 2mM Mg2+ ions was appropriate for PCR. Detection limits of RT-PCR system were determined to be 38 Cell Culture Infective Dose50(CCID50)by serial ten-fold dilutions of viral Effect of reverse transcriptase particles(Fig. 5). Through tested on source water, secondary effluent M: DNA marker: and sewage, the detection limit in water sample with seeded virus was 1: positive control with MLV 38 CCID50(Fig. 6) 2: source water with MLy 3: sewage with ML\ 4: positive control with AMY M: DNA marke 5: source water with AMv 1:380000cc|D50 2:38000cc|D50 750bp 3:3800cc|D50 4:380cc|D50 b Effect of annealing temperature 5:38cc|D50 M: DNA marker: 1000bp 1:52 2:526℃C;3:532℃C; 4:54C:5:55℃ Fig 5 Detection limits of RT-PCR 6:562C;7:57.2°C 8:58C;9:59C M: DNA marker a: source water b: secondary effluent C Effect of magnesium ion C: sewage sample 1:380000cc|D50 M: DNA marker: 2:38000cc|D50 1:0mMMg2+;2:0.5mMMg2+ 3:3800ccD50; 3:1mMMg2+:4:1.5mMMg2+ 4:380Cc|D50 5:20mMMg2+:6:2.5mMMg2+ 7:30mMMg2+;8:4.5mMMg2+ 5:38cc|D50 ig 2 Optimization of operational conditions of RT-PCR Fig. 6. RT-PCR detection of enterovirus in different water samples Conclusions accuracy. It can provide a reliable approach for surveillance and risk assessment of enteroviruses in water environme stage of simpleness, sensitivity and Comparison of other methods, the RT-PCR with universal primers detection method established by this study has adval Acknowledgement: This study is supported by the National Natural Science Foundation of China( Grant No. 50478048)
Optimized RT-PCR with universal primers detection of enteroviruses from water samples Because enteroviruses have been associated with outbreaks of waterborne diseases, their presence in these waters is of important concern for public health. Enteroviruses have been found in various waters including natural water, raw sewage and treated effluents, and even in treated drinking water. However, the conventional methods have disadvantages of being inaccurate, time consuming and expensive. In order to establish a rapid and sensitive method to simultaneously detect enterovirus from different water samples, the detection method of RT-PCR with universal primers combined the modified adsorptionelution was described in this study. The operational conditions of detection method were optimized. By tested source water, secondary effluent and sewage sample with seeded virus, detection limits of RT-PCR system was determined. Introduction Results & Discussion AMV reverse transcriptase was adopted for use with sewage sample. Annealing temperature seems to be no obvious effect to the results of detection. From 0 to 4.5mM Mg2+ ions concentration were tested in PCR reaction system with same other components, the 2mM Mg2+ ions was appropriate for PCR. Conclusions The universal primers tested by RT-PCR on RNA templates prepared from Poliovirus type1, Poliovirus type2, Poliovirus type3 and Coxsackievirus B3. As expected, four limpid specific straps all appeared at 400bp for fours kinds of enteroviruses on the electrophoregrams, respectively (Fig.1). Analysis results verified that the universal primers showed specificities only for their corresponding target genes. Direct sequencing was conducted to further confirm the identity. Fig. 2 Optimization of operational conditions of RT-PCR Comparison of other methods, the RT-PCR with universal primers detection method established by this study has advantage of simpleness, sensitivity and accuracy. It can provide a reliable approach for surveillance and risk assessment of enteroviruses in water environment. Fig.1 RT-PCR amplification of four virus sample. M: DNA marker; 1: CVB3; 2: PV1; 3: PV2; 4: PV3；5：negative control Analysis of RT-PCR amplification Optimization of operational conditions of RT-PCR Virus concentration and RT-PCR analysis The water sample seeded with PV1 was filtered through cellulose mixedester filter with different nominal pore size, respectively. The water sample seeded with PV1 was filtered through 0.22μm filters with different materials. The result showed that the concentrated effect of enterovirus with cellulose mixed-ester filter with nominal pore size of 0.22μm was superior than others. The isolation of the enteroviruses from water sample depends on virus adsorption to the filter by multi-action including electrostatic attraction, hydrogen bond and hydrophobic bond. a. Effect of reverse transcriptase M: DNA marker; 1: positive control with MLV; 2: source water with MLV; 3: sewage with MLV; 4: positive control with AMV; 5: source water with AMV; 6: sewage with AMV. b. Effect of annealing temperature M: DNA marker; 1: 52 ℃; 2: 52.6℃; 3: 53.2℃; 4: 54 ℃; 5: 55 ℃; 6: 56.2℃; 7: 57.2℃; 8: 58 ℃; 9: 59 ℃. c. Effect of magnesium ion M: DNA marker; 1: 0mM Mg2+; 2: 0.5mM Mg2+; 3: 1 mM Mg2+; 4: 1.5 mM Mg2+; 5: 2.0mM Mg2+; 6: 2.5mM Mg2+; 7: 3.0mM Mg2+; 8: 4.5mM Mg2+. Fig. 3 different nominal pore size M: DNA marker; 1: concentrated with 0.22μm filter; 2: concentrated with 0.8μm filter; 3: concentrated with 5μm filter. Fig. 4 Different materials of filters M: DNA marker; 1: NC membrane; 2: Nylon membrane 3: Cellulose mixed-ester membrane (not adjust Mg2+ and pH in solution); 4: Cellulose mixed-ester membrane; 5: negative control M: DNA marker; 1: 380000 CCID50; 2: 38000 CCID50; 3: 3800CCID50; 4: 380CCID50; 5: 38 CCID50. Detection limits of RT-PCR system were determined to be 38 Cell Culture Infective Dose50 (CCID50) by serial ten-fold dilutions of viral particles (Fig. 5). Through tested on source water, secondary effluent and sewage, the detection limit in water sample with seeded virus was 38 CCID50(Fig. 6). M: DNA marker; a: source water b: secondary effluent c: sewage sample. 1: 380000 CCID50; 2: 38000 CCID50; 3: 3800 CCID50; 4: 380 CCID50; 5: 38 CCID50. Fig. 6. RT-PCR detection of enterovirus in different water samples Fig.5 Detection limits of RT-PCR Detection limits of RT-PCR with universal primers Acknowledgement: This study is supported by the National Natural Science Foundation of China (Grant No. 50478048)