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Optimized RT-PCR with universal primers detection of enteroviruses from water samples

Optimized RT-PCR with universal primers detection of enteroviruses from water samples. Introduction.

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Optimized RT-PCR with universal primers detection of enteroviruses from water samples

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  1. 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 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 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 Virus concentration and RT-PCR analysis Analysis of RT-PCR amplification 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. The water sample seeded with PV1 was filtered through cellulose mixed-ester 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. 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 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.1 RT-PCR amplification of four virus sample. M: DNA marker; 1: CVB3; 2: PV1; 3: PV2; 4: PV3；5：negative control Optimization of operational conditions of RT-PCR 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. Detection limits of RT-PCR with universal primers 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). a. Effect of reverse transcriptase M: DNA marker; 1: positive control withMLV; 2: source water withMLV; 3: sewage withMLV; 4: positive control with AMV; 5: source water with AMV; 6: sewage with AMV. M: DNA marker; 1: 380000 CCID50; 2: 38000 CCID50; 3: 3800CCID50; 4: 380CCID50; 5: 38 CCID50. 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 ℃. Fig.5 Detection limits of RT-PCR 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. 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. 2 Optimization of operational conditions of RT-PCR Fig. 6. RT-PCR detection of enterovirus in different water samples Conclusions 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. Acknowledgement: This study is supported by the National Natural Science Foundation of China (Grant No. 50478048)

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