Optimization of RT-PCR methods for enterovirus detection in groundwater

Figures

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  Fig. 1. Propagation of Enterovirus in cell culture. (20× magnification). *Enterovirus = Coxsackievirus B5, Echovirus 30. Images of Vero cells (20× Magnification) taken after infection enterovirus 0 h–72 h.

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  Fig. 2. Comparison of conventional RT-PCR detection limits. The 10-fold serially diluted Coxsackievirus B5 and Echovirus 30 RNAs was amplified using the reference detection methods. Lanes: M, 100 bp DNA marker; 1–10, Coxsackievirus B5 (CVB5) and Echovirus 30 (E30) 1012–103 copy/mL; 11, Negative control.

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  Fig. 3. Comparison of conventional RT-PCR detection limit by primer concentration. (A) The 10-fold serially diluted Echovirus 30 (E30) and Coxsackievirus B5 (CVB5) RNAs were amplified with 10 pmol primers by reference detection methods. Lanes: M, 100 bp DNA marker; 1–10, Echovirus 30 and Coxsackievirus B5 1012–103 copy/mL; 11, Negative control (B) The 10-fold serially diluted Echovirus 30 (E30) and Coxsackievirus B5 (CVB5) RNAs were amplified with 20 pmol primers by reference detection methods. Lanes: M, 100 bp DNA marker; 1–10, Echovirus 30 and Coxsackievirus B5 1012–103 copy/mL; 11, Negative control.

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  Fig. 4. Phylogenetic tree analysis of Enterovirus using the optimized RT-PCR detection method. (A)The phylogenetic tree showed that the enterovirus genotypes detected in the Enterovirus B reference group were aligned to Coxsackievirus B5 and Echovirus 30. (B) Enterovirus A, B, C, and D reference groups were aligned to Coxsackievirus B5 and Echovirus 30.

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  Fig. 5. Specificity verification for optimized conventional RT-PCR and real-time RT-qPCR methods to detect Enterovirus. (A) The specificity of Conventional RT-PCR was verified using the other viruses and bacteria. Lanes: M, 100 bp DNA marker; 1, Coxsackievirus B5; 2, Echovirus 30; 3, Rotavirus; 4, HAV; 5, Norovirus; 6, Aichivirus; 7, Vibrio parahaemolyticus; 8, Staphylococcus aureus; 9, Clostridium perfringens; 10, Escherichia coli; 11, Bacillus cereus; 12, negative control. (B) Specificity of real-time RT-qPCR for other viruses and bacteria.

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  Fig. 6. Applicability verification for conventional RT-PCR and real-time RT-PCR methods to detect Enterovirus. (A) After artificially inoculating Enterovirus into food water, it was detected in triplicate by the optimized conventional RT-PCR method. Lanes: M, 100 bp DNA marker; 1–3, spiking sample of Coxsackievirus B5; 4–6, spiking sample of Echovirus 30; 7, positive control; 8, negative control. (B) Applicability test for real-time RT-qPCR method using spiking samples.