摘要：Objective To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses(HEVs)from clinical samples and to contribute to etiological surveillance of HEV-related *** A panel of RT-nPCR assays,consisting of published combined primer pairs for VP1 genes of HEV A–C and in-house designed primers for HEV-D,was established in this *** sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID50 perμL and copies perμL,and the newly established methods were tested in clinical specimens collected in recent *** The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID50 perμL and 10 virus copies perμL,and for the complete VP1 gene was 1 CCID50 perμL and 100 virus copies perμL,using serially-diluted virus stocks of five *** a proof-of-concept,25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance *** This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A–D,providing rapid,sensitive,and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

摘要：[Objective] This study aimed to optimize the PCR amplification conditions for random ssDNA pool in SELEX technology. [Method] L16（45） orthogonal experimental design was adopted for optimization of five important factors affecting PCR reaction system for random single-stranded DNA pool including Mg2＋ concentration, dNTP concentration, amount of Taq DNA polymerase, primer concentration and amount of random single-stranded DNA pool at four levels. Meanwhile, the annealing temperature and number of PCR reaction cycles were optimized to establish the optimal reaction system and PCR procedure. [Result] The optimal combination of PCR reaction system for random ssDNA pool was obtained, with a total system volume of 20 μl containing 2.0 μl of 10 × Buffer, 0.5 ng of random ssDNA pool, 2.5 mmol/L Mg2＋, 0.25 mmol/L dNTP Mixture, 0.6 μmol/L upstream and downstream primers and 1.5 U of Taq DNA polymerase; the optimal annealing temperature was 68 ℃ and the optimal number of cycles was 12. Under the above conditions, clear and stable bands with high specificity for random ssDNA pool were amplified. [Conclusion] This study laid the foundation for selection of parameters with higher specificity in SELEX technology.