The method exploration of multiplex real-time quantitative PCR for detection of type G1-G4 rotaviruses#br#
Wang Yunjin, Zhou Xu, Bai Muqun, Fu Shengfang, Hu Guanghong, Wang Mingqiang, Li Xiongxiong, Kou Guiying, Bao Hong, Ma Chao#br#
The Second Research Department, Lanzhou Institute of Biological Products Co., Ltd., Center for Gansu Provincial Vaccine Engineering Research, Lanzhou 730046, China; Shanghai Institute of Biological Products Co., Ltd., Shanghai 201403, China
Abstract：Objective To screen reaction system and explore method of multiplex real-time quantitative PCR (qPCR) for rapid genotyping and quantitative testing VP7 genes of G1-G4 rotaviruses. Methods Primers, probes and in vitro transcripted RNAs specific for VP7 genes of G1-G4 rotaviruses were used to screen multiplex qPCR reaction systems and establish multiplex qPCR methods. The detection results between multiplex qPCRs and simplex qPCR were compared by one-way ANOVA and paired-sample t-test. Results After screening, 6 reaction systems of triplex qPCR and 1 reaction system of quadruplex qPCR were obtained. The triplex qPCR and quadruplex qPCR were established with determination coefficients (R2) of standard curves＞0.99, amplification efficiencies ranging from 90% to 110%, except type G1 in quadruplex qPCR (R2=0.982, amplification efficiency=89.221%) slightly below the requirement. The detection sensitivity was 102 copies/μl. The correlation was good (R2 >0.95) when the multiplex qPCRs and simplex qPCR used to detect same sample. There was no significant difference in detection results between triplex qPCR and simplex qPCR (t=1.420-25.786, all P>0.05) and between quadruplex qPCR and simplex qPCR (t=2.505-4.851, all P>0.05). Conclusion The multiplex qPCR established is capable of typing and quantitative detection of more than 3 target genes in the same reaction tube, providing a reference for the establishment of rapid typing and quantitative detection of multivalent rotavirus vaccine and mixed virus samples in the future.
2020, Vol. 43 
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