Establishment Of Real-time TaqMan RT-PCR For Quantitation Rotavirus VP7 Gene Of Serotype G4

Object To establish TaqMan-based real time RT-PCR assay for detection and quantitation rotavirus G4 serotype VP7 gene.Methods The most common cause of severe diarrhea in children under 5 years of age is rotavirus; rotavirus infections are responsible for 5% of all childhood deaths. Ninety percent of these deaths occur in developing countries. World Health Organization (WHO) has recommended that rotavirus vaccination be included in all national immunization programs to provide protection against rotavirus infection. Real-time PCR is the continuous collection of fluorescent signal from one or more polymerases chain reactions over a range of cycles. The fluorescent signals from each reaction correlate with copy number of template. A perfect assay would have a slope of-3.32 which correspond to 100% efficiency, a intercept between 33 and 37 cycles and R2 of 1.00.The purpose of this study is establishment a quickly, accurate and specific TaqMan-based real time RT-PCT assay for detection and quantitation rotavirus G4 serotype VP7 gene. Design primer and probe which locate rotavirus G4 serotype VP7 gene. There has 8 bases between probe and upper stream primer, the 3'terminatio of probe was tagged with JOE as donor fluorescent dye and the 5'terminatio with Eclipse as reporter fluorescent dye. Amplification VP7 gene from RV G4 serotype, construct a plasmid includes this gene and clone culture. Amplification the VP7 gene from above mentioned plasmid and purification the PCR production. In vitro transcription purified VP7 gene to RNA. Purify the RNA and quantitative it by photometry, dilute the RNA to 102-107 copies/ul as standard RNA of RT-qPCR. Screen primer and probe, optimize parameters of PCR system, and quantify rotavirus G4 serotype copy number by absolute assay. Results optimized reaction for detection of RV G4 serotype VP7 gene had a wide dynamic range from 102-107 copies/μl. It had good amplification efficiency greater than 90%. The R2's of standard curve were greater than 0.98. Compared with convention RT-PCR, RT-qRCR had higher sensitive, approximately 10 fold of the former. RT-qPCR can detection target RNA from light concentration sample lower than 10 copes/μl. It is repeatability, specific and sensitive assay for quantitation and detection RV G4 serotype VP7 gene. Conclusion The real-time (TaqMan-based) PCR assay can provide sensitive, specific and repeatable method for detection and quantitation rotavirus G4 serotype VP7 gene...