Establishment Of Fluorescent Quantitative PCR For Detecting Five Important Murine And Rat Viral Diseases

Rats and mice are most popular laboratory animals used for biomedical researches.However,infectious diseases have been a big problem for the health of laboratory rats and mice.In this study,rat parvovirus,rat minute virus,encephalomyocarditis virus,murine rotavirus and Theiler's-like virus of rats were selected,which were not listed in the national laboratory animal testing standards.According to the potential threat to the health of laboratory animals,rapid detection methods are requested to be established.Among the different methods,TaqMan probe real-time fluorescent quantitative polymerase chain reaction(qPCR)method has good specificity and sensitivity,which will be a powerful technical support for the monitoring of these diseases.1.Development of a double TaqMan real-time PCR assay for quantitative and differential detection of Rat minute virus and Rat parvovirusThe purpose of this study was to establish TaqMan real-time fluorescent quantitative PCR method which can detect Rat minute virus(RMV)and Rat parvovirus(RPV)quickly and accurately in clinic.According to the whole genome sequence of strain RMV NTU1(Accession No.JX627317.1 in Genbank),the primers and TaqMan probe were designed from the 3389?3589 bp region.And according to the whole genome sequence of strain RPV NTU1(Accession No.JX827169 in Genbank),the primers and TaqMan probe were designed from the 863~967 bp region.The linearity,specificity,and sensitivity of the method were investigated.50 clinical samples were detected using this fluorescence quantitative method,which compared with the traditional serological test method.The real-time PCR for detecting RMV and RPV showed a perfect linear relationship of standard curve,and R2 value reached 0.99 with a high specificity.The sensitivity of the real-time PCR was 10 copies/pL.The accuracy of test results for 50 clinical samples 100%.The TaqMan probe-based real-time PCR method is established with good specificity and sensitivity,which can make a powerful technical support for RMV and RPV investigation and detection.2.Establishment of TaqMan Real-time RT-PCR Method to Detect Mice Encephalomyocarditis virusTo develop a real-time RT-PCR assay for efficient detection of Murine Encephalomyocarditis virus(EMV).According to the genomic sequences of EMV strain PEC9(DQ288856.1)download from NCBI and to find the conserved sequences.One pair of the specific primers and one TaqMan probe were designed.Then reaction parameters were optimized to develop a real-time RT-PCR assay.The method showed a good linear relationship of standard curve with a R2 value of 0.99.The sensitivity of the real-time PCR was less than 1 copies/?L.1000 times higher than ordinary PCR method,and it's specificity is strong,common mice strains had no specific amplification,between batch and batch of variation coefficient is less than 2%.120 mice feces samples epidemic materials provided by the Shanghai institute of experimental animals were detected by the real-time PCR.All the 120 mice samples were negative.We find the conclusion that the real-time RT-PCR method is good in specificity and sensitivity,which can make a powerful technical support for EMV epidemiological investigation and detection.3.Establishment of TaqMan Real-time RT-PCR Method to Detect Murine RotavirusTo establish and preliminarily apply the real-time PCR for detecting Murine Rotavirus(MRV),one pairs of primers and TaqMan probe were designed according to the genomic sequence of MRV strain EB-C8/G16P VP1 gene(Genbank:KJ477127.1).The linearity,specificity,and sensitivity of the method were investigated.The results showed a perfect linear relationship of standard curve,in which R2 value reached up to 0.99.The sensitivity of the TaqMan-probe real-time PCR was as lest as 10 copies/?L.100 times higher than ordinary PCR method,and it's specificity is strong,common mice strains had no specific amplification,between batch and batch of variation coefficient is less than 2%.120 mice samples were detected by the method.However,the mice samples were negative by the method.The real-time RT-PCR method is good in specificity and sensitivity,which can make a powerful technical support for MRV epidemiological investigation and detection.4.Establishment of real-time quantitative PCR to detect rat Theiler's-like virusTo accurately establish TaqMan probe real-time quantitative PCR(qRT-PCR)method for Theiler 's-like virus of Rats(TLV).We use fluorescent quantitative polymerase chain reaction technology,specific for the TLV virus nucleic acid testing.The primers and TaqMan probe specific to 3622?3729 bp region was designed according to the whole genomic sequence of TLV representative strain in GenBank NGS910(Accession No.AB090161.1).And we synthesis a plasmid gene as the DNA standard template,the stability,specificity,and sensitivity of the qRT-PCR method were investigated.The results showed that,in the standard curve,R2 value could reach up to 0.99 with a high specificity.The sensitivity of the real-time PCR was less than 30 copies/?L.100 times higher than ordinary PCR method,and its specificity is strong,common rat strains had no specific amplification,between batch and batch of variation coefficient is less than 1%.The method is used the TaqMan probe advantages to establish the TLV detection method,ensure the method with simple operation,high sensitivity,good specificity.This research make for the prevention and control of TLV infection in rats.and provides reliable data and monitoring methods for establish national standards and local standards.Finally,in the advantage of the fluorescent quantitative PCR technique in virus detection,five mice and rat virus detection methods are established respectively and validated the accuracy and sensitivity,which were compared with the serological detection and ordinary PCR detection.