Preparation Of Nucleic Acid Reference Materials For Peste Des Petits Ruminants Virus And Establishment Of Real-time Fluorescence Quantitative RT-PCR Detection Method

Peste des petits ruminants(PPR)is a serious,virulent,contact infectious disease and is one of the epidemics developed globally for control and eradication.The high morbidity and mortality of PPR is a serious threat to animal health and safety and the development of the livestock industry in China,and has caused huge economic losses.Therefore,it is important to explore a rapid,effective and standardised test for the diagnosis and detection of Peste des petits ruminants virus(PPRV).However,there is currently no common nucleic acid standard for PPRV,which makes its qualitative and quantitative analysis difficult.Therefore,the establishment of a standard substance for PPRV nucleic acid detection is the key to improving the accuracy of detection and ensuring the quality and safety of animal products.And when establishing the PPRV fluorescence quantitative RT-PCR method,the inclusion of the internal reference gene GAPDH can provide quality control of the sample and improve the accuracy of the test,which can be used for rapid detection of PPR,epidemiological investigation and epidemic surveillance,etc.It is conducive to promoting the standardisation of the PPRV real-time fluorescence quantitative RT-PCR assay.The research in this thesis covers the following two aspects:(1)Preparation of nucleic acid reference material for Peste des petits ruminants virusIn this study,nucleic acid standards for PPRV were prepared using inactivated viral fluids.PPRV was cultured using VERO cells,and the viral fluid was harvested and inactivated in a water bath at 56℃for 4 h.The inactivated cells were passed blindly for three generations.Viral fluids were harvested and tested for inactivation.The PPRV inactivated viral solution was subjected to a preliminary homogeneity test,and the lyophilised protectant was mixed with the viral solution and dispensed,and the resulting samples were taken for lyophilisation.Short-term,long-term and re-dissolution stability were evaluated by physical characterisation,sterility test,mycoplasma test and other virus test,and by homogeneity evaluation test.Standards were also calibrated through collaboration with other laboratories,and measurements were counted and processed for confidence in their accuracy.On this basis,the uniformity,stability and uncertainty of the collaborative calibration measurements were taken into account to obtain the final calibration results.The test results showed no increase in viral load after blind transmission of the inactivated viral solution to three generations of VERO cells.The PPRV nucleic acid standards were lyophilised as pale yellow solid and were fully dissolved as a clear yellow liquid after dilution with sterile water.The inactivated standards were sterile;all tested negative for mycoplasma;and free of exogenous viruses such as bluetongue,foot-and-mouth disease type O,goat pox,sheep mouth sores and deer epidemic haemorrhagic disease.The homogeneity and stability of the nucleic acid standards were good.Analysis of variance for uniformity test data showed that there was no significant difference in uniformity between groups and within groups(P>0.05).Stored at room temperature,4℃,-20℃and 37℃for 9 days and at room temperature,4℃,-20℃and-80℃for 6 months,no significant changes were observed.The calibration work is completed jointly by the external laboratory,and the calibration results are statistically analyzed and processed.When the uniformity,stability and uncertainty were introduced into the determination result,the determination result of this reference material was(4.8±0.45)×10~4copies/μL with the confidence rate of 95%.The standard nucleic acid of PPR virus prepared in this study has good uniformity and stability,which can be used to evaluate laboratory detection methods and select detection reagents.(2)Establishment of a quantitative real-time fluorescence RT-PCR method for ruminant virus pest with internal referenceIn this study,a real-time quantitative fluorescent RT-PCR method for Petit Ruminant virus containing internal reference genes was established.Primers and probes of quantitative fluorescent RT-PCR from the GB/T 27982-2011 Diagnostic Technique for Petit Ruminant were selected.Based on the GAPDH gene sequences of small ruminants such as sheep,goats and deer,a conserved sequence was selected to design a pair of primers and probes for reference gene GAPDH,and a real-time fluorescence quantitative RT-PCR reaction system was established and optimized to detect specificity,sensitivity and repeatability.The common RT-PCR method in GB/T 27982-2011 Diagnostics for Peste Petit Ruminant and the method established in this study were used to detect 92 clinical sheep blood samples,and the coincidence rate of the detection results of the two methods was compared.The experimental results showed that after optimization,the optimal primers concentration of PPRV and probe concentration of 0.4μmol/L,and the optimal primers concentration of GAPDH were 0.4μmol/L and probe concentration of 0.2μmol/L.The method could amplify PPRV specifically,and had no cross reaction with common small ruminant susceptible viruses such as bluetongue disease,foot-and-mouth disease,goat pox,sheep mouth sore and deer epidemic hemorrhagic disease,showing good specificity.The standard curves of PPRV and GAPDH show a good linear relationship.The minimum detection limit was 6.8 copies/μL of PPRV and 190 copies/μL of GAPDH,showing good sensitivity.The coefficient of variation(CV%)of PPRV and GAPDH were both lower than 2%,indicating good stability.The results of 92 sheep blood clinical samples showed that GAPDH was positive,PPRV was positive in 4 samples,and PPRV was negative in 88 samples.The coincidence rate was 100%with the positive and negative results of PPRV detected by national standard ordinary RT-PCR method.The real-time fluorescence quantitative RT-PCR method of PPR virus containing internal reference genes established in this study has strong specificity,high sensitivity and good repeatability.The addition of internal reference genes for sample quality control has improved the accuracy of detection.In summary,this study prepared a standard material for nucleic acid detection of PPR virus,and carried out collaborative calibration of the standard material to provide reliable reference values.A real-time fluorescence quantitative RT-PCR method containing the reference gene was established to detect Petit ruminant virus,and the reference gene was added for sample quality control,which improved the accuracy of detection.This study is conducive to improving the nucleic acid detection system of PPR virus and promoting the standardization of detection methods of PPR virus.