| Orf virus(ORFV),goatpox virus(GTPV),and Peste des petits ruminants virus(PPRV)are the main viral pathogens that cause acute and febrile infectious diseases in ruminants.These pathogens can cause single infections or multiple infections in cattle and goats,with high morbidity and serious harm.Because these diseases have similar clinical symptoms such as herpes and ulcers on the skin and mucous membranes,and mixed infections often occur,it is often difficult to make a fast,accurate and effective differential diagnosis in clinical practice.Therefore,it is of great significance to establish a rapid,accurate,and highly sensitive joint pathogens detection method for the rapid diagnosis and effective prevention and control of these diseases.In this study,a triple TaqMan fluorescence quantitative PCR detection method targeting ORFV,GTPV and PPRV was established,optimized,and used to conduct detection and analysis of ORFV,GTPV,and PPRV on 279 clinical samples collected from 11 goat farms in 4 counties and cities in Sichuan Province from 2019 to 2022,hoping to provide a reference for the improvement of the prevention and control of such diseases in the future.The results of the study are as follows:Ⅰ.Establishment,optimization and evaluation of triple TaqMan fluorescence quantitative PCR detection method targeting ORFV,GTPV and PPRV1.A triple TaqMan fluorescence quantitative RT-PCR method targeting ORFV,GTPV and PRRV was established and optimized.With the complete gene sequences of ORFV,GTPV and PRRV as templates,the ORFV-B2 L,PPRV-N and GTPV-p32 gene sequences were cloned and the nucleic acid standards of the three pathogens were constructed.Three pairs of probes were designed and the triple TaqMan fluorescence quantitative RT-PCR detection method targeting ORFV,GTPV and PRRV was successfully prepared.The detection conditions of the constructed method were optimized,and it was found that the amplification effect was the best when the total reaction volume was 25 μL,the Prime Script RT Enzyme Mix II was 1 μL(20 U/μL),the PPRV and GTPV primers and probe concentrations were 0.2 μmol/L,the ORFV primer and probe concentration was 0.8μmol/L,the volume of standard mixture was 3 μL,and the annealing temperature was55℃.2.The quality of the constructed detection method was evaluated.The specificity test results showed that there was no cross-reaction among ORFV,GTPV and PRRV,with good specificity,and there was no amplification of the genomes of BPIV-3 type,BVDV,BCV,FMDV-O type,FMDV-A type,goat mycoplasma pneumoniae,mycoplasma capricolum,goat mannheimia haemolytica,goat mycoplasma arginini,pseudotuberculosis corynebacterium;the sensitivity test showed that the lowest copy number was 2.98copies/μL for ORFV,3.81 copies/μL for PPRV,the lowest copy number detected by GTPV was 6.68 copies/μL;the stability test showed that the coefficient of variation(CV)within and between groups was less than 4%,indicating that the detection method had good stability.Then,the triple TaqMan fluorescence quantitative PCR detection method was used to detect the known positive and negative nucleic acid samples of 3 pathogens.Then the detection coincidence rates were compared with fluorescence quantitative PCR commercial kits for ORFV and GTPV and OIE standards for Peste des petits ruminants.The results showed that the coincidence rates of detection using triple TaqMan fluorescence quantitative RT-PCR designed in this study with the commercial detection kits and OIE standard were 100 %(k = 1)for ORFV,100 %(k = 1)for PPRV and 100%(k= 1)for GTPV,respectively,indicating that the designed triple TaqMan fluorescence quantitative RT-PCR method has high accuracy.Ⅱ.Epidemiological survey of ORFV,PPRV and GTPV in some areas of Sichuan from 2019 to 2022The triple TaqMan fluorescence quantitative PCR detection method designed in this study was used to detect 279 clinical samples collected from 11 goat farms in 4 counties and cities in Sichuan Province from 2019 to 2022.The real-time fluorescence quantitative detection results were compared between different feeding stages,different types of clinical samples,different varieties,and different seasons.The results showed that the detection rates of positive ORFV,PPRV and GTPV were 34%,4.3% and 2.5%,respectively;the detection rates of mixed infection with ORFV and GTPV was 2.5%.PPRV was only found in samples from Jianyang goat farm;after sequencing,it was found that the homology with the vaccine strains published in NCBI was 99.2%.There were different degrees of ORFV and GTPV infections in goats in the four different areas in Sichuan Province.The infection rate of ORFV in backup goats(38.2%)was relatively higher than that in multiparous ewes(27.5%).PPRV and GTPV were only found out in multiparous ewes,and the detection rates were 11% and 6.4%,respectively.The detection rate of ORFV in goats with scabs on the mouth(66.2%)was significantly higher than that in whole-blood samples(25.6%),but it was not found in feces.PPRV and GTPV were only detected in whole-blood samples,with detection rates of 6.7% and 3.9%,respectively.ORFV can infect goats of different breeds,and the infection rate in pockmarked goats is the highest(37.9%),which is higher than that in Jintang black goat(28.7%)and Jianzhou big-ear sheep(35.7%).PPRV and GTPV were only detected in Jianzhou big-ear sheep,and the detection rates were 12.2% and 7.1%,respectively.The detection rates of ORFV in spring(46.3%)and autumn(43.5%)were significantly higher than that in summer(24.6%)and winter(22.7%).PPRV and GTPV were detected only in autumn,with detection rates of 4.3% and 2.5%,respectively.In conclusion,this study successfully constructed a triple TaqMan fluorescence quantitative PCR detection model,which has good specificity,sensitivity and stability,and can be effectively applied to the clinical sample detection of the above-mentioned goat diseases.In addition,an epidemiological investigation was carried out on ORFV,PPRV and GTPV infections in some areas in Sichuan Province,which can provide an important epidemiological theoretical basis for the prevention and control of these three types of diseases in these areas in the future. |
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