| Feline parvovirus disease and feline herpesvirus type 1 virus disease are main infectious diseases in felines,which seriously endanger the life and health of pets and wild animals.Feline Parvovirus(FPV)infection causes panleukopenia in felines,characterized by high fever,intractable vomiting,diarrhea,dehydration,and severe leukopenia.Feline herpesvirus 1(FHV-1)infection causes fever,mucosal damage to the eyes and nose,and typical symptoms of upper respiratory tract infection.At the early phase of infection,the clinical symptoms of these two diseases are similar,and mixed infection usually occurs in clinical practice.Although,vaccines that can simultaneously immunize these two diseases,there is no effective treatment to cure the two diseases.Sensitive detection methods are effective to early diagnosis,but most detection methods can only identify one pathogen.And the reaction conditions are different for unique tests.Therefore,a rapid and accurate method for simultaneous detection of these pathogens is urgently needed for identification and epidemiological investigation of FPV and FHV-1.Dual qPCR and MAOPA(Magnetic beads mediated All-in-One Polymerase Amplification)assay for FPV and FHV-1 were developed.The preliminary clinical application of the two methods was carried out,and the specificity,sensitivity and repeatability of these methods were evaluated in this study.A dual qPCR method was developed to detect FPV and FHV-1 simultaneously.Specific primers and TaqMan probes were designed based on VP2 gene of FPV and UL21 gene of FHV-1,respectively.By optimizing the reaction conditions,a dual standard curve was established,and the correlation coefficients(R2)of FPV was 0.997 and FHV-1 was 0.998,indicating good linear relationship.The results showed that the lowest detection limits of FPV and FHV-1 were 4.87×103 copies/mL and 2.21×103 copies/mL,respectively.And there was no non-specific reaction to common viral and bacterial disease of cats.Intra and inter-batch coefficients of variation were both less than 2%.This assay was proved to be sensitive,specific and reproducible,indicating that it can be applied to the early diagnosis,quantitative detection and epidemiological investigation of FPV and FHV-1 infection.A dual MAOPA method for simultaneous detection of FPV and FHV-1 was developed.Specific primers and probes were designed for conserved sequences of GAPDH,and recombinant plasmids harboring internal standard genes were constructed.And DH5α-pMD18T-FPV-VP2,DH5α-pMD18T-FH V-1-UL21,DH5α-pMD18T-GAPDH bacterial pools were successfully constructed based on the research results.And the optimal reaction conditions including optimal internal control primers and probes were selected by testing.The DH5α-pMD18T-GAPDH bacterial solution with 106 dilution concentration was selected as the internal control for MAOPA detection.A dual MAOPA detection method for FPV and FHV-1 was developed and the kit was filled and assembled.The specificity,sensitivity and repeatability of MAOPA kits were evaluated.The minimum detection limits of FPV and FHV-1 were 4.37×102 copies/mL and 3.72×102 copies/mL,respectively.And there was no non-specific reaction to common viral and bacterial disease of cats.Intra and inter-batch coefficients of variation were both less than 2%.These results indicated that this dual MAOPA kits of FPV and FHV-1 had high sensitivity,specificity and good repeatability,and there was no false negative result in the reaction analyzed by internal control in the whole process.Therefore,it is suitable for the early diagnosis,identification and epidemiological investigation of FPV and FHV-1 infection.Statistical compliance rate,sensitivity and repeatability tests were performed on 845 clinical samples collected from various places in China during 2019-2022 with preliminary laboratory detection results using dual qPCR methods and MAOPA kits for FPV and FHV-1.The detection rate of FPV was higher than that of FHV-1.560 FPV positive samples were identified by dual qPCR,with a positive rate of 66.27%(560/845),and 566 FPV positive samples were identified by MAOPA kits,with a positive rate of 66.98%(566/845).A total of 165 FHV-1 positive samples were identified by dual qPCR,with a positive rate of 19.52%(165/845),172 FHV-1 positive samples were identified by MAOPA kits,with a positive rate of 20.35%(172/845),and 136 mixed infections were detected by both methods.The mixed infection rate was 16.09%(136/845).The compliance rates of these two detection methods developed in this study compared to the known results were calculated respectively.The FPV compliance rates of positive,negative and total by qPCR were 100%,96.6%and 98.8%.And the FHV-1 compliance rates of positive,negative and total by qPCR were 100%,99.3%,and 99.4%,respectively.Meantime,the FPV compliance rates of positive,negative and total by MAOPA were 100%,94.6%and 98.1%.And the FHV-1 compliance rates of positive,negative and total by MAOPA were 100%,98.2%and 98.6%respectively.Five positive clinical samples were selected for 10-fold gradient dilution,and the results showed that the lowest detection limits were 105 times dilution for MAOPA and 103 times dilution for qPCR,respectively,which indicated that the sensitivity of FPV and FHV-1 dual MAOPA kits was 10-100 times higher than that of dual qPCR method.30 positive and 30 negative clinical samples consistent with known results were selected for repeatability test.The results showed that FPV,FHV-1 dual qPCR method and MAOPA kits had the same results for three independent repeated tests of 60 samples,and the coefficient of variation for positive samples was less than 5%.In conclusion,this study successfully developed dual qPCR and MAOPA detection methods for FPV and FHV-1.Both of dual qPCR and MAOPA have good sensitivity,specificity and repeatability,and can be applied to clinical detection,identification and rapid diagnosis of FPV and FHV-1.Remarkably,MAOPA kits have more advantages than qPCR assay in clinical application. |
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