Research And Application Of High Resolution Melting Curve Analysis In The Genotyping And Detection Of Animal Viruses

High resolution melting (HRM) is an emerging technique for detection of nucleic acid sequence variation that has been widely used in medical fields. However, for the veterinary medicine, especially for rapid detection and diagnostic genotyping of animal virus, the HRM technique was less pronounced. Therefore the aim of this study was to develop rapid diagnostic genotyping assays of high resolution melting curve analysis by evaluating different methods of HRM in six animal virus and to provide referencs for the application of HRM technique in the veterinary medicine.Three DNA virus (Canine Parvovirus,CPV; Muscovy Duck Parvovirus, MDPV and Goose Parvovirus,GPV), two RNA virus (Classical Swine Fever Virus, CSFV and Porcine Reproductive and Respiratory Syndrome Virus, PRRSV) and one retrovirus (Avian Leukosis Virus, ALV) were included in this study. Different HRM methods, including small ampicon high resolution melting (SA-HRM/SNP or SA-HRM) and unlabeled probe high resolution melting (UP-HRM) analysis, were utilized to provide diagnostic genotyping assays.To distinguish three subtypes of CPV (CPV-2a, CPV-2b and CPV-2c), two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that flanked closely a SNP of interest426and87amino acid residues, respectively. The result of PCR-HRM showed that29of30samples were genotyped correctly using PCR-HRM. Of the30clinical canine fecal samples,19of30were CPV-2a (GCP>98%),8of30wereCPV-2b (GCP>90%),and2of30were CPV-2c (GCP>98%) and1of30was mixed infection (CPV-2a/2b). MGV-P1/P2specific primer pairs for VP3gene were designed for detection and differentiation of MDPV and GPV. The PCR-HRM result revealed that in total of12specimens from allantoises fluid,2of12were classified as MDPV with high GCP (GCP>95%),2/12were classified as GPV with high GCP (GCP>95%),1/12was identified as recorribined strain of GPV, and7/12were mixed infection samples. To distinguish CSFV wild strain and attenuated live vaccine strain, the region of12nucleotide difference in the3’UTR was used to designed primer CSFV-F3/F4, and the potential of PCR-HRM were assessed for detection and differentiation of CSFV wild strain and attenuated live vaccine strain in clinical specimens. The result of PCR-HRM analysis showed that all of12clinical specimens were identified as CSFV wild strain, no sample was found positive for CSFV attenuated live vaccine strain. A value of GCP-3SD was used to establish the GCP range, and the GCP range for related field samples was determined to be45-100%in the PCR-HRM analysis. UP-HRM analysis was developed based on the Nsp3gene of PRRSV for rapid detection and classification of C-PRRSV and H-PRRSV. The result of clinical UP-HRM analysis indicated that4of21samples were identified as C-PRRSV strain with the melting peak of unlabeled probe characterized by65.1±0.28℃, and17of21samples were positive for H-PRRSV strain with the melting peak of unlabeled probe characterized by75.3±0.37℃. Three specimens positive for H-PRRSV were identified as vaccine-related H-PRRSV strains using JXA1-R SNP loci. Two sets of primers, H5/H7b for ALV subgroup J and Gp85U3/Gp85L3for ALV subgroups A, B, C, D, E and K, binding to the GP85gene were designed for detection and differentiation of ALV different subgroups using PCR-HRM. The result of PCR-HRM analysis showed that82/276specimens were classified as ALV-K subgroup from clinical albumen samples, and15/200specimens were classified as ALV-J subgroup from clinical cloacal swabs. Due to the endogenous ev element which was part of the chicken genome, the melting profiles of ALV-ev clinical samples were relatively unchangeable, characterized by two melting peaks with the first peak being shorter than the second. We have developed a new method which is useful for discrimination of endogenous and exogenous ALV from clinical samples without the need of tissue culture or sequencing, and provides an interesting means for future studies to determine the epidemiology and persistence of various viral subgroups and to detect newly emerging viral variants in the fields.In this study, we have developled simple and rapid diagnostic genotyping assays of six different animal viruses using high resolution melting curve analysis.The accuracy and sensitivity of high resolution melting analysis can provide appropriate and cost-effective molecular diagnostic methods for the veterinary medicine.