Rapid identification, pathotyping and quantification of infectious bursal disease virus by high-resolution melting curve quantitative reverse transcription PCR analysis: An innovative technology well-suited for real-time large-scale epidemiological surveillance

Abstract

With the virus continuing to evolve, very virulent IBDV (vvIBDV) and novel variant IBDV (nvIBDV) have become the predominant epidemic strains in China, exacerbated by the widespread use of attenuated vaccine strains (attIBDV), making a complex infection situation of IBDV in the field. Therefore, developing a rapid and accurate high-resolution melting curve quantitative reverse transcription PCR (HRM-qRT-PCR) for the identification and pathotyping of IBDV is crucial for clinical monitoring and disease control. Extensive data analysis and genome-screening of the three dominant IBDV pathotypes identified a specific region (nucleotides 2450-2603 in segment A) with distinct GC content as the detection target. Experimental testing of HRM-qRT-PCR revealed distinct melting curves and high sensitivity, with the detection limits of 61.2 copies/μL, 61.1 copies/μL and 67.5 copies/μL for vvIBDV, nvIBDV and attIBDV, respectively. The method exhibited excellent specificity, with no inter-genotypes cross-reactivity among the three pathotypes and no reactivity to other common avian pathogens. Applied to samples with double and triple co-infections of different IBDV pathotypes, the method displayed specific melting peaks corresponding to the viruses present in the samples, with an accuracy rate of 100 %. This method precisely identifies and differentiates all the single or co-infected samples, generating distinct peaks corresponding to the Tm values of each virus pathotype in traditional melting curve plots. Furthermore, the method overcomes the limitations of traditional pathotyping methods, requiring only one reaction to achieve rapid viral pathotyping and facilitating quantitative analysis of viruses within the samples. This study introduces an innovative HRM-qRT-PCR method, offering new technology to rapid and accurate identification, pathotyping and quantification of vvIBDV, nvIBDV, and attIBDV. With strong discriminatory power, user-friendliness and a short processing time, this method is highly attractive for the rapid IBDV pathotyping in real-time large-scale epidemiological surveillance during outbreaks.

Keywords: Epidemiological surveillance; High-resolution melting curve (HRM); Infectious bursal disease virus (IBDV); Pathotyping; Quantitative reverse transcription PCR (qRT-PCR).

Fig. 1

Graphical representation of the IBDV pathotyping by the HRM-qRT-PCR analysis.

Fig. 2

Nucleotide sequence comparison of the PCR amplification region between the different pathotypes of IBDV.

Fig. 3

Normalized melting curves (panel A) and melting peak (panel B) plots for the amplicons of vvIBDV strain NN1172, nvIBDV strain GX-QZ191002 and attenuated vaccine strain B87. IBDV of different genotypes displayed unique melting curves and melting temperatures.

Fig. 4

Specificity of the HRM-qRT-PCR Analysis. Panel (A) Genotype-specific melting peak curves. Panel (B) the target fragment amplicons ran on 2 % agarose gel after RT-qPCR-HRM analysis. Lane 1–10: ALV-J, MDV, NDV, ILTV, FAdV-4, REV, IBV, AIV, aMPV, and ARV; Lane 11–13: vvIBDV strain NN1172, nvIBDV strain GX-QZ191002 and attIBDV strain B87; Lane 14: ddH₂O.

Fig. 5

Sensitivity, melting peak and standard curve of the HRM-qRT-PCR analysis. Panel (A), (B) and (C): the sensitivity analysis of the ten-fold dilutions (10¹⁰ to 10⁰ copies/μL) of the standard plasmids of the three pathotypes. Panel (D), (E) and (F): the melting peak curves of the ten-fold dilutions (10¹⁰ to 10⁰ copies/μL) of the standard plasmids of the three pathotypes. Panel (G), (H) and (I): the standard curve of the ten-fold dilutions (10⁸ to 10⁴ copies/μL) of the standard plasmids of the three pathotypes.

Fig. 6

Identification and genotyping by the melting peak curves of the mixed-samples of the three standard plasmids of different pathotype. Panel (A): the melting peak curves of double mixed plasmid samples of pMD18-T-NN1172 and pMD18-T-B87; Panel (B): the melting peak curves of double mixed plasmid samples of pMD18-T-GX-QZ191002 and pMD18-T-NN1172; (C): the melting peak curves of double mixed plasmid samples of pMD18-T-B87and pMD18-T-GX-QZ191002; (D): the melting peak curves of triple mixed plasmid samples of pMD18-T-NN1172, pMD18-T-B87 and pMD18-T-GX-QZ191002.

Fig. 7

Identification and genotyping by the melting peak curves of the co-infection samples from artificial infection models. Single infected groups A, B and C: the birds in each group only infected with NN1172, B87 and GX-QZ191002, respectively; Double co-infected groups D, E and F: the birds in each group infected with NN1172 and B87, NN1172 and GX-QZ191002, and B87 and GX-QZ191002, respectively; Triple co-infected group G: the birds in the group infected with NN1172, B87 and GX-QZ191002 simultaneously.