Ababneh Mustafa, Ababneh Ola, Al-Zghoul Mohammad Borhan
Department of Basic Medical Veterinary Sciences, Jordan University of Science and Technology, Irbid 22110, Jordan.
Vet World. 2020 Mar;13(3):400-406. doi: 10.14202/vetworld.2020.400-406. Epub 2020 Mar 3.
Belonging to the family, avian infectious bronchitis virus (IBV) causes respiratory, reproductive, and renal diseases in poultry. Preventative measures lie mainly in vaccination, while the gold standard for IBV classification and differentiation is based on the sequence analysis of the spike 1 (S1) gene. In this study, we tested a new assay for IBV strain classification that is less expensive and requires reduced time and effort to perform. We carried out a quantitative real-time polymerase chain reaction followed by high-resolution melting (qRT-PCR/HRM) curve analysis.
In this study, qRT-PCR was conducted on a partial fragment S1 gene followed by a high resolution melting curve analysis (qRT-PCR/HRM) on 23 IBV-positive samples in Jordan. For this assay, we utilized the most common IBV vaccine strains (Mass and 4/91) as a reference in the HRM assay. To evaluate the discrimination power of the qRT-PCR/HRM, we did the sequencing of the partial S1 gene.
It was shown that HRM was able to classify IBV samples into four clusters based on the degree of similarity between their melting points: The first cluster exhibited the highest similarity to the 4/91 strain, while the second was similar to the Mass-related IBV strain. Although the third cluster contained the highest number of samples, it displayed no similarity to any of the reference vaccine strains, and, after comparing them with the sequencing results, we found that the samples in the third cluster were similar to the variant II-like (IS-1494-06) IBV field strain. Finally, the fourth cluster comprised one unique sample that was found to belong to the Q1 IBV strain.
Our developed qRT-PCR/HRM curve analysis was able to detect and rapidly identify novel and vaccine-related IBV strains as confirmed by S1 gene nucleotide sequences, making it a rapid and cost-effective tool.
禽传染性支气管炎病毒（IBV）属于冠状病毒科，可引起家禽的呼吸道、生殖和肾脏疾病。预防措施主要在于接种疫苗，而IBV分类和鉴别 的金标准基于刺突1（S1）基因的序列分析。在本研究中，我们测试了一种用于IBV毒株分类的新方法，该方法成本较低，所需时间和精力较少。我们进行了定量实时聚合酶链反应，随后进行高分辨率熔解（qRT-PCR/HRM）曲线分析。
在本研究中，对23份约旦的IBV阳性样本的S1基因部分片段进行qRT-PCR，随后进行高分辨率熔解曲线分析（qRT-PCR/HRM）。对于该检测，我们使用最常见的IBV疫苗毒株（Mass和4/91）作为HRM检测的参考。为了评估qRT-PCR/HRM的鉴别能力，我们对S1基因部分片段进行了测序。
结果表明，HRM能够根据熔点之间的相似程度将IBV样本分为四类：第一类与4/91毒株的相似性最高，而第二类与Mass相关的IBV毒株相似。虽然第三类包含的样本数量最多，但它与任何参考疫苗毒株都没有相似性，在将它们与测序结果进行比较后，我们发现第三类中的样本与变异II样（IS-1494-06）IBV田间毒株相似。最后，第四类包含一个独特的样本，发现它属于Q1 IBV毒株。
我们开发的qRT-PCR/HRM曲线分析能够检测并快速鉴定出与新的和疫苗相关的IBV毒株，S1基因核苷酸序列证实了这一点，使其成为一种快速且经济高效的工具。
