A novel and cost-effective real-time RT-PCR targeting 24 nucleotides deletion to differentiate PEDV wild-type and classical attenuated vaccine strains

IF 2.2 4区 医学 Q3 BIOCHEMICAL RESEARCH METHODS Journal of virological methods Pub Date : 2024-06-22 DOI:10.1016/j.jviromet.2024.114986
Zhilin Wang, Xuerui Li, Youjun Shang, Jinyan Wu, Xi Lan
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Abstract

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the swine industry, causing severe disease and resulting in substantial economic losses. Despite China’s implementation of a large-scale vaccine immunization strategy in recent years, various strains of PEDV, including classical attenuated vaccine strains, continue to emerge in immunized pig herds. Here, we established a one-step real-time fluorescent reverse transcription PCR (one-step real-time RT-PCR) assay targeting a 24-nucleotide deletion in the ORF1 region of three PEDV classical attenuated vaccine strains, derived from classical strains. This assay effectively distinguishes between PEDV classical attenuated vaccine strains and wild-type strains, and we also explore the causes of this discriminatory target deficiency of this method through phylogenetic and recombination analysis. We found that these three classical attenuated vaccine strains exhibit closer phylogenetic relationships and higher sequence similarity with five cell-adapted strains. Recombination analysis revealed that although recombination is widespread in the PEDV genome, the 24-nucleotide deletion site remains stable without undergoing recombination and can be utilized as a target for identification. Further analysis revealed there are no enzyme cleavage sites near the 24-nucleotide site, suggesting that this deletion may have been lost during the process of culturing these viral strains in cells.The detection method we have established exhibits high specificity and sensitivity to PEDV, without cross-reactivity with other viruses causing diarrheal diseases. A total of 117 swine fecal samples were analyzed using this established one-step real-time reverse transcription PCR assay, indicating the presence of classical attenuated vaccine strains in pig herds in Gansu province, China. Additionally, the designed primer pairs and two probes can be placed in a single reaction tube to differentiate between these two types of strains, effectively reducing detection costs. These findings offer an efficient and cost-effective technological platform for clinical rapid identification testing of both wild-type and classical attenuated vaccine strains of PEDV, as well as for precise investigation of clinical data on natural infections and vaccine immunity in pig herds.

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以 24 个核苷酸缺失为目标的新型、经济高效的实时 RT-PCR 技术可用于区分 PEDV 野生型和经典减毒疫苗株。
猪流行性腹泻病毒(PEDV)对养猪业构成重大威胁,可导致严重的疾病,造成巨大的经济损失。尽管近年来中国实施了大规模疫苗免疫战略,但包括经典减毒疫苗株在内的各种 PEDV 株系仍不断在免疫猪群中出现。在此,我们建立了一种针对三种PEDV经典减毒疫苗毒株ORF1区24核苷酸缺失的一步实时荧光反转录PCR(one-step real-time RT-PCR)检测方法。这种检测方法能有效区分 PEDV 经典减毒疫苗株和野生型株,从而通过系统发育和重组分析初步探究造成这种缺陷的原因。我们发现,这三种经典减毒疫苗株与五种细胞适应株的系统发育关系更密切,序列相似度更高。重组分析表明,虽然 PEDV 基因组中普遍存在重组现象,但 24 核苷酸缺失位点仍保持稳定,没有发生重组,可作为鉴定目标。进一步分析发现,24 核苷酸位点附近没有酶裂解位点,这表明该缺失位点可能是在细胞培养这些病毒株的过程中丢失的。我们建立的检测方法对 PEDV 具有高度的特异性和灵敏度,与其他引起腹泻病的病毒没有交叉反应。利用这种一步法实时逆转录 PCR 检测方法共分析了 117 份猪粪便样本,结果表明中国甘肃省猪群中存在经典减毒疫苗毒株。此外,所设计的引物对和两个探针可以放在一个反应管中来区分这两种毒株,从而有效降低了检测成本。这些发现为PEDV野生型毒株和经典减毒疫苗毒株的临床快速鉴定检测,以及猪群自然感染和疫苗免疫临床数据的精确调查提供了一个高效、经济的技术平台。
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来源期刊
CiteScore
5.80
自引率
0.00%
发文量
209
审稿时长
41 days
期刊介绍: The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. The methods may include, but not limited to, the study of: Viral components and morphology- Virus isolation, propagation and development of viral vectors- Viral pathogenesis, oncogenesis, vaccines and antivirals- Virus replication, host-pathogen interactions and responses- Virus transmission, prevention, control and treatment- Viral metagenomics and virome- Virus ecology, adaption and evolution- Applied virology such as nanotechnology- Viral diagnosis with novelty and comprehensive evaluation. We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.
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