[1]付琦媛?,严世杰?,王春芳,等.鉴别猪流行性腹泻病毒经典株与变异株双重纳米 RT-PCR 检测方法的建立和应用[J].中国预防兽医学报,2021,(02):145-149.[doi::10.3969/j.issn.1008-0589.202003032]
 FU Qi-yuan,YAN Shi-jie,WANG Chun-fang,et al.Development and application of duplex Nano RT-PCR methoddistinguishing the classical and variant PEDV strains[J].Chinese journal of preventive veterinary medicine,2021,(02):145-149.[doi::10.3969/j.issn.1008-0589.202003032]
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鉴别猪流行性腹泻病毒经典株与变异株双重纳米 RT-PCR 检测方法的建立和应用

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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2021年02
页码:
145-149
栏目:
诊断和检测技术
出版日期:
2021-03-16

文章信息/Info

Title:
Development and application of duplex Nano RT-PCR method
distinguishing the classical and variant PEDV strains
文章编号:
:1008-0589(2021)02-0145-05
作者:
 付琦媛?严世杰?王春芳李文傲马增军*宋涛芮萍*
(河北科技师范学院动物科技学院/河北省预防兽医学重点实验室,河北秦皇岛066600)
Author(s):
 FU Qi-yuan? YAN Shi-jie? WANG Chun-fang LI Wen-ao MA Zeng-jun* SONG Tao RUI Ping*
 (College of Animal Science andTechnology, Hebei Normal University of Science and Technology/Hebei Key Laboratory of Preventive
Veterinary Medicine, Qinhuangdao 066600, China)
关键词:
:猪流行性腹泻病毒纳米RT-PCR鉴别诊断
Keywords:
: porcine epidemic diarrhea virus Nano RT-PCR differential diagnosis
分类号:
S854.4
DOI:
:10.3969/j.issn.1008-0589.202003032
文献标志码:
A
摘要:
为了建立快速高效鉴别猪流行性腹泻病毒(PEDV)经典株与变异株的检测方法,本研究参照
GenBank中PEDV经典株CV777和变异株CH-HB1-2018的S基因序列,设计了3条特异性引物,并以纳米金颗粒
作为热导介子,通过优化PCR反应条件,建立了能够区分PEDV经典株和变异株的双重纳米RT-PCR检测方法。
利用该方法同时检测PEDV经典株和变异株、猪伪狂犬病病毒(PRV)、猪圆环病毒2型和3型(PCV2和PCV3)、
猪繁殖与呼吸综合征病毒(PRRSV)和猪德尔塔冠状病毒(PDCoV),结果显示,该方法能特异性鉴别PEDV经典株
(747 bp)和变异株(442 bp),且与其他病原均无交叉反应,特异性较强;将重组质粒标准品10倍倍比稀释后分别
利用该方法和普通RT-PCR同时检测,结果显示,本研究建立的双重纳米RT-PCR方法能检测出经典株CV777和
变异株CH-HB1-2018 重组质粒标准品的下限分别为5.68×102 拷贝/μL 和4.93×102 拷贝/μL,其敏感性较普通RTPCR
的敏感性高100倍。利用本研究建立的方法对不同地区采集的阳性病料提取基因组后分别于同一时间和不同
时间进行检测,结果显示该方法稳定性和重复性良好。利用该双重纳米RT-PCR方法对34份采集自河北省腹泻
仔猪的样品进行检测,结果显示与普通RT-PCR检测结果符合率为97.1%,且PEDV变异株的阳性率高于PEDV经
典株。本研究建立的双重纳米RT-PCR方法为PEDV病原鉴别检测及流行病学调查提供了可行的技术手段。
Abstract:
In order to develop a rapid and efficient detection method to distinguish the classical and variant PEDV strains,
three specific primers was designed based on S gene sequences of classical strain (CV777) and variant strain (CH-HB1-2018) in
GenBank, and a duplex Nano RT-PCR method was developed based on using the gold nanoparticles as thermal conduction mesons
and optimized PCR conditions. Using this method to detect classical and variant PEDV strains, PRV, PCV2, PCV3, PRRSV, and PDCoV, the results showed that the fragments from classical PEDV (747 bp) and variant PEDV (and (442 bp) were amplified,and
had no cross-reaction with other five swine viruses. The diluted recombinant plasmid samples detected by this method and RT-PCR
to detect the sensitivity, and the limited detection of this Nano RT-PCR was 5.68×102 copies/μL and 4.93×102 copies/μL for CV777
and CH-HB1-2018, respectively, which is 100 times higher than that of RT-PCR. The recombinant plasmid samples were detected
at same and different time, which showed that the duplex Nano RT-PCR possesses the features of high repeatability and
repeatability. Thirty- four samples of diarrheal piglets collected from Hebei province were detected using this method, the results
showed that the coincidence rate with RT-PCR was 97.1% and the positive rate of PEDV variant strains was higher than that of
classical PEDV strains. The developed duplex Nano RT- PCR can be used for the PEDV pathogen differential diagnosis and
epidemiological investigation.

相似文献/References:

[1]王华俊,赵雪丽,闫若潜*,等.猪流行性腹泻病毒时间分辨荧光免疫层析检测方法的建立[J].中国预防兽医学报,2021,(01):40.[doi::10.3969/j.issn.1008-0589.202003050]
 WANG Hua-jun,ZHAO Xue-li,YAN Ruo-qian*,et al.Establishment of a time-resolved fluorescenceimmunochromatographic assay for detection of PEDV[J].Chinese journal of preventive veterinary medicine,2021,(02):40.[doi::10.3969/j.issn.1008-0589.202003050]

更新日期/Last Update: 2021-03-17