[1]蒲 静,乔彩霞*,尹 羿,等.蓝舌病病毒核酸标准样品的制备及荧光定量RT-PCR方法的研究[J].中国预防兽医学报,2017,(10):799-803.[doi:10.3969/j.issn.1008-0425.201509045]
 PU Jing,QIAO Cai-xia*,YIN Yi,et al.Preparation of standard materials of bluetongue virus and research on real-time RT-PCR for the detection of bluetongue virus[J].Chinese journal of preventive veterinary medicine,2017,(10):799-803.[doi:10.3969/j.issn.1008-0425.201509045]
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蓝舌病病毒核酸标准样品的制备及荧光定量RT-PCR方法的研究()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2017年10
页码:
799-803
栏目:
诊断技术
出版日期:
2017-11-15

文章信息/Info

Title:
Preparation of standard materials of bluetongue virus and research on real-time RT-PCR for the detection of bluetongue virus
文章编号:
1008-0589(2017)10-0799-05
作者:
 

蒲 静乔彩霞*尹 羿高志强汪 琳任 彤张 伟

 (北京出入境检验检疫局 检验检疫技术中心, 北京101113)
Author(s):
 

PU Jing QIAO Cai-xia* YIN Yi GAO Zhi-qiang WANG Lin REN Tong ZHANG Wei

 (Inspection and Quarantine Technical Center of Beijing Entry-exit Inspection and Quarantine Bureau, Beijing 101113, China)
关键词:
蓝舌病病毒NS3基因标准样品荧光定量RT-PCR
Keywords:
bluetongue virus  NS3 gene  standard materials  real-time RT-PCR
分类号:
S852.65
DOI:
10.3969/j.issn.1008-0425.201509045
文献标志码:
A
摘要:
为制备蓝舌病病毒(BTV)NS3基因标准样品并建立BTV通用型荧光定量RT-PCR检测方法,本研究首先从BTV-1型核酸中扩增完整的NS3基因,经克隆测序后,采用体外转录方法制备其cRNA。使用RNA保存液稀释至含量约108拷贝/μL,分装后进行均匀度和稳定性检验,并通过联合定值确定标准样品的量值。结果表明,BTV NS3标准样品的瓶间差异<5 %,均匀度和稳定性良好;定值结果为(1.032±0.02)×108拷贝/μL。此外,使用该BTV标准样品作为模板,通过优化反应体系和条件,建立了BTV通用型荧光定量RT-PCR快速检测方法,并评价其特异性和敏感性。结果显示,该方法能够对目前流行的26个血清型的BTV核酸检测均为阳性,而与其它相关的反刍动物病毒均无交叉反应,特异性良好,且最低可检测到10拷贝/μL病毒核酸。采用建立的BTV荧光定量RT-PCR方法对送检的临床样品进行检测,并与国家标准规定的套式RT-PCR方法进行比较,结果表明两种方法一致性很高,但荧光定量RT-PCR方法操作更为简便,结果准确,更适用于大量临床样品的检测。
Abstract:
The complete sequence of NS3 gene amplified from serotype 1 of bluetongue virus was transcribed in vitro to synthesize cRNA and the homogeneity and stability of the cRNA were tested. The average value of gene copies determined by commissioned laboratories was used as unified standard for quantity of prepared cRNA standard material. The results showed that the value of prepared cRNA standard material was 1.032±0.02×108 copies/μL, and vial variation was less than 5%, which indicated that the prepared cRNA was stable. Furthermore, based on BTV-NS3 standard materials, a real-time RT-PCR detection method using TaqMan technology for bluetongue virus was developed. The result of sensitivity test showed that the detection limit of the developed method was 10 copies/μL, which indicated that the methord had high sensitivity. The results of specificity test showed that the developed methods was highly specific for detected and identified bluetongue virus, and had was no cross reactions with other related ruminants virus. The results of clinical test indicated that the consistency between the developed method and the standard nested RT-PCR methord, but the former was more simple and accurate, and was more suitable for clinical diagnosis than the latter.

参考文献/References:

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                                   (本文编辑:李 娜)

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更新日期/Last Update: 2017-11-06