Improved detection of bovine coronavirus N gene in faeces of calves infected naturally by a semi-nested PCR assay and an internal control.
Improved detection of bovine coronavirus N gene in faeces of calves infected naturally by a semi-nested PCR assay and an internal control.
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DOI：
10.1016/j.jviromet.2005.08.005
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发表时间：
2006-03
影响因子：
3.1
通讯作者：
Alfieri AA
中科院分区：
文献类型：
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作者：
Takiuchi E;Stipp DT;Alfieri AF;Alfieri AA
牛冠状病毒（BCoV）是一种单股正链RNA病毒，是世界范围内引起肉牛和奶牛新生犊牛腹泻的重要病原。BCoV的常规检测和诊断主要依赖于低灵敏度的检测方法。本研究的目的是建立和评价半巢式PCR（SN-PCR），以扩增251 bp的片段BCoV N基因从新鲜（n = 25）和冷冻（n = 25）的自然感染小牛的粪便样品。为了提高通过SN-PCR检测粪便样品中的BCoV，开发了内部对照，并将结果与常规RT-PCR测定进行了比较。SN-PCR和RT-PCR的阳性率分别为24%（12/50）和8%（4/50）（K = 0.43）。RT-PCR仅对新鲜样品呈阳性反应，而SN-PCR在新鲜和冷冻粪便样品中均检出BCoV。SN-PCR的灵敏度通过将BCoV卡克川株（HA滴度：256）连续稀释10倍直至稀释10−7来测定。通过限制性片段长度多态性和序列分析评估扩增子的特异性。纳入内部对照提供了一种检测粪便样本中试验抑制和核酸提取失败的方法，可减少假阴性结果的数量。
Bovine coronavirus (BCoV), a positive sense single-stranded RNA virus, is an important causative agent of neonatal diarrhoea in calves from beef and dairy cattle worldwide. The routine detection and diagnosis of BCoV have been mainly dependent on assays with low sensitivity. The aim of the present study was to develop and evaluate a semi-nested PCR (SN-PCR) to amplify a 251 bp fragment of BCoV N gene from fresh (n = 25) and frozen (n = 25) diarrhoeic faecal samples of naturally infected calves. To improve detection of BCoV in faecal samples by the SN-PCR an internal control was developed, and the results were compared with a conventional RT-PCR assay. The rates of positive samples by SN-PCR and RT-PCR were 24% (12/50) and 8% (4/50), respectively (K = 0.43). Only fresh samples were positive in RT-PCR while the SN-PCR detected BCoV in both fresh and frozen faecal samples. The sensitivity of SN-PCR was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) that was detected until 10−7 dilution. The specificity of the amplicons was assessed by restriction fragment length polymorphism and sequence analysis. The inclusion of an internal control provides a way to detect assay inhibition in faecal samples and failure of nucleic acid extraction that allow reduction of the number of false-negative results.
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影响因子：
3.1
作者：
Hale, AD;Green, J;Brown, DWG
通讯作者：
Brown, DWG
影响因子：
9.4
作者：
Dingle, KE;Crook, D;Jeffery, K
通讯作者：
Jeffery, K
影响因子：
1.5
作者：
Kapil, S;Richardson, KL;ChardBergstrom, C
通讯作者：
ChardBergstrom, C
DOI：
10.1007/s002160000641
发表时间：
2001-02-01
期刊：
FRESENIUS JOURNAL OF ANALYTICAL CHEMISTRY
影响因子：
--
作者：
Butler, JM;Ruitberg, CM;Vallone, PM
通讯作者：
Vallone, PM
DOI：
10.1111/j.1439-0450.2004.00737.x
发表时间：
2004-04-01
期刊：
JOURNAL OF VETERINARY MEDICINE SERIES B-INFECTIOUS DISEASES AND VETERINARY PUBLIC HEALTH
影响因子：
--
作者：
Barreiros, MAB;Alfieri, AF;Alfieri, AA
通讯作者：
Alfieri, AA