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A RT-PCR assay for the rapid recognition of border disease virus 总被引：1，自引：0，他引：1
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Fluorogenic RT-PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus 总被引：12，自引：0，他引：12
A single tube fluorogenic RT-PCR-based 'TaqMan' assay was developed for detection and classification of bovine viral diarrhea virus (BVDV). TaqMan-PCR was optimized to quantify BVD virus using the ABI PRISM 7700 sequence detection system and dual-labeled fluorogenic probes. Two different gene specific labeled fluorogenic probes for the 5' untranslated region (5' UTR) were used to differentiate between BVD types I and II. Sensitivity of the single tube TaqMan assay was compared with two-tube TaqMan assay and standard RT-PCR using 10-fold dilutions of RNA. Single tube TaqMan assay was 10-100-fold more sensitive than the two-tube TaqMan assay and the standardized single tube RT-PCR. Specificity of the assay was evaluated by testing different BVD virus strains and other bovine viruses. A total of 106 BVD positive and negative pooled or single serum samples, field isolates and reference strains were tested. Quantitation of cRNA from types I and II BVD virus was accomplished by a standard curve plotting cycle threshold values (C(T)) versus copy number. Single tube TaqMan-PCR assay was sensitive, specific and rapid for detection, quantitation and classification of BVD virus. 相似文献
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In this study, genomic sequences of pestiviruses available in GenBank were aligned to design three primer pairs and TaqMan probes: two targeting the NS5A region of the viral genome of classical swine fever virus (CSFV) for the differentiation of wild-type CSFV and hog cholera lapinized vaccine (HCLV) vaccine, and one targeting the 5'-untranslated region of bovine viral diarrhea virus 1 (BVDV-1). With these primers and probes, a triplex TaqMan real-time RT-PCR assay was developed for differentiating wild-type CSFV, the HCLV strain, and BVDV-1. The detection limit of the assay was 4.5 TCID(50) for wild-type CSFV, 10 TCID(50) for HCLV-strain CSFV, and 3.2 TCID(50) for BVDV-1. The triplex real-time RT-PCR had at least 98% (248 samples) agreement with other RT-PCR methods. The assay provides a sensitive tool for simultaneous detection and differentiation of wild-type CSFV and HCLV from BVDV-1. 相似文献
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Nested reverse transcriptase-polymerase chain reaction (RT-PCR) for typing ruminant pestiviruses: bovine viral diarrhea viruses and border disease virus. 总被引：1，自引：1，他引：0 
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下载免费PDF全文 R W Fulton J M d'Offay J T Saliki L J Burge R G Helman A W Confer S R Bolin J F Ridpath 《Canadian journal of veterinary research》1999,63(4):276-281
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牛病毒性腹泻病毒RT-PCR检测方法的建立及应用 总被引：1，自引：1，他引：0
根据GenBank中登录的牛病毒性腹泻病毒(BVDV)基因序列,设计合成了1对特异性引物,建立了检测BVDV的RT-PCR方法。通过对该方法的特异性、敏感性和重复性进行试验,结果显示,该方法可从BVDV标准毒株Oregon C24V中扩增出471 bp的特异性片段,而对猪瘟病毒、牛传染性鼻气管炎病毒、牛呼吸道合胞体病毒、牛副流感病毒、MDBK正常细胞的扩增结果均为阴性。经对标准毒株的细胞毒进行检测,其敏感度达10-1 TCID50/mL。应用该方法对临床腹泻病牛各脏器样品进行检测,结果比病毒分离方法更为敏感,操作简便。表明建立的RT-PCR方法具有特异、灵敏、高效、快速的特点,可用于BVDV的临床检测及流行病学监测。 相似文献
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牛病毒性腹泻病毒一步法RT-PCR检测方法的建立 总被引：1，自引：0，他引：1
根据牛病毒性腹泻/粘膜病病毒5'端非结构蛋白基因序列,设计合成1对特异性引物,建立检测牛病毒性腹泻/粘膜病病毒244 bp片段的RT-PCR一步法。该方法对牛病毒性腹泻/粘膜病病毒NADL、OregonC24V和长春184毒株各标准毒株检测,结果均为阳性。对标准毒株的细胞毒进行检测,其敏感度达0.1 TCID50;而对牛传染性鼻气管炎病毒、猪瘟病毒、牛轮状病毒和牛冠状病毒的细胞培养物进行检测,结果均为阴性。检测460份不同样品,与病毒分离试验比较,符合率100%,证明该方法特异性强,敏感性高。初步结果表明所建立的一步法RT-PCR技术可用于牛病毒性腹泻/粘膜病的检测及流行病学调查。 相似文献
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MA Baoyi SHENG Chenyan LIU Hongying MA Qingxia WANG Tingting LI Jianming SHI Kun DU Rui LENG Xue 《中国畜牧兽医》2022,49(6):2326-2335
【Objective】 This study was aimed to establish a duplex TaqMan Real-time PCR method for rapid detection of Bovine viral diarrhea virus types 1(BVDV1) and 2(BVDV2).【Method】 Specific primers were designed based on the 5'-non-coding region of 95 strains of BVDV1 and BVDV2 in GenBank.The positive plasmids containing the target fragments of BVDV1 and BVDV2 were constructed.The reaction conditions were optimized, the standard curve was constructed, the specificity, sensitivity and reproducibility of the duplex TaqMan Real-time PCR method were tested, and the established duplex TaqMan Real-time PCR assay was used to detect the clinical samples collected from Jilin province.【Result】 The results showed that the optimal annealing temperature of the duplex TaqMan Real-time PCR was 57.0 ℃, the optimum primer concentration was 0.5 μmol/L, and the optimum probe concentration was 0.3 μmol/L.The standard curves of BVDV1 and BVDV2 were Y=-3.54X+37.36 (R2=0.990) and Y=-3.18X+35.95 (R2=0.997), respectively.There was no specific amplification of Infectious bovine rhinotracheitis virus (IBRV), Bovine respiratory syncytial virus (BRSV) and Bovine parainfluenza virus type 3 (BPIV3), with intra- and inter-batch CV less than 3%, and the lower limit of detection was 10 copies/μL.The results of clinical samples showed that the overall positive rate was 23.1% (36/156), of which 17.9% (28/156) were positive for BVDV1 and 5.1% (8/156) were positive for BVDV2.【Conclusion】 In this study, a duplex TaqMan Real-time PCR method was established, which could identify BVDV types 1 and 2 simultaneously, quickly and accurately, and provided technical support for the prevention, control and purification of BVDV. 相似文献
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牛病毒性腹泻病病毒荧光定量PCR检测体系的建立与评价 总被引：2，自引：0，他引：2
基于实时荧光定量PCR技术建立了一种有效地检测牛病毒性腹泻病病毒(Bovine viral diarrhea virus,BVDV)核酸的方法.对BVDV基因组进行同源比对,选取5'UTR区作为扩增目的区,经软件分析后设计特异扩增引物,扩增片段长度为203 bp.选用SYBR染料作为扩增时信号指示剂,经扩增曲线分析表明,建立的方法可有效地检测BVDV.检测体系可检测到10~2 copies/μL的样品拷贝数.故本研究建立的BVDV实时定量检测体系可用于易感动物,牛源血液生物制品及其他可能感染或污染BVDV样品的检测. 相似文献
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本研究按照牛轮状病毒(BRV)结构蛋白VP6基因序列,设计合成引物和探针,经各反应条件的优化,建立了BRV TaqMan实时荧光定量RT-PCR技术。对BRV进行了特异性、敏感性和重复性试验。结果表明,TaqMan实时荧光RT-PCR最低可检测到100个拷贝病毒RNA;与牛病毒性腹泻病毒(BVD)、猪瘟病毒(CSFV)、牛结核杆菌(MB)和牛传染性鼻气管炎病毒(IBRV)不发生交叉反应;所制作的标准曲线在102~109拷贝/μL浓度范围内有极好的线性关系且线性范围宽,相关系数为0.997;与常规的RT-PCR相比,该方法具有快速、特异、敏感、重复性好、可同时检测大量样品等优点。可对样品中微量BRV进行准确检测,对BRV的诊断有重要意义。 相似文献
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为建立一种快速检测1型、2型牛病毒性腹泻病毒(BVDV)的通用RT-PCR方法,根据GenBank上收录的64株1型、2型BVDV以及1株猪瘟病毒(CSFV)的全基因组序列,应用Primer 6.0软件设计针对5'-UTR区域的1型、2型BVDV特异性通用引物对,扩增目的片段,并对该方法进行特异性、敏感性、重复性试验及利用该方法开展临床样品检测。结果显示:扩增的目的片段长度约为302 bp;该方法的灵敏度为2.09×102 copies/μL,无非特异性扩增,且重复性良好。对采自山东省发病牛场的13份鼻腔棉拭子和9份牛血清临床样品进行检测,发现有8份鼻腔棉拭子和8份血清为BVDV阳性,随机抽取4份阳性样品送测序,发现3份样品毒株为1型BVDV,1份样品还需进一步鉴定。本研究建立的RT-PCR方法可实现对1型、2型BVDV核酸的特异性检测,也可用于该病的流行病学调查研究。 相似文献
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根据牛病毒性腹泻病毒(BVDV)5'端非编码区基因序列,设计合成了1对特异性引物,参考本实验室针对猪繁殖与呼吸综合征病毒(PRRSV)N蛋白设计的引物,经过PCR反应条件的优化,建立了BVDV和PRRSV双重RT-PCR的检测方法。对于PRRSV和BVDV的cDNA最低检测量分别为3.8×10-4 ng和7×10-4 ng,对于猪瘟病毒(CFSV)、脑心肌炎病毒(EMCV)和猪圆环病毒2型(PCV-2)的PCR扩增结果均为阴性;用该方法对江苏省不同地区采集的75份仔猪的肺脏、脾脏和淋巴结等病料进行了检测,结果PRRSV有55份阳性,BVDV有14份阳性,PRRSV和BVDV混合感染的有12份,与PRRSV和BVDV单一RT-PCR的检测结果符合率分别为89.3%和92%。证明建立的双重RT-PCR检测方法可用于临床样品中BVDV和PRRSV的检测。 相似文献
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N. Mishra B.S. Mathapati K. Rajukumar R.K. Nema S.P. Behera S.C. Dubey 《Research in veterinary science》2010,89(1):130-132
The aim of this study was to determine the kinetics of noncytopathic bovine viral diarrhoea virus (BVDV) multiplication and synthesis of BVDV specific RNA and proteins in ovine cells (SFT-R) during a one-step growth curve. The virus titre and RNA level were determined by focus-forming assay and real time RT-PCR. The RNA synthesis was detected by Northern blot while synthesis of E2 and NS3 proteins was assayed by immunohistochemistry and Western blot. The results showed that synthesis of viral RNA is initiated at 4 h, NS3 and E2 proteins are detectable at 6-7 h and the replication cycle is complete at 10-12 h. Additionally, we provide evidence that NS2-3 protein was cleaved in ovine cells early during infection and in proliferated leukocytes of acutely infected sheep. This study showed that synthesis of BVDV RNA and proteins in ovine cells occurs at similar times as found in bovine cells. 相似文献
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猪源牛病毒性腹泻病毒JLS-01株的分离鉴定及致病性研究 总被引：1，自引：1，他引：0
为了解猪源牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的分子特征及致病性,本研究利用RT-PCR从吉林省某猪场出现严重腹泻症状的仔猪病料中检测到BVDV核酸阳性,将处理后的BVDV阳性样品接种于MDBK细胞,分离到1株病毒,命名为BVDV JLS-01。通过免疫荧光检测、5′UTR与Npro RT-PCR扩增对其分子进化特征进行分析。结果显示,该分离毒株在MDBK细胞上盲传至8代未出现细胞病变,在免疫荧光试验中呈阳性荧光信号。RT-PCR扩增获得大小分别为280和735bp的5′UTR和Npro片段。BVDV JLS-01株5′UTR与Npro序列遗传进化分析表明,其与LN-1和ZM-95亲缘性最近,与牛源毒株LN-1基因同源性达99.3%,提示该毒株可能来源于牛源毒株。将BVDV JLS-01株F8代细胞培养液人工感染BVDV和猪瘟病毒(CSFV)抗体阴性猪,感染猪未表现出明显的体温升高,但白细胞数量下降,并在感染猪的白细胞提取物中分离到该毒株,表明该毒株具有一定的致病性。该毒株的成功分离对进一步开展BVDV流行病学调查及致病机理等方面的研究具有重要意义。 相似文献
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牛病毒性腹泻病毒一步法RT-PCR检测方法的建立与应用 总被引：2，自引：1，他引：1
为建立一种快速检测牛病毒性腹泻病毒病原的方法,本研究根据GenBank上登录的牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)基因组序列,设计1对引物,建立了检测BVDV的一步法RT-PCR方法。该方法对牛传染性鼻气管炎病毒、猪瘟病毒、牛副流感病毒3型的扩增结果均为阴性,检测的敏感性达1 ng RNA。该一步法RT-PCR方法具有良好的特异性、敏感性、重复性,可以准确快速检测出极低含量的BVDV,将为BVDV的病原检测及分子流行病学调查等提供一种快速、灵敏、特异、准确的分子生物学检测方法。 相似文献
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为了建立检测(番鸭)呼肠孤病毒(DRV)感染的方法,本研究根据DRV的σNS基因核苷酸序列设计引物及TaqMan探针,经过反应条件的优化,建立了该病毒的TaqMan探针实时荧光RT-PCR检测方法.用已知浓度的重组质粒为标准品,构建的标准曲线的相关系数达到99%.试验表明,该方法的重复性、稳定性、特异性良好,且灵敏度高,可以检测到1.0×10~2拷贝/μL的标准品,比普通PCR至少高100倍.该检测方法对DRV人工感染番鸭肝脏组织检出率达到100%.本实验所建立的TaqMan探针荧光RT-PCR为的快速检测以及进行分子流行病学的调查提供了新的技术手段. 相似文献
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Zhao JJ Cheng D Li N Sun Y Shi Z Zhu QH Tu C Tong GZ Qiu HJ 《Veterinary microbiology》2008,126(1-3):1-10
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), one of OIE listed diseases. Most of the currently available detection methods do not allow discrimination between wild-type CSF viruses and the vaccine strains. This study was designed to develop a multiplex real-time RT-PCR for the quantitative and differential detection of wild-type viruses and C-strain vaccine widely used in China. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C-strain vaccine were designed in the 5'-untranslated region of the viral genome of CSFV. The two TaqMan probes specifically hybridize wild-type viruses of different subgroups and C-strain vaccine, respectively, in the multiplex real-time RT-PCR, with no cross-reaction to a number of non-CSFV porcine viruses. The sensitivity of the assay for detecting wild-type and C-strain-type vaccine viruses was determined to be 41.8 and 81.5copies/microL viral RNA, respectively. Completely correct differentiation of wild-type viruses from C-strain vaccine was achieved when testing reference strains and characterized field isolates of CSFV in China. The multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples of experimentally infected pigs as early as 2 days post-infection, 3 to 4 days prior to the onset of clinical signs in co-housed pigs. The agreements between the multiplex real-time RT-PCR and a multiplex RT-nested PCR for detection of wild-type and C-strain-type viruses were 96.9% and 100%, respectively, when detecting 106 different field samples. There is a positive correlation between the titers of C-strain vaccines titrated in rabbits and RNA copies quantitated by the multiplex real-time RT-PCR. The novel assay described here is rapid and sensitive, and is useful for differentiating field strains and C-strain of CSFV in China. 相似文献