逆转录-聚合酶螺旋反应检测结核分枝杆菌的应用

目的:建立并应用逆转录-聚合酶螺旋反应(RT-PSR)快速检测结核分枝杆菌(MTB)。方法:针对结核分枝杆菌16S rRNA基因设计特异性引物,通过反应条件的优化初步建立结核分枝杆菌的RT-PSR扩增方法;随后,用2株结核分枝杆菌和11株其他致病菌进行RT-PSR、RT-LAMP和荧光定量PCR法的特异性与敏感性试验;利用RT-PSR法、罗氏培养法、RT-LAMP法和荧光定量PCR法对83名结核病患者的痰液标本进行诊断对比。结果:成功建立并优化了MTB的RT-PSR检测方法,与RT-LAMP法相比,两者特异性均为100%;RT-PSR法的检测灵敏性为1 CFU/mL,为荧光定量PCR法的10倍,且检测下限可达0.1 pg/μL;临床患者痰液样本检测结果表明,与罗氏培养法相比,RT-PSR法和RT-LAMP法的阳性率分别为98.80%(P0.05)和96.39%(P0.05),差异均具有统计学意义。结论:与传统检测法相比,RT-PSR法对于诊断临床样本中的MTB具有良好的特异性和敏感性,适合基层医疗单位防治MTB的推广和应用。     

肾综合征出血热病毒基因检测及分型的研究

根据流行于我国的两型ＨＦＲＳＶ代表株汉滩型７６１１８株及汉城型Ｒ２２株Ｍ节段的核酸序列,设计两型共同引物,建立了逆转录－聚合酶链反应（ＲＴＰＣＲ）方法,检测３９株从不同地区、不同宿主分离的ＨＦＲＳＶ感染鼠脑及细胞培养物;同时还建立了捕捉ＥＬＩＳＡ法（ｃＥＬＩＳＡ）,检测了３９株中的３６株,每份样本设复孔,以Ｐ／Ｎ≥２．１０且Ｐ－Ｎ≥０．１０者判为阳性。ＲＴＰＣＲ及ｃＥＬＩＳＡ两法的检出率分别为９７．６％与８２．４％,二者符合率８４．６％。此外,对ＲＴＰＣＲ产物进行酶切分型,３８份扩增产物中的１５份可被ＡｌｕＩ切开。根据所获酶切图谱的差异,可分为汉滩型及汉城型两型,显示了酶切分型的潜在价值     

Detection of Flaviviruses by Reverse Transcriptase-Polymerase Chain Reaction with the Universal Primer Set

Using a universal primer set designed to match the sequence of the NS1 gene of flaviviruses, the virus RNA of dengue (DEN), Japanese encephalitis (JEV), powassan and langat of Flaviviridae were successfully amplified by polymerase chain reaction (PCR) via cDNA; and with different internal primers, the serotypes of the dengue viruses were identified. Of the 78 clinically diagnosed dengue fever patients, 18 patients were positive for DEN 1, 48 patients for DEN 2 and 8 patients concurrently infected with DEN 4. Of the 52 patients admitted with Japanese encephalitis (JE), 45 were determined to be JEV infections. By nested PCR, we completed the identification of flaviviruses within 2 days. The results show that seven primers have a potential value for rapid clinical diagnosis of flavivirus infections.     

猴泡沫病毒（SFV）RT-nestedPCR检测方法的建立及初步应用

目的建立实验猴群及相关生物制品猴泡沫病毒（SFV）的PCR检测方法。方法选择SFV-1、SFV-3、SFVCPZ前病毒序列的pol基因同源性较高的区域设计嵌套引物对SFV-1毒种进行RT-nestedPCR扩增并克隆测序,以确定其准确性,通过验证方法的特异性和敏感性,初步应用该方法对恒河猴外周血淋巴细胞（PBLs）,常用猴肾传代细胞及猴源性生物制品进行检测。结果经RT-nestedPCR扩增出的片断与SFV-1 cDNA序列同源性达到99%,对10只恒河猴的检测结果为5只阳性,5只阴性,对常用猴肾传代细胞及脊髓灰质炎疫苗的检测结果均为阴性。结论所建立的SFV RT-nestedPCR检测方法能准确的检测出恒河猴SFV的感染情况,对控制实验猴群的质量具有重要意义。该方法可用于检测猴源性生物制品中SFV的污染情况,为保证生物制品应用的安全性提供一定依据。     

逆转录-聚合酶链反应检测鼠肝炎病毒方法的建立

建立特异、灵敏的逆转录聚合酶链反应（RT－PCR）技术,结合碱性磷酸酶标记的探针杂交检测鼠肝炎病毒（MHV）,采用MHV－3,MHV－A59病毒株感染DBT细胞,37℃培养,待细胞出现病变时收集提取病毒RNA。依据MHV基因序列设计一对高度保守区特异性引物,进行RT－PCR扩增,结果可见147bp的鼠肝炎病毒产物特异扩增带。敏感性实验检测到10pg的鼠肝炎病毒RNA,同时用ELISA方法对照。结果提示应用RT—PCR技术结合探针杂交检测鼠肝炎病毒。具有简便、快速、灵敏等优势。本研究在国内尚未见报道。     

Use of Polymerase Chain Reaction for the Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus in Penaeid Shrimp

A rapid and reliable polymerase chain reaction (PCR) method was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. The oligonucleotide primers amplify a 1681-bp fragment of IHHNV, which encompasses the coding sequence for one of the viral coat proteins. The PCR method detects IHHNV in hemolymph and homogenized tissue obtained from the cephalothorax or pleopods of infected shrimp. The technique was also successfully applied to tissue samples preserved in 70% ethanol. The correct size fragment was amplified using IHHNV obtained from six different geographic regions in three different species of penaeid shrimp. No DNA extraction method was necessary for this technique. The use of hemolymph or pleopods provides a nondestructive screening method by which to test juveniles and adult broodstock for the presence of IHHNV. Received September 21, 1999; accepted January 21, 2000     

Detection of Garlic Viruses Using SYBR Green Real‐time Reverse Transcription‐Polymerase Chain Reaction

SYBR Green real‐time RT‐PCR assay was developed and optimized for the sensitive detection of Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Garlic common latent virus (GCLV), Shallot latent virus (SLV) and Mite‐borne filamentous virus (MbFV). The polyvalence of the designed primers was tested on 50 genotypes of garlic (Allium sativum L.) which originated from different countries. Plasmid standards were prepared and used as positive standards. The efficiencies of all reactions were 97, 93, 99, 98 and 87% for OYDV, LYSV, SLV, GCLV and MbFV standards, respectively. The detection limit for OYDV, LYSV and GCLV was as low as five gene copies, for SLV it was 15 gene copies and for MbFV it was 130 gene copies. In comparison with ELISA, more virus‐positive garlic accessions were detected with LYSV and GCLV by SYBR Green‐based real‐time RT‐PCR assay. This method was shown to be a more suitable tool for the detection of highly variable pathogens, such as garlic viruses.     

细胞内RT－PCR扩增免疫球蛋白重链可变区基因

通常,逆转录ＰＣＲ（ＲＴ－ＰＣＲ）需要高质量的ｍＲＮＡ,操作过程复杂,效率低且易受到ＲＮａｓｅ的破坏,为了简化操作,提高效率,用１０％甲醛盐溶液固定杂交瘤细胞,以ＮＰ－４０渗透化处理细胞后做ＲＴ－ＰＣＲ,获得了大约３５０ｂｐ的特异性免疫球蛋白重链可变区基因片段,与用同一对引物得到的常规ＲＴ－ＰＣＲ扩增产物一致。这项技术可用于获得特定结构基因片段,连结并扩增嵌合蛋白基因及构建多克隆免疫球蛋白文库等。