反向杂交膜片检测结核菌耐利福平基因突变

目的利用一种新型的反向点杂交膜片,快速检测结核分枝杆菌耐利福平rpoB基因突变.方法根据结核分枝杆菌rpoB基因序列设计寡核苷酸探针并固定于尼龙膜上,用生物素标记的引物增扩结核分枝杆菌rpoB基因片断,与膜片上探针杂交,膜片检测结果与常规药敏试验结果和测序结果进行对照.结果对63株临床分离株进行检测,10株利福平敏感株未检测到突变,53株耐利福平株,其中48株检测到突变.灵敏度为90.6%,特异度为100%,阳性预测值100%,阴性预测值66.7%.用反向点杂交膜片检测27份肺结核患者的痰标本,17份非结核的痰液标本.12份耐利福平的痰标本中,11份检测到突变,膜片检测与药敏结果的符合率为92.3%;15份利福平敏感的痰标本中,膜片检测结果有6份出现突变信号;17份非结核病的其他患者的痰液标本中有1份检测到突变.检测痰标本的灵敏度为91.7%,特异度为78.1%,阳性预测值61.1%,阴性预测值96.1%.结论反向点杂交膜片可简便、快速、较准确地检测出大多数结核分枝杆菌耐利福平分离株的rpoB基因突变.     

结核特异性抗体结合肽的筛选研究

目的 利用纯化的结核病患者血清IgG从噬菌体展示随机7肽库中筛选结核特异性抗体结合肽,为下一步开发新的结核病血清学检测试剂提供思路.方法 以纯化后结核病患者血清IgG作为固相配基,按吸附、洗脱、扩增的生物淘洗(简称淘选)过程对噬菌体展示随机7肽库进行筛选,并于第2至3轮的筛选中加入纯化后健康人血清IgG进行反筛,经过3轮淘选使噬菌体得到富集,分别从滴度测定平板上各个方位挑取结核病患者IgG和健康人IgG洗脱单噬菌体各20个进行扩增纯化,提取单链DNA作为模板进行测序,间接ELISA法检测不同单噬菌体与结核病患者和健康人IgG的结合情况,鉴定出阳性克隆.取收集的47份结核病患者和37份有卡介苗接种史的健康人血清标本,采用phage-ELISA法对获得的阳性单噬菌体进行初步验证,并对其中24份筛选用血清标本的检测结果进行分别统计.结果 通过3轮淘选,能与靶分子特异性结合的噬菌体得到明显富集.单噬菌体测序分析共获得12种共同序列.12种序列各取1个克隆扩增后进行间接ELISA检测,结果表明单噬菌体H12与结核患者IgG具有较高的亲和力(S/N≥2.1),确定为阳性克隆.间接ELISA检测发现,单噬菌体H12对47份结核病患者血清标本的检出值(0.105±0.010)明显高于对37份健康人的检出值(0.070±0.005),t=2.912,P<0.0001.对其中筛选用12份结核病患者和12份健康人血清标本的检出值分别为0.144±0.016、0.052±0.004,差异同样具有统计学意义(t=5.69,P<0.0001).结论 利用噬菌体展示随机肽库淘选获得了结核特异性抗体结合肽,显示出与结核病患者IgG较特异的结合活性,为以多肽为基础探索新的结核病血清学诊断方法提供了依据.

Abstract：

Objective To screen the specific antibody binding peptides of tuberculosis from phagedisplayed random phage display(Ph. D.)-7 peptide libraries with purified IgG from tuberculosis serum ,and to provide the basis for the development of serological detection reagent of tuberculosis. Methods Purified IgG of tuberculosis serum was used as solid ligand to screen the binding peptide from the Ph. D.-7 peptide library,according to the biopanning process of absorption, elution and amplification. Purified IgG of health people serum was used as the molecule of counter selection during the second and third selection. Phages were enriched after 3 rounds of screening, then 20 single phages separately eluted by IgG of tuberculosis and health people were randomly selected on each direction of the determination plates and amplified. The single chains DNA were extracted as template for sequencing. The combination abilities of selected clones to IgG of tuberculosis and health people were tested by indirect enzyme linked immunosorbent assay (ELISA) ,and the positive clone was identified. Serum samples, from 47 patients with tuberculosis and 37 healthy people vaccinated with BCG, were verified by positive phage clones using phage-ELISA. The verifying results of 24 serum samples used for panning were separately analyzed statistically. Results After 3 rounds of panning, remarkable enrichment of phages that could specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 12 sequences were obtained.12 phage clones with different sequences were amplified and detected with indirect ELISA and single phage H12 showed higher affinity with IgG of tuberculosis(S/N ≥2. 1)and was identified as the positive clone. It was found that,in indirect ELISA with singe Phage H12,the A450 value of tuberculosis patients (0. 105 ±0. 010) was significantly higher than that of healthy individuals (0. 070 ± 0. 005), and the t value was 2.912 (P< 0. 0001). The A450 value of 12 serum samples of tuberculosis patients and 12 samples of health individuals used for panning were 0. 144 ± 0. 016 and 0. 052 ± 0. 004, and the t value was 5. 69 (P <0. 0001). Conclusion By using phage-displayed random peptide libraries, we obtained the specific antibody binding peptides of tuberculosis,which showed specific binding activity with IgG of tuberculosis. It can provide a basis for the establishment of a new serological detection method of tuberculosis with peptide.     

结核分枝杆菌耐RFP快速检测的方法研究

目的应用PCR—SSCP技术直接快速检测痰标本中结核分枝杆菌rpoB基因突变，评价此方法用于结核分枝杆菌利福平（RFP）耐药性的意义。方法采用PCR—SSCP检测痰标本和菌株中结核杆菌rpoB基因突变。结果50例耐RFP菌株中有45例PCR—SSCP条带与结核分枝杆菌H37Rv条带有明显差别，与药敏试验结果做对比，PCR—SSCP检测敏感性为90％，特异性为100％；12例敏感菌株与H37Rv条带一致，35份肺结核病人痰标本，21份痰PCR扩增阳性，15份SSCP图谱与H37Rv图谱有差别，而且PCR—SSCP28份图谱与H37Rv有差别，阳性率为80％。结论PCR—SSCP检测结核分枝杆菌rpoB基因突变快速、简便、易行，有望用于结核分枝杆菌RFP耐药性的快速测定，并将成为直接检测临床标本中结核分枝杆菌耐RFP快速方法。     

煤工尘肺肺结核患者结核分枝杆菌L型耐药基因rpoB突变特点

目的 分析淮南矿区煤工尘肺结核患者感染的结核分枝杆菌L型利福平耐药基因rpoB突变特点.方法 收集尘肺并发肺结核患者的痰标本97份,对检出的结核分枝杆菌L型临床分离株52株进行分析,抽提临床分离株DNA和H37Rv标准菌株DNA,PCR法扩增rpoB基因,并应用全自动DNA测序仪对rpoB基因的突变集中区域进行测序分析.结果 52株临床分离株中,47株耐药株中rpoB基因突变率为91.49%(43/47),主要集中在531位点(48.94%,23/47)和526位点(25.25%,12/47)碱基置换突变,其次是511、516位点,1株535位点突变,其中41株单位点突变(87.23%,41/47),2株双位点突变(4.26%,2/47);5株敏感株未见rpoB基因单链构象异常.结论 煤工尘肺结核患者感染的结核分枝杆菌L型利福平耐药的分子基础是高度保守的rpoB基因突变,其突变位点呈多样性.     

微量液体培养法快速鉴别分枝杆菌

目的 建立微量液体培养法快速鉴别MTB和非结核分枝杆菌(NTM)方法,探讨其临床应用价值.方法 将不同浓度的对硝基苯甲酸(PNB)及噻吩-2-羧酸肼(TCH)和液体培养基加入96孔板中,种入待检菌株,37 ℃培养7～10 d观察结果.根据15种分枝杆菌标准株和30株已知MTB临床分离株不同PNB和TCH浓度时生长试验结果,确定PNB和TCH最佳浓度.用建立的微量液体培养法对424株分枝杆菌临床分离株进行菌种鉴定,并与PCR鉴定及测序鉴定结果进行比较.结果 微量液体培养法中PNB浓度200μg/ml时可有效区分MTB和NTM标准株;TCH浓度0.5 μg/ml时可有效区分结核分枝杆菌与牛结核分枝杆菌标准株.本法鉴定424株分枝杆菌临床分离株,以PCR法鉴定结果为标准,可鉴定出313株结核分枝杆菌复合群菌株中306株和全部NTM菌株,鉴定结核分枝杆菌复合群灵敏度为97.8%(306/313),特异度为100.0%(107/107);鉴定NTM的灵敏度为100.0%(107/107),特异度为96.5%(306/317);可鉴定出全部的MTB菌株,鉴定MTB的灵敏度可以达到100.0%(305/305),特异度尚待研究.结论 微量液体培养法可以在7～10 d内对分枝杆菌菌种进行快速鉴定,结果准确度高,操作简便,成本低廉,符合临床快速诊断的需要,适合在我国各级医疗机构推广使用.

Abstract：

Objective This research was to establish a method for fast identification of mycobacteria in microtiter liquid culture and to evaluate its clinical value. Methods 2-thiophenecarboxylic acid hydrazide (TCH) and paranitrobenzoic acid(PNB) at different concentrations were added into liquid culture in 96-well plate. Different mycobacterium standard strains were incubated in liquid culture with PNB and TCH for 7 to 10 days. According to the growth assay for 15 mycobacterium strains and 30 mycobacterium tuberculosis strains, the best PNB and TCH concentration were determined. A total of 424 clinical mycobacterium isolates were identified by microtiter liquid culture at the best PNB and TCH concentration. The results of microtiter liquid culture were compared with those of PCR and DNA sequencing. Results The best concentration of PNB was 200 μg/ml in microtiter liquid culture. Compared with the results of PCR, the sensitivity and specificity for identification of mycobacterium tuberculosis complex in microtiter liquid culture were 97. 8%(306/313) and 100. 0% (107/107) respectively and those for non-tuberculosis mycobacteria in microtiter liquid culture were 100. 0% (107/107) and 96. 5% (306/317) respectively. The best concentration of TCH was 0. 5 μg/ml. Compared with the results of PCR,the sensitivity of mycobacterium tuberculosis in microtiter liquid culture was 100.0% (305/305). The specificity remained under and more studies were needed. Conclusion In microtiter liquid culture with PNB and TCH, mycobacteria can be identified in 7 to 10 days. The results were accurate and the process was simple without expensive equipments. This method meets clinical needs and can be used in all level hospitals in China.     

结核分枝杆菌利福平耐药株rpoB基因突变特征研究

目的了解郑州市结核分枝杆菌耐利福平临床分离株的相关耐药rpoB基因突变特征。方法应用PCR方法对40株结核分枝杆菌利福平耐药、40株结核分枝杆菌利福平敏感临床分离株及1株H37Rv结核分枝杆菌标准株中包含"利福平耐药决定区(RRDR)"在内的rpoB基因片段(385bp)进行扩增,并将扩增产物进行测序,以H37Rv结核分枝杆菌标准株的DNA序列作为参比序列,分析结核分枝杆菌临床分离株的突变特征。结果 40株结核分枝杆菌利福平敏感临床分离株及1株H37Rv结核分枝杆菌标准株均未检测到突变,结核分枝杆菌耐利福平临床分离株中有36株检测到突变,突变率92.3%,且突变均位于RRDR区域内;突变位点为8个,共有8个突变类型;最常见的突变热点依次为531位点,该位点突变率为64.1%;526位点,其突变率为12.8%和516位点,其突变率为10.3%。结论 rpoB基因是郑州市结核分枝杆菌对利福平耐药的最相关基因,其中在RRDR内的531位点基因突变率最高,并可通过对rpoB基因的突变检测,对耐多药结核病进行预测。     

结核分枝杆菌利福平耐药基因突变的研究

目的评价检测rpoB基因突变在结核分枝杆菌耐利福平（I肝）耐药性测定中的应用价值。方法采用DNA序列分析法和聚合酶链反应-单链构象多态性（PER—SSCP）法对91株结核分枝杆菌临床分离株（其中药物敏感株35株，耐RFP或含耐RFP耐多药株56株）rpoB基因核心区域的突变情况进行检测。结果以结核分枝杆菌H37RV为对照，所有35株药物敏感株的rpoB基因均无突变，用DNA序列分析方法检测56株耐药株中53株发生突变，敏感性为94．6％（53／56），其中最常见的突变位点是531位丝氨酸和526位组氨酸。PER—SSCP检测56株耐RFP分离株中，52株rpoB基因SSCP图谱异常，其敏感性为92．9％（52／56）。结论DNA序列分析对判断结核分枝杆菌耐利福平非常有价值。PER—SSCP可初步筛选结核分枝杆菌RFP耐药性。     

焦磷酸测序技术在利福平rpoB基因突变检测中的应用评价

目的 应用焦磷酸测序技术检测结核分枝杆菌rpoB基因突变,并探讨其在结核分枝杆菌利福平耐药性检测中的应用价值.方法 利用焦磷酸测序技术,测定150株结核分枝杆菌临床分离株rpoB基因81个碱基的利福平抗药性决定区(RRDR),分析rpoB基因突变特点,并与绝对浓度法和MIC法药敏结果进行比较.结果 150株结核分枝杆菌中66株耐药84株敏感,54株为耐多药菌株,rpoB基因突变涉及6个密码子12种突变基因型,最常见的突变位点及突变形式分别为531位TCG突变为TYG,占60.6%,526位CAC突变为TAC、GAC,占22.7%.耐药性检测结果表明,焦磷酸检测法与绝对浓度法二者的符合率为92.7%,与MIC法的符合率为97.8%.结论 焦磷酸测序检测技术不仅是一种快速、准确、高通量的利福平分子药敏检测方法,同时也是结核分枝杆菌耐药性研究的有用工具.     

PCR-反向斑点杂交技术检测结核分枝杆菌及其耐药基因的突变

目的探讨PCR-反向斑点杂交技术用于检测结核分枝杆菌及其耐药基因突变的效果,了解绍兴地区结核分枝杆菌耐药基因的突变情况。方法根据结核分枝杆菌基因特征,运用反向斑点杂交技术构建特异性的PCR反应体系,并用10株非结核分枝杆菌、3株其他菌株（金黄色葡萄球菌、大肠埃希菌和鲍曼不动杆菌）和1株H37Rv结核分枝杆菌对该反应体系进行验证,同时用该体系对来源于绍兴市立医院的125份抗酸杆菌涂片阳性痰液样本及150株结核分枝杆菌进行5种耐药突变基因（rpoB、katG、inhA、embB、rpsL）的检测。结果构建的PCR-反向斑点杂交体系用于10株非结核分枝杆菌、3株其他菌株的检测结果均为阴性,H37Rv标准株可检出,但未检出耐药突变位点。275份结核分枝杆菌样本中对4种抗结核药物均敏感的菌株有201份（73．1％）,单药耐药20份（7．3％）,耐多药54份（19．6％）;共检出133个耐药突变位点,其中对利福平、异烟肼、链霉素和乙胺丁醇的耐药突变位点数分别为41个、51个、30个、11个;前5位突变位点为315M、43M、S531L、15M和H526Y。结论绍兴地区耐药结核分枝杆菌以耐多药为主。构建的PCR．反向斑点杂交体系用于检测结核分枝杆菌特异性强、灵敏度高,可快速检测结核分枝杆菌的耐药基因。     

淮南矿区煤工尘肺结核患者耐多药结核分枝杆菌耐药基因rpoB序列分析

目的 分析淮南矿区尘肺结核患者感染的耐多药结核分枝杆菌(multiple drugs resistant bacillus tuberculosis,MDR-TB)利福平耐药基因rpoB突变特点.方法 收集结核分枝杆菌临床分离株114株,采用药敏试验鉴定MDR-TB,抽提MDR-TB DNA和H37Rv标准菌株DNA,PCR法扩增rpoB基因,并应用全自动DNA测序仪对rpoB基因的突变集中区域进行测序分析.结果 从114株结核分枝杆菌临床分离株中检出MDR-TB31株(27.19％,31/114),31株MDR-TB中rpoB基因突变率为93.55％(29/31),主要集中在531(45.16％,14/31)和526位点(29.03％,9/31)碱基置换突变,其次是516(3.23％,1/31)、535位点(3.23％,1/31);1株526位和531位同时突变,1株511位和526位同时突变,2株未发现rpoB基因突变.随机检测的10株利福平敏感株未见rpoB基因单链构象异常.结论 高度保守的rpoB基因突变是导致尘肺结核患者MDR-TB利福平耐药的分子基础,其突变位点呈多样性.     

用DNA芯片检测结核分枝杆菌耐利福平基因突变的初步研究

目的研制一种新型DNA芯片，用于快速检测结核分枝杆菌耐利福平rpoB基因突变。方法根据结核分枝杆菌rpoB基因序列设计探针并制作DNA芯片，PCR掺入法荧光标记根据结核分枝杆菌rpoB基因突变热点的目的片断，与DNA芯片杂交，同时以DNA测序法为对照。结果18株结核分枝杆菌临床分离株中，1株敏感株DNA芯片杂交结果与标准株完全相同；17株耐RFP临床分离株中有17株检测到rpoB基因突变，其中14株单位点突变，1株511和516位双位点突变，与测序结果完全一致。另有两株为517和518、526和517位双位点突变，因为芯片上未点517位突变的探针，所以只检测到518和526位的突变，该突变与测序结果完全一致、结论用DNA芯片可快速、特异地检测出大多数结核分枝杆菌耐利福平分离株的rpoB基因突变，可用于临床耐药性的检测，指导临床用药。     

Discordant results between genotypic and phenotypic assays (Xpert MTB/RIF vs. BACTEC MGIT 960 system) for detection of RIF-resistant Mycobacterium tuberculosis isolates in a high burden region

Clinical isolates with discordant phenotypic and genotypic results were submitted to DNA sequencing to identify which were genuinely resistant to rifampin and determine the frequency of silent and disputed mutations in our region. We present the retrospective analysis of all the culture-proven TB cases tested with the Xpert®MTB/RIF assay at the Tuberculosis Clinic and Laboratory of the Tijuana General Hospital, Mexico. Clinical isolates showing a discrepancy between phenotypic and molecular tests were analyzed by DNA sequencing. Thirteen isolates tested as rifampin susceptible on the MGIT system were rifampin-resistant according to Xpert®MTB/RIF assay. DNA sequencing showed that seven (53.8%) isolates had a silent (P514P) mutation; three isolates showed different missense (L511P, D516Y, and S531L) mutations. Three isolates showed no mutations.The existence of heteroresistance and silent or disputed mutations warrants that all rifampin-resistance cases diagnosed with the Xpert®MTB/RIF should be referred to specialized centers for DNA sequencing.     

结核分枝杆菌利福平耐药基因rpoB突变特征初步分析

目的 阐明结核分枝杆菌利福平耐药株rpoB基因突变特点。方法 对110株结核分枝杆菌临床分离株包括rpoB基因利福平耐药决定区（RRDR）81个碱基在内的286bp片段进行聚合酶链反应．单链构象多态性（PCR．SSCP）分析，并对经检测显示有突变的结核分枝杆菌rpoB基因此扩增区域进行序列测定。其中利福平耐药株73株，非利福平耐药株11株，敏感株26株。结果 PCR—SSCP检测显示利福平耐药株中有47株有突变存在，经直接测序分析其中76．6％的菌株存在rpoB基因的单位点突变，主要集中在531位（61．1％）和526位（25．0％）；联合突变发生率为23．4％。此外，经PCR—SSCP检测，11株非利福平耐药株中2株有突变存在，26株敏感株中1株有突变存在。经直接测序分析突变涉及位点为511位、526位和535位。结论 rpoB基因耐药决定区发生突变是结核分枝杆菌利福平耐药的主要原因，其中531位丝氨酸和526位组氨酸是最常见的突变位点。     

荧光PCR探针熔解曲线法检测结核分枝杆菌耐利福平突变研究

目的对荧光PCR熔解曲线法检测结核分枝杆菌耐利福平突变方法进行临床研究,评价检测能力及在国境口岸的应用价值。方法应用荧光PCR熔解曲线法检测结核病人临床分离结核分枝杆菌,以传统药物敏感性试验为标准,获得该方法的灵敏性、特异性。结果对347例结核病人临床分离培养样本,传统药物敏感性试验检出271例利福平敏感标本,76例利福平耐药标本。荧光PCR熔解曲线法检出269例利福平敏感标本,78例利福平耐药标本,灵敏性为93.42%,特异性为97.42%,符合率为96.54%。结论荧光PCR熔解曲线法检测速度快速、灵敏度高、特异性强,可用于结核分枝杆菌利福平耐药突变的快速检测,适于国境口岸对耐多药结核病的快速筛查。     

Prevalence of non-tuberculous mycobacteria in a hospital environment

In recent years, non-tuberculous mycobacteria (NTM) have emerged as an important cause of opportunistic nosocomial infections but there is little known about the isolation and identification of NTM in Korea. The aim of this study was to assess the prevalence of NTM in the hospital environment and identify the species. A total of 150 samples were collected from different parts of the hospital. NTM were isolated and identified by restriction fragment length polymorphism analysis of the gene encoding rpoB and partial sequencing analysis of hsp65 and rpoB. In this study, 60 strains of NTM were isolated from 50 of the 150 samples. Half of the tap water samples (50 of 100) were positive for mycobacteria. An estimated 73.3% of the isolates were saprophytic, 21.7% were potentially pathogenic and 5% were unidentified. The presence of NTM in hospital tap water is not uncommon. Such water isolates might cause true nosocomial infection in immunocompromised patients, in addition to the risk of false-positive culture results.     

rpoB Gene mutations in rifampin-resistant Mycobacterium tuberculosis identified by polymerase chain reaction single-stranded conformational polymorphism.

The use of polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) to study rpoB gene mutations in rifampin-resistant (RIFr) Mycobacterium tuberculosis has yielded contradictory results. To determine the sensitivity of this method, we analyzed 35 RIFr strains and 11 rifampin-susceptible (RIFs) strains, using the DNA sequencing of the core region of rpoB for comparison. Of the RIFr, 24 had a PCR-SSCP pattern identical to that of H37Rv; the other 11 had four different patterns. The 11 RIFs had PCR-SSCP patterns identical to that of H37Rv. The sensitivity of the assay was 31.4%; its specificity was 100%. We observed a strong correlation between the degree of resistance and the type of mutation.     

非结核分枝杆菌快速检测方法的评价

目的评价针对结核分支杆菌复合群(Mycobacterium Tubercu losis Comp lex,MTC)MPB64抗原,快速检测非结核分枝杆菌(Nontubercu lous mycobacteria,NTM)的方法。方法与对硝基苯酸(PNB)生长实验检测NTM的方法比较。结果检测结核分枝杆菌(M.tubercu losis)H37RV、蟾蜍分枝杆菌(M.xenop i)、偶然分枝杆菌(M.fortu itum)和86例临床分离菌株,快速检测非结核分枝杆菌与PNB方法符合率100%。结论与PNB方法相比,快速检测非结核分枝杆菌有较高的特异性和灵敏性,更适于基层实验室和大规模调查时的初筛实验。     

反向斑点杂交技术在结核分枝杆菌耐药性检测中的应用研究

目的利用反向斑点杂交技术，建立结核分枝杆菌对利福平（RFP）、异烟肼（INH）、链霉素（SM）和乙胺丁醇（EMB）耐药相关基因突变的快速检测方法。方法根据结核分枝杆菌标准株H37Rv序列，自行设计针对rpoB、katG、inhA、rpsL和embB基因中常见耐药突变区的系列寡核苷酸探针，制成膜芯片，并对64例结核分枝杆菌临床株的基因突变情况进行检测。结果在36株RFP耐药菌中，有32株被检出在rpoB基因上发生突变，突变检出率为88．89％（32／36）；在39株INH耐药菌中，有30株被检出在katG或inhA基因上发生基因突变或缺失，检出率为76．92％（30／39）；在34株SM耐药菌中，有25株被检出在rpsL基因分析部位发生突变，检出率为73．5％（25／34）；在32株EMB耐’药菌中，有19株在embB基因分析部位发生突变，检出率为59．4％（19／32）。膜芯片检出的突变与基因测序结果一致。结论用膜芯片检测结核分枝杆菌对利福平（RFP）、异烟肼（INH）、链霉素（SM）和乙胺丁醇（EMB）四种主要一线抗结核药的耐药性，具有较高的特异性和敏感性，可作为常规药敏方法的补充，用于指导开展早期、有效的临床化疗。     

HBV耐药基因的实验研究

目的:利用多色荧光PCR技术,建立快速检测HBV耐药基因的方法,并与基因测序法进行对照研究。方法:建立2个反应体系,每个反应体系包含四个耐药位点。对新建的多色荧光PCR法进行灵敏度和特异性分析,并对42例临床收集的服药患者血清进行耐药检测。结果:构建了多色荧光PCR检测HBV耐药位点的体系,其特异性较好,分析灵敏度可达1×103copies/ml。42例样本中检测到YVDD 3例(占7.14%),YIDD 1例(占2.38%);1896变异7例,占总数的16.67%。其他位点未检测到耐药突变。结论:所构建的多色荧光PCR检测体系为临床医院提供快速、简便的HBV耐药位点突变检测手段,较基因测序法方便、快捷。     

牡蛎和粪便中GⅠ、GⅡ型诺如病毒实时聚合酶链反应检测方法的建立

目的 建立适用于牡蛎和粪便中快速、特异、灵敏的GⅠ、GⅡ型诺如病毒(NV)定量分型诊断方法.方法 通过对GⅠ、GⅡ型NV基因组保守序列的比对分析,设计高度特异的引物和探针,建立以TaqMan探针为基础的实时聚合酶链反应方法(TaqMan Real-time RT-PCR).结果 该方法对NV核酸检测高度特异,且GⅠ和GⅡ型之间无交叉反应,最低检出限达102 copies/μl.对90份新鲜牡蛎样品和37份腹泻粪便标本分别用常规RT-PCR和笔者建立的TaqMan Real-time RT-PCR进行NV检测,发现牡蛎样品中后者的检出率明显高于前者,而粪便标本中两者无明显差别.同时对阳性标本的测序分析证实结果准确可靠.结论 研究中建立的TaqMan Real-time RT-PCR方法可用于海产品标本及粪便中NV定量及分型检测,可作为应对NV胃肠炎暴发的有效诊断方法.