Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization

Objective A PCR-reverse dot blot hybridization （RDBH） assay was developed for rapid detection of rpoB gene mutations in ＇hot mutation region＇ of Mycobacterium tuberculosis （M. tuberculosis）. Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing （DST） and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2% （165/181） and 98.3% （117/119）, respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value （PPV） and negative predictive value （NPV） of the RDBH assay were 97.7% （293/300）, 98.2% （164/167）, and 97.0% （129/133）, respectively. Furthermore, the results indicated that the most common mutations were in codons 531 （48.6%）, 526 （25.4%）, 516 （8.8%）, and 511 （6.6%）, and the combinative mutation rate was 15 （8.3%）. One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

Comparison of the Tellgenplex HPV DNA test with the PCR-reverse dot blot assay for human papillomavirus genotyping

Objective: To access the performance of the Tellgenplex human papillomavirus(HPV) DNA test compared to the polymerase chain reaction-reverse dot blot(PCR-RDB) assay for the HPV genotyping.Methods: Sixty cervical swab samples were genotyped by the Tellgenplex HPV DNA test and the PCR-RDB assay.The Tellgenplex HPV DNA test and the PCR-RDB assay can detect 26 and 23 HPV genotypes, respectively.Each sample showed discrepancy was genotyped using sequencing.Results: The percent agreement between the two tests ranged from 83.3% to 100.0% according to different genotype.This showed perfect agreement(>0.81) for high-risk HPV genotypes(35, 39, 45, 53, 56, 59, 66, 68, and 82), substantial agreement(>0.65) for high-risk HPV genotypes(16, 18, 33, 52, and 58) and low-risk HPV genotype 43 between the two assays by the kappa analysis.The positive rates of the two assays for frequent HPV genotypes(16, 35, 39, 45, 52, 53, 58, 59, 66, and 82) were not statistically different, but the PCR-RDB assay showed higher positive rates than the Tellgenplex HPV DNA test for HPV genotypes 81(P<0.05).As for more than 10 positive results by the Tellgenplex HPV DNA test and/or the PCR-RDB assay, the PCR-RDB assay showed higher relative sensitivity and specificity than the Tellgenplex HPV DNA test for the three HPV genotypes(16, 52, and 81).All HPV genotypes that can be detected by only the Tellgenplex HPV DNA test(HPV genotypes 44 and 55) were confirmed by sequencing.Conclusions: In conclusion, our results demonstrated that the PCR-RDB assay which can detect more multiple HPV genotypes in each specimen shows higher relative sensitivity and specificity than the Tellgenplex HPV DNA test, which makes it a better option for routine clinical use.

Dot-Blot Hybridization for Detection of Five Cucurbit Viruses by Digoxigenin-Labelled cDNA Probes

Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops, Zuccini yellow mosaic virus （ZYMV）, Watermelon mosaic virus （WMV）, Cucumber mosaic virus （CMV）, Papaya ringspot viruswatermelon strain （PRSV-W） and Squash mosaic virus （SqMV）, as a good alternative assay in seed health test and epidemiological and transgenic research. Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves. And three SqMV probes of different lengths （0.55, 1.6, and 2.7 kb, respectively） were designed to investigate the effect of hybridization. The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV, WMV, CMV, PRSV-W, and SqMV was down to 1：160, 1：160, 1：320, 1：160, and 1：320, respectively. Three SqMV probes of different length showed no differences on the sensitivity and specificity. The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities, sensitivities, specificity, and reproducibilifies.

A rapid reverse dot blot assay for all 18 β-thalassemia mutations in Chinese population

A set of allele-specific oligonucleotide (ASO) probes used for detecting all 18 β-tha-lassemia mutations found in Chinese was immobilized on two strips of Biodyne C membrane;one containing 7 pairs of oligonucleotide probes specific for the most commonly found mutant al-leles,and the other containing the remaining 11 pairs of ASO_s specific for the less commonlyfound.The membranes were hybridized with β-globin sequences amplified by polymerase chainreaction (PCR) with biotinylated primers,and then treated with Streptavidin-POD conjugateand substrates for color development.The method has been applied successfully to the detectionof all 18 Chinese β-thalassemia mutations and prenatal diagnosis of two high-risk pregnancies ofβ-thalassemia.Patients with homozygous,heterozygous and compound heterozygous alleles ofthese mutations and normal individuals could be easily distinguished by the present method.Us-ing the immobilized-probe format (reverse dot blot),it was able to screen simultaneously multi-ple β-thalassemia mutations of a DNA sample by performing hybridization only once.This assayis simple,rapid and independent of radio-isotopes and can be appplied for all 18 β-thalassemiamutations so far found in Chinese population.It is considered that this method may be usefulfor gene frequency investigation of large numbers of β-thalassemia DNA samples and used as aroutine method in the clinic laboratory.

PCR-Dotblot杂交法直接检测临床病原菌的报告

目的为了寻找临床病原菌系统，检测和鉴别的有力手段。方法用真细菌保守的１６ＳｒＲＮＡ基因为模板，以ＰＣＲ－Ｄｏｔｂｌｏｔ杂交的方法检测临床病原菌。结果它将１６ＳｒＲＮＡ基因的广谱性和变异性并存之特点和ＰＣＲ－Ｄｏｔｂｌｏｔ杂交的敏感性结合起来，对该法的建立进行了探讨，并初步用于临床感染的检测。结论为细菌通用检测法的建立提供了基础。

BALF Xpert MTB/RIF和PCR-反向斑点杂交技术在涂阴肺结核诊断中的价值

目的:对比支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)结核分枝杆菌(MTB)/利福平(RIF)耐药实时荧光定量核酸扩增检测技术(Xpert MTB/RIF)和聚合酶链反应(PCR)-反向斑点杂交技术在涂阴肺结核诊断中的价值。方法:纳入2021年6月—2023年1月于江西省胸科医院接受治疗的肺结核患者共200例,所有患者均留取BALF送检,进行Xpert MTB/RIF、PCR-反向斑点杂交技术检测。以病理检查结果为金标准,统计确诊涂阴肺结核的占比情况,并计算Xpert MTB/RIF与PCR-反向斑点杂交技术两种检测方法的阳性率、与金标准结果的一致性,并评估两种检测方法对涂阴肺结核的诊断价值。结果:Xpert MTB/RIF检测涂阴肺结核的阳性率(76.50%)与PCR-反向斑点杂交技术(69.50%)比较,差异无统计学意义(χ^(2)=2.486,P=0.115)。200例肺结核患者中确诊为涂阴肺结核有137例,占比68.50%;Xpert MTB/RIF的符合率(94.89%)高于PCR-反向斑点杂交技术(74.45%),差异有统计学意义(P<0.05)。Xpert MTB/RIF诊断涂阴肺结核的敏感度、特异度、阳性预测值、阴性预测值、准确度均高于PCR-反向斑点杂交技术(P<0.05)。结论:Xpert MTB/RIF、PCR-反向斑点杂交技术均可用于涂阴肺结核诊断中,其中Xpert MTB/RIF的诊断价值更高,可辅助临床早期诊断筛查。

应用RT-PCR、斑点杂交法和SDS-PAGE检测水稻黑条矮缩病毒

用RT PCR技术、PCR标记的探针点杂交和SDS PAGE检测了生产上严重危害玉米和水稻的水稻黑条矮缩病毒(RBSDV)。由RT PCR扩增的RBSDV第 7片段第 92 1～ 14 11碱基作探针 ,用PCR法DIG标记后点杂交 ,可以从 10 0ng玉米病叶中检测到RBSDV ,灵敏度是RT PCR的 1/10 ;10 %SDS PAGE只能检测到从 1g玉米病叶中提取的病毒dsRNA ,但对江苏玉米田间分离的不同的RBSDV样品电泳发现 ,该病毒基因组dsRNA有差异 。

Accuracy of a reverse dot blot hybridization assay for simultaneous detection of the resistance of four anti-tuberculosis drugs in Mycobacterium tuberculosis isolated from China

Background:Drug resistant tuberculosis poses a great challenge for tuberculosis control worldwide.Timely determination of drug resistance and effective individual treatment are essential for blocking the transmission of drug resistant Mycobacterium tuberculosis.We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization(RDBH)assay to simultaneously detect the resistance of four anti-tuberculosis drugs in M tuberculosis isolated in China.Methods:In this study,we applied a RDBH assay to simultaneously detect the resistance of rifampicin(RIF),isoniazid(INH),streptomycin(SM)and ethambutol(EMB)in 320 clinical M.tuberculosis isolates and compared the results to that from phenotypic drug susceptibility testing(DST) and sequencing.The RDBH assay was designed to test up to 42 samples at a time.Pearson's chi-square test was used to compute the statistical measures of the RDBH assay using the phenotypic DST or sequencing as the gold standard method,and Kappa identity test was used to determine the consistency between the RDBH assay and the phenotypic DST or sequencing.Results:The results showed that the concordances between phenotypic DST and RDBH assay were 95%for RIF,92.8%for INH,84.7%for SM,77.2%for EMB and the concordances between sequencing and RDBH assay were 97.8%for RIF,98.8%for INH,99.1%for SM,93.4%for EMB.Compared to the phenotypic DST results,the sensitivity and specificity of the RDBH assay for resistance detection were 92.4 and 98.5%for RIF,90.3 and 97.3%for INH,77.4 and 91.5%for SM,61.4 and 85.7%for EMB,respeaively;compared to sequencing,the sensitivity and specificity of the RDBH assay were 97.7 and 97.9%for RIF,97.9 and 100.0% for INH,97.8 and 1OO.O% for SM,82.6 and 99.1%for EMB,respectively.The turnaround time of the RDBH assay was 7 h for testing 42 samples.Conclusions:Our data suggested that the RDBH assay could serve as a rapid and efficient method for testing the resistance of M. tuberculosis against RIF,INH,SM and EMB,enabling early administration of appropriate treatment regimens to the affected drug resistant tuberculosis patients.

PCR-反向点杂交法在外阴阴道念珠菌病诊断及白念珠菌耐药基因突变检测中的应用

目的评价PCR-反向点杂交法用于念珠菌菌种鉴定及白念珠菌耐药基因突变检测的应用价值。方法收集念珠菌性阴道炎患者及体检健康妇女分泌物各285份和50份。用生物梅里埃酵母菌鉴定卡进行菌种鉴定,用最低抑菌浓度(MIC)法(郑州安图真菌快速培养鉴定药敏试剂)进行药敏试验;采用PCR-反向点杂交法(深圳亚能念珠菌菌种鉴定及白念珠菌耐药基因突变检测试剂)进行菌种鉴定和耐药基因突变检测;采用PCR及测序方法进行耐药基因的检测。分别以培养鉴定法、MIC法、核酸序列测定法为对比方法,评价PCR-反向点杂交法念珠菌菌种鉴定及白念珠菌耐药基因突变检测的敏感性、特异性及准确性。结果与培养鉴定法相比,PCR-反向点杂交法检测6种念珠菌菌种的敏感性、特异性、阳性预测值、阴性预测值和总符合率分别为95%、96%、96%、98%、97%以上,2种方法检测6种念珠菌(白念珠菌、光滑念珠菌、热带念珠菌、近平滑念珠菌、克柔念珠菌和季也蒙念珠菌)结果的差异无统计学意义(χ~2值分别为0.44、0、0、0、0和0,P均>0.05),一致性较好(Kappa均>0.9)。与MIC法相比,PCR-反向点杂交法检测白念珠菌耐药的敏感性、特异性、阳性预测值、阴性预测值和总符合率分别为98%、88%、98%、88%和96%,2种方法检测结果的差异无统计学意义(χ~2=0.17,P>0.05),一致性较好(Kappa>0.8)。PCR-反向点杂交法与核酸序列测定法相比,对6种白念珠菌耐药基因突变位点的检测结果完全一致。结论PCR-反向点杂交法在念珠菌菌种鉴定及白念珠菌耐药基因突变检测上与培养鉴定法以及核酸序列测定法的一致性高,比传统检测方法更早期更快速,可应用于外阴阴道念珠菌病(VVC)的辅助诊断。

应用PCR-反向点杂交法检测β-地中海贫血基因点突变

目的对疑似β-地中海贫血的4例患者进行基因诊断。方法针对β-珠蛋白基因上17个位点突变,使用特异性带有生物素标记的引物进行PCR扩增,扩增产物与固定在膜条上的寡核苷酸探针进行杂交,并通过一系列显色反应,明确检测样品在17个位点是否发生了突变,以及是否为突变纯合子或杂合子。结果 1号和2号患者均为CD17M(A→T)/N,βEM(GAG→AAG)/N双重杂合子;D3号患者为IVS-Ⅱ-654M(C→T)/N杂合子;D4号患者为IVS-Ⅱ-654M/IVS-Ⅱ-654M(C→T)纯合子。结论利用PCR-反向点杂交法技术,能对β-地中海贫血患者进行基因诊断,对其产前诊断、临床治疗以及携带者检出具有重要意义。

实时PCR和PCR-RDB法检测人乳头瘤病毒的比对研究

目的：比较实时 PCR 法和 PCR-反向点杂交法（PCR-RDB）检测 HPV 的一致性。方法收集109份女性生殖道样本，利用实时 PCR 法和 PCR-RDB 法分别检测 HPV 感染和基因型分布情况，不一致样本采用 PCR-悬浮芯片杂交法复检。结果83.5％（91／109）的样本两种方法结果一致（kappa＝0.671），18例不一致样本 PCR-悬浮芯片杂交法复检显示7例与实时 PCR相符，11例与 PCR-RDB 相符；高、低病毒载量组间 PCR-RDB 法的检测结果差异无统计学意义（χ2＝1.476，P ＝0.224）。结论实时 PCR 和 PCR-RDB 两法用于 HPV 检测一致性一般；HPV 病毒载量在103～108范围内时 PCR-RDB 法的阳性率较稳定。

应用PCR-RDB杂交技术检测人类乳头瘤病毒基因型的临床意义

目的采用聚合酶链反应(PCR)和反向斑点杂交(RDB)技术检测西安地区人类乳头瘤病毒(HPV)感染流行状态和HPV基因型分布特征。方法共收集266例脱落细胞标本,其中尖锐湿疣86例、宫颈上皮内瘤变144例、宫颈癌36例,应用PCR-RDB杂交技术进行23种HPV基因型分型检测。结果全部标本HPV总阳性率为71.43%(190/266),宫颈癌HPV阳性率明显高于其他疾病,差异有统计学意义(χ2=6.297,P<0.05);HPV阳性标本基因分型全部成功,HPV16型为主要感染基因型,其次为HPV18、6、11型;单基因型总感染率91.58%(174/190),多基因型总感染率8.42%(16/190)。不同疾病单基因型感染和多基因型感染构成比差异无统计意义(χ2=0.3511,P>0.05)。结论 HPV16型是西安地区主要感染基因型,应用PCR-RBD技术检测HPV基因型对HPV感染的防治具有一定临床意义。

基于PCR-反向点杂交法对江西地区分枝杆菌感染状况分析

目的了解江西地区分枝杆菌感染流行现状,为临床防治提供科学依据。方法运用PCR-反向点杂交法对345例可疑分枝杆菌感染患者不同标本进行分枝杆菌核酸检测。结果 345份标本中共检出68例阳性(阳性率为19.7%),其中结核分枝杆菌复合群(MTC)59例(占86.76%),非结核分枝杆菌(NTM)8例(占11.77%),混合感染(MTC/MIN)1例(1.47%);NTM感染中检测出5种,分别为偶发/猪分枝杆菌(MFO)、戈登分枝杆菌(MGO)、胞内分枝杆菌(MIN)、龟分枝杆菌脓肿亚肿(MAB)、蟾蜍分枝杆菌(MXE),其中以MIN为主,占5.88%;不同标本中分枝杆菌检出率不同,痰液、灌洗液、分泌物、胸水中分枝杆菌检出率分别为:31.5%、20.3%、16.0%、4.7%。结论江西地区分枝杆菌感染主要以MTC为主,且多为单一感染。

Gap-PCR与反向斑点杂交技术检测α-地中海贫血

目的探讨Gap-PCR与反向斑点杂交技术在α-地中海贫血中的应用价值。方法采用PCR(即Gap-PCR)技术检测缺失型α-地中海贫血;用反向斑点杂交技术检测非缺失型α-地中海贫血。结果在3 896份标本中共检出237例α地贫,阳性检出率为6.08%;通过Gap-PCR技术检测出56例α缺失型地贫,基因型分别为东南亚缺失型(--^(SEA)、右侧缺失型(-α^(3.7)和左侧缺失型(-α^(4.2));4例HbH型;通过反向斑点杂交技术检测出8例非缺失型α地贫,基因型分别为αα/α^(QS)和αα/α^(WS)。结论α-地中海贫血是我国南方影响最大的常见遗传病之一,应加强对地中海贫血的血液学筛查和基因诊断,提高诊断符合率。

应用PCR-反向斑点杂交快速鉴定分枝杆菌菌种

目的:应用聚合酶链反应-反向斑点杂交法(PCR-RDBHA)快速鉴定分枝杆菌至种的水平。方法:以分枝杆菌rpoβ基因编码序列为靶基因,应用PCR-RDBHA检测126株分枝杆菌临床分离株。以PCR-DNA测序为对照,对经PCR-RDBHA鉴定为非结核分枝杆菌(NTM)的临床分离株进行PCR-DNA测序。结果:126株临床分离株中,经鉴定115株(91.3%)为结核分枝杆菌复合群(MTC),11株(8.7%)为NTM。NTM中,4株胞内分枝杆菌,1株堪萨斯分枝杆菌,1株戈登分枝杆菌,2株瘰疬分枝杆菌,3株脓肿分枝杆菌。11株NTMPCR-RDBHA与PCR-DNA测序结果一致。结论:PCR-RDBHA能鉴定分枝杆菌临床分离株至种的水平,方法简便、快速,结果准确,具有良好的临床应用价值。

PCR法对HLA-DQ_α基因的分型及其在性犯罪鉴定中的应用

本文应用 PCR 法及 ASO 探针斑点杂交技术,对100例无关个体血液 DNA 及10例性犯罪案件混合斑中精子 DNA 进行了 HLA-DQα基因的扩增及其 DNA 分型。结果正常人0.1～0.3μgDNA 就能满足 DQα基因扩增的需要。在100例个体中可以观察到由4种等位基因组成的10种 DQα基因型。10例不同条件的混合斑中精子 DNA 经30～60次扩增后与 ASO 探针杂交均能准确地判定 DQα基因型。HLA-DQα是个体识别能力较强的遗传标志。本文为性犯罪中精子来源的个体识别提供了一个新方法,具有一定的实用价值。

血红蛋白电泳及PCR-反向斑点杂交法诊断β-地中海贫血

目的:探讨血红蛋白电泳与突变基因检测诊断β-地中海贫血的临床意义。方法:应用醋酸纤维薄膜电泳对100例小细胞低色素贫血病人进行血红蛋白电泳分析,将检测结果中MCV<80%,HbA2>3.2%,或HbF>3.1%的判为β-地中海贫血表型阳性,对阳性患者进一步应用聚合酶链反应(PCR)结合反向斑点杂交技术(RDB)检测其基因突变类型。结果:47例病人经血红蛋白电泳定量检测为β-地中海贫血表型阳性,其中22例为β-地中海贫血,阳性率为46.8%,检出5种突变基因,依次为:IVS-Ⅱ-654(C-T)、CD41-42(-TTCT)、CD17(A-T)、TATAbos-28(A-G)、CD14-15(+G)。以IVS-Ⅱ-654(C-T)突变类型占首位。结论:血红蛋白电泳联合基因诊断是目前诊断β-地中海贫血最可靠的实验方法。

B组轮状病毒RT-PCR来源的cDNA探针核酸诊断方法的建立

近十年来，国内外学者相继报道在人以及牛、羊、猪、大鼠的腹泻粪样中检出Ｂ组轮状病毒〔１－３〕。洪涛等于１９８３年首次报道成人腹泻轮状病毒（ＡＤＲＶ）属于Ｂ组轮状病毒〔４〕。目前，轮状病毒Ｂ组已被公认为引起人及不同动物腹泻的流行病学的重要因素。由于Ｂ组轮...

PCR-反向斑点杂交法检测HBV YMDD模序混合感染

目的为HBV感染临床个体化治疗、指导临床用药寻找一种能更好地检测HBV YMDD模序混合感染的方法。方法28份已知乙肝病人血清,其中10份为HBV YMDD模序感染,5份为HBV YIDD模序感染,5份为HBV YVDD模序感染,2份为HBV YMDD和YIDD模序混合感染,2份为HBV YMDD和YVDD模序混合感染,2份为HBV YIDD和YVDD模序混合感染,2份为HBV YMDD、YIDD和YVDD模序混合感染,都用PCR产物直接测序和PCR-流过式反向斑点杂交(Flowthrough-RDB)方法检测。结果HBV YMDD模序单一感染标本用两种方法检测准确性都达到100%,对混合感染标本PCR-流过式反向斑点杂交方法检测准确性也可达100%,然而用PCR产物直接测序方法只能检测其中一种或两种型别。结论PCR-流过式反向斑点杂交方法是一种敏感、特异和快速的检测HBV YMDD野生型及其突变型模序单一感染和混合感染的有效方法,比PCR产物直接测序更优越,更有利于实现临床个体化治疗和指导临床用药。

采用单引物AP-PCR技术对胃癌相关基因片段的分离分析

目的 分离与胃癌发生相关的基因片段并予以鉴定。方法 运用单引物AP -PCR技术 ,对 2 0例配对的胃癌组织和非癌胃组织的基因DNA进行扩增 ,分离出差异片段 ,进行克隆 ,测序和杂交鉴定。结果 分离出三条差异片段 ,其中一条片段 ( 8ndf)基因文库内无同源序列存在 ,斑点杂交显示该片段存在于非癌组织中而癌组织中缺如。结论 单引物AP -PCR技术对于分离差异基因行之有效 ;