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.

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.

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.

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。

DETECTION OF STRAND BREAKS OF DNA IN HUMAN EARLY CHORIONIC VILLUS CELLS INDUCED BY DIAGNOSTIC ULTRASOUND USING ^(32)P-LABELED ALU HYBRIDIZATION

Objective To explore if strand breaks of DNA in human early chorionic villus cells in uterus were induced by diagnostic ultrasound and to evaluate the method used for detection of single-stranded breaks and double-stranded breaks in human DNA. Methods 60 normal pregnant women aged 20-30, who underwent artificial abortion during 6-8 weeks of gestation, were randomly divided into 2 experimental groups: All 30 cases were exposed to diagnostic ultrasound in uterus for 10 minutes, and 24 hours later chorionic villi were extracted; the other 30 cases were taken as the control group. Single-stranded DNA and double-stranded DNA in villus cells in all cases were isolated by the alkaline unwinding combined with hydroxylapatite chromatography, and were quantitatively detected using 32 P-labeled Alu probe for dot-blotting hybridization. Results There was no significant difference in quantity and percentage in single-stranded DNA and double-stranded DNA between 2 groups (P>0.05). 32 P-Alu probe could only hybridize with human DNA, and could detect DNA isolated from as few as 2.5×10 3 chorionic villus cells and 0.45ng DNA in human leukocytes. Conclusion The results suggested that there were no DNA strand damages in human chorionic villus cells when the uterus was exposed to diagnostic ultrasound for 10 minutes. The method,^(32)P-Alu probe for dot-blotting hybridization, was even more specific, sensitive and accurate than conventional approaches.

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法制备地高辛标记DNA探针斑点杂交检测对虾传染性皮下及造血组织坏死病毒(IHHNV)

利用非放射性标记物地高辛(DIG),通过PCR方法制备了对虾传染性皮下及造血组织坏死病毒(IHHNV)DNA探针,探针长度705bp,标记产量为20ng/μL。通过核酸探针斑点杂交检测方法对此探针特异性及灵敏度进行验证,结果表明,该探针具有较高的灵敏度和较强的特异性,检测IHHNVDNA的检出灵敏度为24.8pg,可检出26.6ng患病对虾组织DNA中的IHHNV,与250.4ng健康虾组织DNA、202.5ng健康虾匀浆液,白斑综合症病毒(WSSV)DNA和肝胰腺细小病毒(HPV)DNA均不发生交叉反应。本方法可应用于健康亲虾、苗种的培育和无特定病原(SPF)对虾种群的选育及IHHNV流行病学调查,并具有较高的应用价值。

对虾WSSV人工感染螯虾及其检测

用对虾白斑综合症病毒(WSSV)人工感染淡水克氏原螯虾(Procambarusclarkii) ,感染组螯虾3～6d内全部死亡。核酸探针点杂交两次检测感染螯虾的鳃、胃、血淋巴 ,阳性检出率分别为92% (92% ) ,89 % (86 % ) ,81 % (75 % ) ,对照为11 % (3% ) ,5 % (5 % ) ,0(0) ;光镜下可观察到感染螯虾的胃、鳃组织的靶细胞核肿大、嗜酸性着染 ,电镜下病变组织细胞核可见形态大小与WSSV一致的病毒粒子 ;病毒核酸原位杂交两次检测感染螯虾的胃、鳃 ,阳性检出率均为100 % ,对照阳性检出率均为0。结果表明 :对虾WSSV可感染淡水克氏原螯虾 。

一氧化氮在迟发性神经元坏死中与程序性细胞死亡关系的研究

本文采用分子生物学方法，观察了ＮＯＳｍＲＮＡ在Ｗｉｓｔａｒ大鼠ＤＮＤ模型中的表达，对ＮＯ与ＤＮＤ模型中ＰＣＤ的关系进行了研究。结果发现：ＮＯＳｍＲＮＡ在正常组织中即存在，缺血期可能是表达高峰期。提示：ＮＯ在ＤＮＤ过程中可能是启动ＰＣＤ死亡基因，导致选择性易损神经细胞发生ＰＣＤ的重要外在因素之一。

基因芯片对人乳头瘤病毒的快速检测和分型

目的 针对HPV DNA亚型的异质性,建立HPV DNA亚型的基因芯片快速检测方法,为HPV感染病例提供诊断依据。方法 18 种HPV DNA亚型(HPV6、16、18、31、33、35、39、11、45、51、52、53、56、58、42、59、66、68)特异性探针,固定于硝酸纤维素膜制备成基因芯片,生物素标记引物经通用引物介导PCR(GD PCR)扩增HPV DNA, PCR产物与基因芯片经反向点杂交检测HPV亚型;同时采用荧光定量PCR检测HPV6、11、16 和18亚型。结果 31 例标本中,基因芯片的阳性检出率为74.2%,其中HPV6/18、HPV11/16、HPV33/58 和HPV6/11/33多重感染各1 例(3.2%);荧光定量PCR 阳性检出率为67.7%,与前种方法相比较,漏诊率为6.5%。结论 HPV分型基因芯片可1次检测HPV多种亚型,灵敏度高和特异性强,有利于对HPV多重感染的诊断。

核酸斑点杂交分析法检测对虾皮下及造血组织坏死杆状病毒(HHNBV)

于1995年7月在青岛市城阳对虾养殖场采集患暴发性流行病的中国对虾样品，提纯HHNBV并研制了一组地高辛标记的HHNBV核酸探针。采用核酸斑点杂交方法研究了此组核酸探针的灵敏度和特异性。结果表明，此组探针检测HHNBVDNA的灵敏度为62.5pg，对469ng病虾胃中的病毒可以检出；此组探针与3．75ug健康对虾组织和2ng健康对虾DNA均无交叉反应。1997年，应用此组特异性核酸探针检测了发病虾池对虾及紧急抢捕虾，检测结果显示为强烈的HHNBV阳性，表明此组核酸探针在对虾杆状病毒的检测、对虾暴发性流行病的诊断和抗特种病原对虾育种等方面具有很高的应用价值。

反向斑点杂交法快速检测甘薯羽状斑驳病毒和甘薯G病毒

【目的】以克隆得到的甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)和甘薯G病毒(Sweet potato virus G,SPVG)基因片段为探针建立反向斑点杂交技术体系,应用于甘薯组培脱毒苗带毒情况检测,为生产优质甘薯组培脱毒苗提供保障。【方法】根据已公布的侵染甘薯的SPFMV和SPVG的核苷酸序列设计两对特异引物,以RT-PCR法从染病的甘薯叶片扩增SPFMV和SPVG的两个片段,并以克隆到的两个病毒片段及内参基因Actin片段为探针建立反向斑点杂交体系。【结果】分别克隆出长度约310和500 bp的片段,经BLAST比对,所获得的310 bp片段为SPFMV的外壳蛋白基因片段,同源性为97%,500 bp片段为SPVG的外壳蛋白基因片段,同源性为99%。分别用带有SPFMV和SPVG片段的重组质粒pUC-SPFMV和pUC-SPVG进行反向斑点杂交,发现不同病毒能获得单一信号,无交叉现象。以染病甘薯和脱毒甘薯苗为样品,用该种病毒的探针进行反向斑点杂交,染病植株样品中能获得单一信号,而脱毒苗甘薯样品未见任何信号,杂交结果与RT-PCR检测结果一致。【结论】用建立的反向斑点杂交技术体系能有效检测甘薯中的SPFMV和SPVG,无交叉信号,可用于甘薯组培脱毒苗的前期检测。

用DNA斑点杂交法检测对虾及其饵料和环境生物携带白斑综合症病毒状况的调查

于 1996～ 1998年采用DNA斑点杂交法 ,完成了共计 836份养殖对虾 ,2 13份对虾饵料和环境生物样品的检测。结果显示 ,对虾在每年的 4～ 10月都可检测出白斑综合症病毒 (WSSV) ,6月出现最高峰 ,阳性检出率达到 4 2 .6 % ;其次 ,为 7月和 5月 ,分别达 31.6 %和 2 4 .5 %。在对虾不同生长阶段 (包括亲虾、各期幼体、仔虾、幼虾及成虾 )均可检出WSSV ,其中以仔虾的带毒率最高 ,达 36 .4 % ,其次为糠虾幼体 ,达 34.6 %。体长范围 1.1～ 2 .0cm的仔虾阳性率最高 ,其次体长为 5 .1～ 6 .0cm的幼虾样品。检测的中国对虾样品(6 2 5份 )带毒率为 16 .8% ,日本对虾样品 (2 11份 )带毒率为 4 5 .0 %。对虾主要饵料及环境生物类群包括鱼类、虾类、蟹类、桡足类、端足类、沙蚕、卤虫均可检出WSSV ,其中小型虾类、端足类、桡足类、蟹类样品的WSSV检出率均高于 2 0 %。

PCR和斑点杂交检测鸡传染性贫血病毒

通过聚合酶链反应（ｐｃＲ）扩增鸡传染性贫血病毒（ｃＡＶ）ＤＮＡ特定片段，将此ｐｃＲ产物用Ｄｉｇｏｘｉｇｅｎｉｎ标记后作为探针进行斑点杂交，以此检测ＣＡＶ并作流行病学调查。对从江苏省不同地市随机采集的２月龄左右不同疾病的病鸡ＤＮＡ样品进行检测，在被检的５２个不同鸡群来源的病料中，有３６个鸡群来源的病料ＤＮＡ显示ＣＡＶ阳性（群阳性率为６９．２％）；备组织中ＣＡＶＤＮＡ含量有所差异，依次为胸腺＞脾、骨髓、肝＞肾、法氏囊、脑。用ＰＣＲ检测得到的阳性鸡的组织病料感染ＳＰＦ鸡，在感染后第２４天检查，出现贫血症状。初步调查表明，在江苏一些地区ＣＡＶ感染已相当普遍，但它与发病的关系还有待于进一步研究。

应用膜反向斑点杂交技术快速检测结核分支杆菌耐乙胺丁醇基因型的研究

应用膜反向斑点杂交技术快速检测结核分支杆菌对乙胺丁醇 (EMB)耐药性。设计与合成用于检测结核分支杆菌耐EMB基因embB的寡核苷酸探针 ,点于硝酸纤维素膜上 ,与结核分支杆菌临床分离株生物素标记的聚合酶链反应 (PCR)产物进行反向斑点杂交 ,并与PCR 单链构象多态性 (PCR SSCP)和PCR 直接测序 (PCR DS)结果比较。对 81株结核分支杆菌临床分离株进行分析 ,31株EMB敏感株中 ,2 6株embB基因的SSCP图谱、膜反向斑点杂交结果与标准株 (H37Rv)完全相同 ;其余 5株SSCP图谱出现泳动变位 ,其中 3株E1b杂交阳性 ,PCR DS分析为embB基因 30 6位密码子ATG→GTG突变 ;2株E1d杂交阳性 ,PCR DS分析为embB基因 30 6位密码子ATG→ATA突变。 5 0株耐EMB菌株中 ,2 4株PCR SSCP图谱与标准菌株相同 ,E1杂交阳性 ;2 6株PCR SSCP图谱出现泳动变位 ,其中 18株E1b杂交阳性 ,2株E1c杂交阳性 ,5株E1d杂交阳性 ,1株E1e杂交阳性 ,未发现E1f杂交阳性 ,与PCR SSCP、PCR DS分析结果一致。突变检出率为 5 2 %。膜反向斑点杂交技术可能成为检测部分结核分支杆菌乙胺丁醇耐药基因型简便、快速的方法。

贵州三都水族β-地中海贫血筛查及基因分析

目的 了解贵州省三都水族β-地中海贫血的发病率、基因突变类型及分布特点,进一步从分子水平揭示β-地中海贫血多态性。方法 采用"红细胞休克一管定量法"测定红细胞脆性,乙酸纤维素薄膜电泳分离测定HbA2,一分钟碱变性法测定HbF。对贵州省三都水族自治县1 090例当地水族居民,进行β-地中海贫血血液学筛查。用常规酚-氯仿抽提法提取β-地中海贫血携带者DNA,经PCR-反向点杂交法对β珠蛋白基因进行突变分析。结果 在受检的1 090人中,共检出β-地中海贫血携带者49例,检出率为4.50％,男性26例,女性23例,男女比值为1.1:1。经基因分析该地人群的β-地中海贫血基因突变类型主要为CD41-42(TCTT)移码突变(20例,40.82％)和CD17(A→T)无义突变(20例,40.82％),尚有9例(18.36％)不在中国人常见的16种β-地中海贫血突变范围内,待测序。结论 贵州省三都地区水族人群中β-地中海贫血有较高的发生率,且其基因突变类型有明显的地区差异和显著的民族特点,是我国一个较为特殊的β-地中海贫血分布区域。

刺参腐皮综合征病原灿烂弧菌检测探针的制备及应用

腐皮综合征是近年来养殖刺参发生的严重疾病。发病时,刺参大批死亡,经济损失惨重。以其主要致病原灿烂弧菌(Vibrio splendidus)DNA为模板,PCR扩增出16S-23S核糖体RNA基因间隔区序列,插入pMD19-T,转化大肠杆菌DH5α并测序。根据该间隔区序列,设计引物扩增177 bp的地高辛标记的探针。该探针对灿烂弧菌呈现特异性,而对其它细菌V.fluvialis,V.anguillarum,V.alginolyticus,Aeromonas hydrophila,V.harveyi,V.parahaemolyticus,V.vulnificus均呈阴性。探针对灿烂弧菌DNA的检出限为6.25 pg,具有较高敏感度。用该探针对人工感染以及从青岛、烟台、威海获取的腐皮综合征发病海参和养殖水体进行斑点杂交,灿烂弧菌检出率达100%。结果表明,该探针具有良好的特异性和敏感性,可用于快速检测养殖刺参腐皮综合征的病原灿烂弧菌。用该方法检测刺参腐皮综合征尚属首次,为刺参腐皮综合征的快速诊断和防治提供技术支撑。

尖锐湿疣人乳头瘤病毒的基因分型

目的 检测尖锐湿疣中人乳头瘤病毒 (HPV )的基因型别。方法 用通用引物进行聚合酶链反应 ,扩增尖锐湿疣中HPVDNA ,用生物素标记的特异性基因分型探针 ,对聚合酶链反应扩增后的HPVDNA进行斑点杂交。结果 5 0例尖锐湿疣标本经PCR扩增后 48例HPVDNA阳性 ,阳性标本中HPV 6、11、16、18型的检出率分别为 16.7% ( 8/48)、3 7.5 % ( 18/48)、14 .6% ( 7/48)、4.2 % ( 2 /48) ,多重型别检出率为 2 2 .9% ( 11/48)。结论 HPV11型感染是尖锐湿疣发病的首要原因。

肉鸡群中呼吸道病原体共感染情况调查

【目的】对肉鸡群中常见的呼吸道病原体共感染情况进行调查,为肉鸡呼吸道疾病的防控提供流行病学资料。【方法】从山东省济南、潍坊、青岛等地区采集421份有呼吸道症状的病(死)鸡肺脏及160份临床健康肉鸡的肺脏作为供检样品,用特异性核酸探针技术,对其进行H9N2亚型禽流感病毒(H9N2AIV)、新城疫病毒(NDV)、传染性支气管炎病毒(IBV)、禽偏肺病毒(aMPV)、禽败血性支原体(MG)、鼻气管鸟杆菌(ORT)和大肠杆菌(E.coli)共感染情况的调查。【结果】421份发病肉鸡肺脏中,H9N2AIV、NDV、IBV、aMPV、MG、ORT和E.coli的检出率分别为53.68%,68.41%,33.49%,54.87%,57.25%,9.02%和41.17%;且存在严重的共感染现象,共感染率高达90.26%,单一感染和阴性个体仅占7.36%和2.38%;发病肉鸡群中,7种呼吸道病原体2重、3重、4重、5重、6重、7重感染的检出率分别达19.48%,26.12%,27.32%,11.88%,4.51%和0.95%,以2重、3重和4重感染较为常见。而临床健康肉鸡肺脏中,H9N2AIV、NDV、IBV、aMPV、MG、ORT和E.coli的检出率分别为12.50%,23.13%,5.63%,36.87%,21.88%,0%和16.88%;呈阴性和单一感染的样品分别占25.00%和43.75%;多重感染样品仅占31.25%,且主要为2重感染。7种病原体中,H9N2AIV、NDV、MG和E.coli在肉鸡呼吸道疾病的共感染中起重要作用。【结论】发病肉鸡和临床健康肉鸡均存在混合感染,但以发病鸡群更为严重和复杂;呼吸道病原体多重感染是导致肉鸡呼吸道疾病日益严重的重要原因。

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 。