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.

A Simple Method for Detection of Multiple Chemical-Specific IgGs in Serum Based on Dot Blotting

Plastic resins are known to cause occupational allergies. Therefore, serum-specific antibodies against plastic resins have been widely investigated as diagnostic markers for occupational allergies. In this study, we aimed to establish a convenient method for detection of multiple chemical-specific IgG antibodies in human serum based on dot blot analysis. Toluene diisocyanate (TDI), phthalic anhydride (PA), and formaldehyde (FA), which are frequently used to synthesize various resins, reacted well with lysine residues of human serum albumin (HSA) under alkaline conditions. Native polyacrylamide gel electrophoresis (PAGE) showed that the structures of chemical adducts of HSA were different from those of native HSA. Therefore, we performed dot blot assays using these adducts as artificial antigens. Serum samples from workers at plants utilizing plastic resins strongly reacted with TDI, PA, and FA adducts in HSA, while reduced signals were detecting using the serum from unexposed workers. These results suggested that dot blot assays using chemical-HSA adducts as antigens could be beneficial for simultaneously measuring multiple chemical-specific IgGs.

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.

番茄斑萎病毒外壳蛋白原核表达及Dot-blotELISA检测方法的建立

将番茄斑萎病毒(Tomato spotted wilt virus,TSWV)全长外壳蛋白基因亚克隆到原核表达载体pET-32a中,经限制性酶切分析及测序结果表明插入方向正确且阅读框架无移码突变.将重组表达质粒转化大肠杆菌BL21(DE3),SDS-PAGE分析结果证实IPTG可诱导1个分子量约为48 kDa的融合蛋白表达.利用6×His标签单抗和TSWV FoPaTs1多抗证实所表达的蛋白为TSWV外壳蛋白.以纯化的重组外壳蛋白免疫小鼠,制备杂交瘤细胞培养上清单抗,并用杂交瘤细胞培养液上清建立了可靠、有效检测TSWV的Dot-blot ELISA方法.

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。

PCR及Dot-blot法对先天感染DHBV筛选的比较

目的比较PCR及Dot-blot法对先天感染DHBV筛选的效果。方法分别利用PCR法及Dot-blot法对先天感染DHBV筛选,并进行比较。结果与Dot-blot法比较,PCR法有简单、快捷、敏感度高等优点,且可避免Dot-blot带来的放射性污染。但PCR法所需费用高,易因交叉污染而导致假阳性结果。结论PCR与Dot-blot法筛选先天感染鸭乙肝的方面各有其优缺点。由于PCR法更为敏感、快捷,很有必要对其加以改进和完善。

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.

Dot-blot对转DAF基因猪外源基因整合的检测

衰变加速因子(DAF) 是补体激活途径中的一个重要膜调节蛋白, 转人DAF 基因猪可以作为人器官移植的供体。采用斑点杂交试验(Dotblot) , 对经显微注射导入了hDAF 质粒片段、用聚合酶链反应(PCR) 鉴定的阳性猪所产仔猪32 头进行了整合检测, 获得转基因阳性仔猪14 头。说明了Dotblot 可作为转基因动物外源基因整合检测的一种手段。

Reverse Dot Blot Analysis: A Rapid Prenatal Diagnostic Approach for β-thalassemia Mutations in Chinese

The recently developed method of in vitro DNA amplification by PCR coupled with radioactive or nonradioactive ASO probe detection provides a simple approach to the prenatal diagnosis of β-thalassemia. However, the DNA diagnosis of β-thalassemia has remained a complicated problem,because β-thalassemia

一种基于手动芯片点样仪的快捷高效的转基因植物PCR-dot-blot鉴定法

本论文报道一种快捷高效的PCR-dot-blot新方法.该方法利用手动芯片点样仪将微量PCR产物(约100 pg DNA)点样,并采用North2 South Biotin Random Prime DNA Labeling and Detection Kit(PIERCE)完成快速斑点杂交.通过鉴定转基因烟草证明这种方法是可行而有效的.这种新方法节省材料,效率极高,特别适合大批量鉴定转基因植物.

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.

高灵敏Dot Blot检测转基因小鼠肝细胞内HBV DNA的初步研究

目的考察dot blot检测HBV转基因小鼠肝细胞内HBV DNA含量的可行性。方法用地高辛标记HBV探针,利用pTHBV1047质粒检测杂交系统灵敏度,同时进行标准曲线研究,采用dot blot检测转基因小鼠肝细胞内HBV DNA含量。结果杂交系统的检测灵敏度为0.1pg;pTHBV1047质粒在100~6.25pg之间呈线性关系,R2为0.976~0.999;同一只HBV小鼠肝3次检测误差范围在±140%,5只鼠HBV DNA含量分别为6.48、3.59、7.83、7.96、5.28pg/μg基因组DNA。结论优化后的dot blot系统灵敏度高、特异性强、重复性好,可应用于转基因小鼠肝细胞内HBV DNA检测。

The Use of the PCR-Based Dot-Blot Hybridization Assay to Detect Resistance Markers to Rifampicin and Streptomycin in Mycobacterium tuberculosis Isolates from the SW Region of Cameroon

Drug sensitivity testing to establish resistance to TB drugs takes many months to arrive at. Public health physicians have difficulties with such an approach due to long wait periods and cannot use it to establish community wide prevalence as a way to understand where resistance may be emerging faster and to limit its spread. The objective of this study was to use the dot-blot hybridization technique in the detection of resistance to rifamycin (RIF) and streptomycin (SM) in South- Western Cameroon and to compare the technique with the routine culture and drug susceptibility testing for detecting resistance in a resource poor country, Cameroon. A hospital-based study was conducted at the Regional hospitals of Buea and Limbe and Tiko Central Clinic. Tuberculosis (TB) patients aged 15 to 50 (mean age: 30.50 ± 8.33 standard deviation) were recruited for the study between December 2006 and April 2007. Cultures from 59 patients were tested for rifampicin and streptomycin sensitivity by the modified proportion method and mutational analysis for rpoB codon 516 and rrs codon 513 was performed by the dot-blot hybridization technique. Of the 59 sputum samples collected (36 were males and 23 were females) came from Buea 19 (32.2%), Limbe 20 (33.9%) and Tiko 20 (33.9%) towns respectively. Amplification for the gene showed that there was (59) 100% amplification with primers used for rpoB genes and 43 (72.9%) amplification with primers used for the rrs gene. Mutational analysis demonstrated that resistance to RIF was common in females (52.1%) than males (41.7%) while 6% of the samples were indeterminate. 12 (20.3%) samples showed phenotypic and genotypic resistance to RIF compared to 34 samples (58.1%) for SM. Phenotypic resistance and genotypic susceptibility were found in 5 (8.5%) RIF and 3 (4.7%) SM compared to phenotypic susceptibility and genotypic resistance that were found in 2 (3.5%) RIF and 3(4.7%) SM. Double mutation on rpoB and rrs genes occurred in 8 (13.6%) DNA samples. Resistance to RIF and SM due to mutations on the rpoB and rrs genes respectively in the SW region was found to be high and comparable to the drug susceptibility testing by 92%, (95% CI: 75.7 - 99.1). The Dot-blot technique will be useful in rapidly assessing the effectiveness of national TB control programs in limiting the spread of resistance strains in Cameroon.

免疫斑点印迹法检测水解乳蛋白婴幼儿配方粉免疫反应

目的:建立一种快速且精准的方法,以确定水解乳蛋白婴幼儿配方粉的免疫反应性。方法:首先采用直接免疫斑点印迹法,利用牛奶过敏患儿血清初步评估待测样品的免疫反应性;然后通过间接竞争免疫斑点印迹法以市售普通牛奶粉为阳性对照,评估水解乳蛋白婴幼儿配方粉与血清免疫球蛋白E(IgE)的结合能力;最后采用间接和间接竞争酶联免疫检测法,以佐证免疫斑点印迹结果。结果:部分水解乳蛋白婴配粉仍可以结合IgE以诱发过敏反应,而深度水解乳蛋白婴配粉不具有免疫反应性。免疫斑点印迹和酶联免疫检测评价免疫反应性结果显著且一致。结论:免疫斑点印迹技术操作性强,周期短,成本低,结果可靠,为利用较难获取的婴幼儿过敏患儿血清初步评估乳蛋白产品致敏性提供了新思路。

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。

Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay （dot-ELISA）, indirect ELISA and Westem blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Westem blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5-6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards （＂marker＂） so that the alterations ofprotease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10^4 cell/mL in the indirect ELISA, while 10^5 cell/mL in the dot-ELISA.

某地区女性HPV感染情况及分型分析

目的调查分析山西地区女性HPV的感染情况和常见感染类型,以及不同年龄组中HPV感染的大体情况。从而为该地区宫颈癌的预防、早期诊断和治疗等提供流行病学参考。方法选取送检于太原金域医学检验中心的7030例女性宫颈脱落细胞样本,进行23种HPV基因分型检测。结果7030例样本中共检测出1406例阳性标本,总阳性率为20%。高危型HPV(HR-HPV)共检出1509例(15.06%),低危型HPV(LR-HPV)共检出347例(4.94%)。HR-HPV各亚型检出率从高到低排前五位的分别为HPV52(3.81%)、HPV16(3.29%)、HPV53(2.35%)、HPV58(2.02%)、HPV56(1.59%)和HPV51(1.51%);LR-HPV感染型主要以HPV81(1.35%)、HPV43(1.17%)和HPV42(1.14%)为主。单一亚型的感染率最高为13.90%,双重感染率为3.54%,三重感染率为1.10%,多重感染率为0.50%。HPV检出率最高的是>60~70岁年龄段的女性人群(64.29%),其次为≤20岁的女性人群(54.55%)。结论山西地区女性人群HPV感染率较高,高危型HPV感染占主导位置。HPV感染高发年龄人群应加强宫颈病变的筛查意识,重视HPV的定期复查,以早期发现和预防宫颈癌。

人参皂甙与免疫核糖核酸对癌基因表达的协同抑制作用

应用斑点杂交技术对癌细胞(Lc株)的两种癌基因(c-Ha-ras和cmyc)进行检测,证明这两种基因表达可被人参皂甙和免疫核糖核酸的协同作用所抑制。单独用人参皂甙处理细胞,两种癌基因无明显受抑制现象;单独用免疫核糖核酸作用2 h,然后除去作用因素,对两种癌基因表达亦无明显影响。但用免疫核糖核酸作用2 h,除去作用因素培养24 h之后再加入人参皂甙继续作用一定时间,则对两种基因的表达均产生明显的抑制作用并呈现时间和剂量效应。RNA的Northern blot分析表明转录物的结构在处理前后未发生改变。

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。