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.

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.

应用显微切割-cDNA PCR-SSH法克隆胃癌前病变相关基因

目的构建胃异型增生组织cDNA消减文库,初步筛选差异表达基因。方法手工显微切割取胃异型增生和正常组织,应用cDNAPCR方法对少量组织全转录组扩增后进行双向抑制性消减杂交(suppressionsubtractivehybridization,SSH),消减后片段与载体连接、克隆、筛选、测序及同源性检索,并用斑点杂交方法验证检测结果。结果正常和异型增生组织互为tester和driver成功构建了2个cDNA消减文库,分别代表在异型增生组织中表达上调和下调基因,测序的26个克隆中15个为已知基因,3个为已知EST,8个为新的EST,GenBank登录号为BQ164614～BQ164616,BQ291516～BQ291520,其中15个片段经证实在胃异型增生阶段有显著表达差异。结论利用本室创建的显微切割-cDNAPCR-SSH法成功构建胃异型增生组织cDNA消减文库,初步筛选部分基因,为寻找胃癌发生相关基因提供重要线索。

Optimization of Multiplex PCR Systems for Gene-chip Detection of Mutations in Exons in cTnI Gene Associated with FHCM

Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (FHCM).A microarray was fabricated to screen mutations in exons 3,5,7,and 8 in cTnI gene.Primers were designed for the PCR (polymerase chain reaction) to amplify the target DNA fragments from fresh blood samples.In order to simplify the PCR process,multiplex PCR technology was investigated in detail.The concentration of Mg^(2+) played an important role in multiplex PCR process,a properly low concentration of Mg^(2+) submitted a better speciality of PCR products.The speciality was also favored when the annealing temperature was reasonably enhanced and 64℃is the optimal annealing temperature for the multiplex PCR systems.When applying the fabricated gene-chip to detect the target fragments from PCR mixture,the signal intensity sequence is in accordance with that from theoretic estimate.

Studies on in situ PCR Detected by the BrdU Antibody Technique

Using the BrdU antibody technique followed by an immuno-chemical staining(BAT),the amplification o f DNA fragments specific to human Y chromosome on cell specimen slides was efficiently detected. Whether direct BrdU incorporation into PCR products or in situ hybridization with PCR products on slides, the amplified targetDNA fragments of specimen were visualized by BAT under the microscope. The availability of BAT and differencesin the sensitivity and efficiency between BAT and dig--if-dUTP labeling in cell in situ PCR were disCussed.

Real-time PCR技术在弧菌DNA-DNA同源性测定中的应用

目前在微生物学领域,研究各级分类单元的最有效手段是多相分类,该法能较客观地反映生物间自然系统进化的关系。DNA-DNA同源性分析在多相分类中扮演重要的角色,常用于亲缘关系密切的微生物种内相似度描述。它在检测错分菌株以及新描述的菌株划归已有分类单元时非常有用。DNA-DNA同源性分析最常用的检测手段是点杂交,杂交值可反映出两基因组间序列的相似度。已有研究证明,基因组碱基对每错配1%,

Real-Time PCR和组织原位杂交法检测enJSRV在绵羊胎儿免疫器官及肺脏中的表达

本研究旨在揭示感染JSRV病羊体内检测不到循环抗体的免疫学机理。用地高辛(DIG)标记制备enJS-RV-env探针,原位杂交法检测妊娠70 d的绵羊胎儿(免疫器官初步形成期)、妊娠130 d胎儿(即将出生的)和出生7日龄羔羊的胸腺、脾脏、肠淋巴结和肺脏内enJSRVmRNA表达情况。并利用Real-Ti me PCR法,对enJSRVmRNA在以上组织中的表达进行定量分析。结果表明,在所检测组织内均有阳性信号出现,而阴性对照组均没有阳性信号;enJSRVmRNA在胎儿和初生羔羊各免疫器官高水平表达,特别是在妊娠130 d胎儿和初生7日龄羔羊的胸腺和脾脏中表达水平比较高,而在各时期的肺脏组织中都检测到低水平表达。本试验结果为机体对exJSRV的感染产生免疫耐受这一假说提供了有力的证据。

RAPID SCREENING OF AN ARRAYED cDNA LIBRARY BY IMPROVED PCR-BASED METHOD

The present study reports an improved PCR-based technique that allows quick and effective screening of cDNA libraries. First, the cDNA library was arrayed as follow: about 3 X 10’ cDNA clones were multiplied as individual plaques on solid medium in 24-well culture dishes at 1 200 plaque forming units per well. The phage suspension of each well was transferred to an individual microcentrifuge tube in 72-tube box. Then, box pool, row pools and column pools were set up that respectively represent a 72-tube box, rows and columns within the box. To screen a specific target cDNA,primers specific for novel ESTs ob- tained in our laboratory were employed to conduct PCR in a hierarchy mode. PCR began with the box pools, resulting in the identification of some Positive box pools. Then PCR went down to the row and col- umn pools of the positive box. The intersection of the positive row (s) and column (s) revealed the candi- date positive tubes. The specificity of PCR products were meanwhile checked by restriction enzyme diges- tion. Finally, hybridization was carried out to get single specific cDNA clomes from the positive tubes. This PCR-based technique features high specificity, high efficiency and less-cost in large-scale cDNA library screening. Our initial implementation of the technique resulted in the isolation of three longer different cD- NA clones from a human fetal brain cDNA library. Thus this improved technique can serve as an alterna-tive to the time-consuming and laborious conventional hybridization-based method for screening cDNA li-brary.

Identification of Genetic Relationship of Hybrid Rice Varieties to Their Parents with PCR Products

[Objectives]To establish a simple,rapid and accurate method for identifying the genetic relationship of hybrid rice varieties to their parents.[Methods]Taking F1 hybrids Liangyou 336,Deliangyou Huazhan,and the parents of Liangyou 336,i.e.,C815S(♀)and R336(♂),as experimental materials,the genetic relationship of the hybrid rice varieties to the parental materials was identified by way of PCR amplification with the 48 pairs of SSR primers of Protocol for Identification of Rice Varieties:SSR Marker Method(NY/T 1433-2014).[Results]The genetic relationship of the hybrid rice varieties could be determined by comparing the PCR amplification products of the mixed DNA of the parents and the DNA of the F1 hybrids.[Conclusions]This method not only reduced the number of samples required but also had a good visual effect and high accuracy.

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 and identification of Vibrio parahaemolyticus by multiplex PCR and DNA-DNA hybridization on a microarray

In this paper, we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains, using multiplex PCR and DNA--DNA hybridization. Multiplex PCR was used to simultaneously amplify three diagnostic genes （tlh, tdh andfla） that serve as molecular markers of V. parahaemolyticus. Biotinylated PCR products were hybridized to primers immobilized on a microarray, and detected by chemiluminesce with avidin-conjugated alkaline phosphatase. With this method, forty-five samples were tested. Eight known virulent strains （tlh＋/tdh＋/fla＋） and four known avirulent strains （tlh＋/tdh /fla＋） of the V. parahaemolyticus were successfully detected, and no non-specific hybridization and cross-hybridization reaction were found from fifteen closely-related strains （tlh-/tdh-/fla＋） of the Vibrio spp. In addition, all the other eighteen strains of non-Vibrio bacteria （tlh-/tdh /fla-） gave negative results. The DNA microarray successfully distinguished V. parahaemolyticus from other Vibrio spp. The results demonstrated that this was an efficient and robust method for identifying virulent strains of V. parahaemolyticus.

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。

应用RT-PCR和dot-ELISA方法检测单头灰飞虱体内水稻条纹病毒

本文比较了RT-PCR和dot-ELISA两种方法对单头灰飞虱携带水稻条纹病毒（Rice stripe virus,RSV）的检出率、检测灵敏度和检测成本。结果表明,两种方法均能检测到单头灰飞虱体内的RSV,RT-PCR检测单头灰飞虱体内RSV的阳性检出率比dot-ELISA方法低11.79%-15.77%,但RT-PCR的检测灵敏度比dot-ELISA高10倍,RT-PCR技术的检测成本比dot-ELISA的高。结果暗示,对于单头介体昆虫中的病毒检测,RT-PCR技术的检出率不一定比dot-ELISA的高。综合考虑后,如对室外大批量灰飞虱进行带毒率检测时可采用dot-ELISA方法,如需准确分析单头灰飞虱体内RSV时可采用RT-PCR方法。

Non-radioactive Hybridization in Microwells Using Enzym eLinked Im m uneSorbentAssay forDetection ofRT-PCR-Am -plified CK19-and CEA-m RNA

PCR enzyme linked immune sorbent assay (ELISA) was developed for detection of RT PCR amplified cytokeratin 19 (CK19) mRNA and carcinoembryonic antigen (CEA) mRNA. The non radioactive hybridization was performed in a streptavidin coated microwell with digoxigenin labeled PCR products and with biotin labeled capture probe. PCR ELISA was proved to be expedient, simple, sensitive and safe for identification of CK19 , CEA RT PCR products. These results were proven by sequencing.

Detection of HBV DNA in Extrahepatic Tissue with PCR Comparison with Immunohistochemistry and in Situ Hybridization

Formalin-fixed paraffin-embedded tissue,including gallbladder,kidney,spleen,adrenal gland,heart,testicle,pancreas,and liver from 18 autopsied cases with HBV infection were studied with nested polymerase chain reaction

Dot-ELISA和RT-PCR检测南方水稻黑条矮缩病毒灵敏度的比较

南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)是近年来在我国南方稻区和越南危害严重的一种新病毒,主要由白背飞虱传播,造成水稻大面积减产。检测田间白背飞虱、杂草和水稻植株的带毒率是南方水稻黑条矮缩病预测预报的基础。为寻找一种适合大量样本的检测方法,以感染南方水稻黑条矮缩病毒的水稻植株和带毒的白背飞虱为材料,比较了斑点免疫测定法(Dot-ELISA)和反转录-聚合酶链式反应(RT-PCR)两种检测方法的灵敏度。试验结果表明:RT-PCR技术的检测灵敏度比Dot-ELISA技术检测灵敏度高。

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.

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.

Detection of mutation in embB gene of Mycobacterium tuberculosis from clinical isolates of tuberculous patients in China by means of reverse-dot blot hybridization

The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol (EMB) resistance of the clinical isolates of tuberculous patients in China was investigated by reverse dot blot hybridization (RDBH) in addition to evaluating the clinical value with application of PCR-RDBH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates (65.9%) showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation (38/91, 41.8%) and the ATA mutation (16/91, 17.6%) were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing for one wide-type probe and 5 probes for specific mutations was 100% . It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.