The mutation characteristics of drug-resistance of Mycobacterium tuberculosis in Qinzhou,Guangxi / 国际检验医学杂志

Objective To analyze the variation characteristics of rpoB,katG,inhA,rpsL and embB related genes of Mycobacterium tuberculosis(MTB)in Qinzhou,Guangxi.Methods PCR reverse point hybridization was used to detect 5 common resistance mutants of Mycobacterium tuberculosis in 237 MTB-DNA positive sputum samples.Results Among 237 cases of tuberculosis patients,72 cases(30.38%)were resistant to the four kinds of anti-TB drugs.The resistance mutation rate of rifampin,isoniazid and streptomycin was 2.53%, 13.92%,3.80%.The top 5 gene mutation detection loci of Mycobacterium tuberculosis were-15M,S531L and 43M.Conclusion The main drug-resistant strains are isoniazid resistance,and the mutation of inhA gene were the major one in Qinzhou,Guangxi.

Application of PCR－reverse dot blot hybridization gene membrane chip technology in genetic testing of children with hereditary nonsyndrome deafness / 中华实用儿科临床杂志

Objective To explore the application value of PCR－reverse dot blot hybridization (PCR－RDB) gene membrane chip technique in genetic diagnosis of hereditary non－syndrome deafness in children.Methods The blood samples(2 mL)were collected from 38 children with congenital deafness,excluding high－risk factors for deaf-ness at Dongguan Rehabilitation School,and then genomic DNA extracted.By using self－designed multiplex－PCR combined with PCR－RDB gene chip technology,20 hot－spot mutations of 4 pathogenic genes of common deafness in Chinese population were detected.Sanger sequencing was used as the gold standard to corroborate the positive samples. Results Among 38 subjects,deafness gene mutations were detected in 16 cases,with a detection rate of 42.11%,and they were all verified by family study.Among 16 cases,6 cases of GJB2 gene mutation(3 cases of homozygote,3 cases of heterozygous),4 cases of SLC26A4 mutation,2 cases of MTRNR (m.1555A＞G)mutation,4 cases of compound muta-tion,but none of GJB3 gene mutations.And their detection rates were 15.79%,10.53%,5.26%,10.53%,and 0,re-spectively.DNA samples from 16 children with deafness gene mutation were corroborated by Sanger sequencing,and the compliance rate was 100%.Conclusions For 20 hot－spot mutations of 4 common deafness pathogenic genes,the matc-hing PCR－RDB gene membroine chip technology was designed and the susceptible gene of congenital deafness children was detected.This technique has some advantages like high detection rate,fast,accurate and economical.It is an ideal method for gene screening on hereditary non－syndrome deafness children and has good clinical application prospects.

Analytical research on HPV infection among women in Xingjiang Kashgar area / 国际检验医学杂志

Objective To investigate the exfoliative cell HPV infection situation,genotype distibution and charactristics among female population in Kashgar area.Methods The cervical exfoliated cells specimens were collected from 1548 women,and 23 genotypes of HPV were detected by using the PCR reverse dot blot hybridization method.The HPV infection situation and its genotypes were analyzed.Results The HPV infection positive rate was 33.33% (485/1 548),in which the HPV-16 infection rate was 10.3%;HPV52 infection rate was 9.9%;HPV58 infection rate was 7.6%,HPV53 infection rate was 7.2%;the single subtype infection rate was was 66.8%,the multiple infection rate was 33.2%;the HPV infection rate was highest(43.3%) in female population aged 41-50 years old,which was extremely higher than that in other age groups(P<0.05).Conclusion The HPV infection among female population of Kashgar area is dominated by HPV16,the HPV infection rate is highest in female population aged aged 41-50 years old,which provides a theoretical basis for the HPV prevention and control work of related departments in Kashgar area.

Application of PCR-reverse dot blot hybridization in the diagnosis of vulvovaginal candidiasis and the detection of Candida albicans drug-resistant genes / 临床检验杂志

Objective To evaluate the application value of PCR-reverse dot blot hybridization in the identification of Candida and the detection of Candida albicans drug-resistant genes.Methods The vaginal secretion samples from 285 patients with candidal vaginitis and 50 healthy women were collected.The identification of Candida species and their drug susceptibility were detected by the bioMérieux Yeast identification cards and MIC method(Zhengzhou Antu kit),respectively.The identification of Candida species and the mutation of Candida albicans,drug-resistant genes were also detected by the Shenzheng Yaneng test kit(PCR-reverse dot blot hybridization).The drug-resistant genes were also identified by PCR and nucleic acid sequencing.Based on the culture identification,MIC method and nucleic acid sequencing as the contrast methods,the sensitivity,specificity and accuracy of PCR-reverse dot blot hybridization in the identification of Candida species and the mutation detection of Candida albicans drug-resistant genes were evaluated.Results Compared with the bioMérieux Yeast identification method,the sensitivity,specificity,positive predictive value,negative predictive value and total coincidence rate of PCR-reverse dot blot hybridization for detecting six kinds of Candida species,including Candida albicans,Candida glabrata,Candida tropicalis,Candida parapsilosis,Candida krusei and Candida guilliermondii,were above 95％,96％,96％,98％ and 97％,respectively.There was no significant difference in detecting six kinds of Candida species between the two methods (x2 =0.44,0,0,0,0 and 0,respectively,P ＞ 0.05),and there was good consistency between them (Kappa ＞ 0.9).Compared with the MIC method,the sensitivity,specificity,positive predictive value,negative predictive value and total coincidence rate of PCR-reverse dot blot hybridization for detecting the drug resistance of Candida albicans were 98％,88％,98％,88％ and 96％,respectively.There was no significant difference in detecting the drug resistance of Candida albicans between the two methods (x2 =0.17,P ＞ 0.05),and there was good consistency between them (Kappa ＞ 0.8).The results of PCR-reverse dot blot hybridization in detecting the mutation sites of six kinds of Candida albicans drug-resistant genes were 100％ of coincidence with that of the nucleic acid sequencing method.Conclusion The PCR-reverse dot blot hybridization has high consistency with the culture method and the nucleic acid sequencing method in the identification of Candida species and the mutation detection of Candida albicans drug-resistant genes,which is more early and rapid than the traditional detection methods,and may be applied to the auxiliary diagnosis of vulvovaginal candidiasis (VVC).

Analysis of HPV subtype infection in 4 037 healthy women in Haikou area / 中华微生物学和免疫学杂志

Objective To investigate the infection of 23 kinds of human papillomavirus ( HPV) subtypes in female cervical epithelium samples and to analyze their relationships with age and results of cyto-logic test in Haikou area. Methods A total of 4 037 local healthy women were enrolled in this study from July 2013 to August 2016, 1 967 of whom received cervical cytology test. Cervical cell samples collected from those women were detected for HPV typing by using PCR-reverse dot blot hybridization. Results (1) The total positive rate of HPV in 4 037 samples was 22. 15% (894 cases), and the detection rates of carci-nogenic, possibly carcinogenic and non-carcinogenic HPV were 16. 13%, 3. 99% and 5. 55% (651, 161 and 224 cases), respectively. The positive rates of 6 genotypes were high, which were HPV52, 53, 81, 51, 16 and 58 in turn. (2) The detection rates of carcinogenic, possibly carcinogenic HPV and some of the gen-otypes (HPV18, 52, 53, 66) increased with age (all P<0. 05). (3) Multiple infection in HPV-positive women accounted for 24. 38% (218/894), in which the infection rates of carcinogenic types declined with age and the numbers of HPV genotypes in carcinogenic infections were negatively correlated with age ( both P<0. 05). (4) Only 2. 49% (49/1 967) of the samples were positive for cervical cytologic test, classified into the ≥ASC-US ( atypical squamous cells of undetermined significance ) group. The detection rates of eight kinds of carcinogenic and two kinds of possibly carcinogenic HPV in≥ASC-US group were significantly higher than those in the negative (no intraepithelial lesion or malignant lesion, NILM) group (all P<0. 05). Conclusion This study indicates that Haikou women have higher rates of HPV infection, especially the eld-erly group. HPV subtype infections present some regional characteristics and are mainly single type infec-tion. Cervical cancer screening should be combined with tests for HPV and cytology analysis to improve its effectiveness.

Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.</p>

Survey and research of common thalassemia genotypes in northeast area of Chongqing / 国际检验医学杂志

Objective To understand the gene carrying rate ,gene mutation types and distribution characteristics of thalassemia in the northeast area of Chongqing .Methods 28 633 specimens collected from the patients and individuals with physical examina‐tion in our hospital from January to December 2013 were performed the RBC parameters detection and hemoglobin electrophoresis screening .The specimens with phenotype positive were definitely verified the thalassemia type by using Gap‐PCR and reverse dot blot(RDB) .Results Among 28 633 specimens ,1 358 specimens were finally diagnosed as thalassemia with the thalassemia carrying rate of 4 .74% ,including 589 cases(2 .06% ) of α‐thalassemia and 741 cases (2 .59% ) of β‐thalassemia cases .Among the α‐thalasse‐mia genotypes ,‐αα/‐‐SEA genotype(1 .38% ) was most common ,the next was ‐αα/‐α3 .7 genotype (0 .37% ) and αα/‐α4 .2 genotype (0 .20% ) .Among the β‐thalassemia genotypes ,CD41‐42 genotype (1 .27% ) had the highest constituent ratio ,followed by IVS‐2‐654 genotype(1 .27% ) and CD17 genotype(0 .30% ) .28 cases were found to be the double heterozygote with α‐thalassemia and β‐thalassemia .Conclusion The northeast area of Chongqing is a region with the high incidence rate of thalassemia and complicated heredity .Therefore this research provides the reference information for the prevention of thalassemia ,genetic counseling and prena‐tal diagnosis .

Comparison of real time PCR and PCRˉreverse dot blot hybridization for detection of Human papillomavirus / 国际检验医学杂志

Objective To compare real time PCR with PCR-reverse dot blot hybridization (PCR-RDB)for detecting human pap-illomavirus (HPV)infection in women.Methods A total of 109 genital specimens from women were collected in the study.All specimens were tested HPV by using real time PCR and PCR-RDB,discrepant samples were tested again by PCR-xMAP.Results The concordant rate was 83.5%(91/109)between real time PCR and PCR-RDB (kappa=0.671),the other 18 discrepant samples were retested by PCR-xMAP,7 of those were identical with real time PCR and 11 with PCR-RDB.No differences of PCR-RDB pos-itive rates were found between the high and low viral load groups (χ2 =1.476,P =0.224).Conclusion It demonstrated moderate consistency between real time PCR and PCR-RDB.The HPV positive rates of PCR-RDB were stable when the viral loads were 103-108 .

Analysis of women′s genital human papillomavirus infection and genotype distribution in Shenzhen / 国际检验医学杂志

Objective To investigate the female human papillomavirus(HPV)infection situation in Baoan district,the HPV pos-itive rates in different age groups and the subtypes distribution.Methods PCR followed with reverse dot blot was performed to ex-amine 23 kinds of HPV genotypes in 2 627 female patients in our hospital from the January 2011 to December 2012.Results In 2 627 samples,the positive rate of HPV was 23.94% (629 cases),in which the infection rate of single low-risk type was 15.1%(95 cases),the main HPV genotype was HPV43 (7.79%);the infection rate of high-risk type was 55.17% (347 cases),the 3 most prevalent HPV genotypes were HPV52 (12.56%),HPV16 (9.86%)and HPV58 (7.79%).The multiple HPV infection ac-counted for 29.73% (187 cases).The HPV infection rates in different age groups were 50.0% in age 15-20 years,24.7% in age 21-30 years,20.8% in age 31-40 years,25.8% in age 41 -50 years,42.1% in age >50 years respectively,the differences had statistical significance.Conclusion The HPV infection rate is 23.94% in Bao′an district.The most prevalent HPV genotypes are HPV 52,16,58,43.Women in age 15-20 years old have a higher infection rate.

Analysis of β-thalassemia mutations in Fujian province / 中国地方病学杂志

ObjectiveTo explore the prevalence and spectrum of β-thalassemia mutations in Fujian province,and to provide a reference for prenatal diagnosis and genetic counseling in this population.Methods Two thousand three hundred and one blood samples were randomly selected from 9 different areas of Fujian province from May 2008 to December 2010.PCR and reverse dot blot hybridization (RDB) were adopted for detection of the 17 common types of mutation,and the frequency of each genotype of β-thalassemia mutations was calculated.The β-globin gene of unknown positive samples were analyzed directly with DNA sequencing.Results Three hundred and fifty-nine cases were detected with β-thalassemia mutations of the 2301 copy blood samples submitted,and the detection rate was 15.60％ (359/2301).Of the mutated genes,12 different mutations were identified,namely IVS-2-654(C→T),CD41-42(-TCTT),CD17(A→T),-28(A→G),CD27-28(+C),CD26(G→A),CD71-72(+A),IVS-1-1(G→T),CD43(G→T),-29(A→G),initiation codon ATG→AGG and CD36(-C).Mutation frequencies were 46.54％ (175/376),33.24％ (125/376),9.31％ (35/376),5.05％ (19/376),2.13％(8/376),1.33％(5/376),0.80％(3/376),0.27％(1/376),0.27％(1/376),0.27％(1/376),0.53％(2/376),and 0.27％(1/376),respectively.The most common mutations were IVS-2-654 (C→T) and CD41-42 (-TCTT),which accounted for 79.78％(300/376) of total genetic mutations.In addition,a novel β-globin gene mutation CD36 (-C) allele was detected for the first time,the deletion of a nucleotide C at code 36 within exon 2 lead to a frameshift mutation that could result in a premature termination at code 60.Conclusions β-thalassemia mutations in Fujian province are complex with significant genetic heterogeneity.We present for the first time the detection of a new β-thalassemia mutation in the population:CD36(-C),which provides valuable information for genetic counseling and prenatal diagnosis in Fujian province.

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3 percent and 98.1 percent, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6 percent and 100 percent, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100 percent consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

Analysis of β-thalassemia mutations in Guangdong province / 中国地方病学杂志

Objective To investigate the prevalence and spectrum of β-thalassemia mutations in C, uangdong province, and provide a reference for prenatal diagnosis and genetic counseling in this population. Methods Three thousand two hundred and forty-seven blood samples were randomly selected from Guangzhou and 2984 blood samples from Shenzhen from January in 2005 to January in 2009. PCR and reverse dot blot hybridization (RDB) were adopted for detection of β-thalassemia mutations in Guangzhou and Shenzhen city. Results Seven hundred and fifty-one individuals in Guangzhou were found to have β-hemoglobin gene mutations, the detection rate was 23.13%(751/3247); 10 different mutations were identified, namely CD41-42(-TCTT), IVS-Ⅱ-654(C→T), -28(A→G), CDI7(A→T), CD71-72(+A), 13E, IVS-I-1(G→T), CD43(G→T), -29(A→G), CDI4-15(+G), which accounted for 42.53% (336/790) ,25.19% (199/790), 12.66% (100/790), 10.89% (86/790) ,3.29% (26/790), 2.15%(17/790), 1.27%( 10/790), 1.14%(9/790) ,0.51%(4/790) ,0.38%(3/790), respectively; the most common mutation was CD41-42(-TCTT), which accounted for 42.53%(336/790). In Shenzhen, 179 individuals were found to have β-thalassemia mutations, the detection rate was 6.00% (179/2984); 8 different mutations were identified excluding CD43 (G→T) and CD14-15 (+G); the most common mutation, however, was IVS-lI--654(C→T), which accounted for 40.44% (74/183). Conclusions The β-thalassemia mutations in Guangdong province are not only frequent, but also obviously heterogeneous, and the mutations differ from region to region. CD41-42 (-TCTT),ⅣS-Ⅱ-654(C→T), -28(A→G), CD17(A→T) were the 4 predominant mutations.

Application and Evaluation of Reverse Dot Blot (RDB) Hybridization Assay for HBV YMDD Motif Mutants / 中山大学学报(医学科学版)

[Objective] To detect HBV YMDD motif mutants using RDB hybridization assay in lamivudine treated patients with chronic hepatitis B virus infection,as well as to evaluate the detection capability for clinical application.[Method] HBV DNA was extracted from serum for a total of 242 cases,after the PCR amplification,the hybridization was performed.By comparing the RDB assay results to sequence analysis,the concordant results were analyzed.The sensitivity and detection capability for mixed infection samples are also evaluated.[Results] There are 236 of concordant results for RDB assay and sequencing were obtained in a total of 242 cases,accounting for 97.5%.For all of the cases,there are 58 cases with coexisting mutant viruses in wild type viruses,accounting for 24%.The sensitivity of RDB hybridization assay for HBV YMDD motif mutants was 103 IU/mL,and approximately 10% mutant type strains can be detected from a mixed infection sample.[Conclusion] The RDB hybridization assay for HBV YMDD motif mutants is a simple,accurate,and economic method and it may be a promising tool for clinical application.

Rapid Detection of Mycobacterium Tuberculosis Streptomycin-resistant Genes by PCR-reverse Dot Blot Hybridization / 中国医师杂志

Objective To study the rapid detection of mycobacterium tuberculosis resistance to streptomycin by reverse dot blot hybridization technique. Methods The oligonucleotide probes of streptomycin-resistant genes (rpsL and rrs) were prepared and dropped on nitrocellulose membrane. The target DNA fragments of M. tuberculosis clinical isolates were labeled with biotin by PCR amplification, and then hybridized with the oligonucleotide probes on the membranes. PCR-SSCP and PCR-direct sequencing (PCR-DS) techniques were used to detect the target fragment of M.tuberculosis as control. Results In 53 M. tuberculosis clinical isolates, the consistent rate of three detection methods was 100%. Both the SSCP mapping of rpsL and rrs genes and the results of membrane hybridization in 9 drug-sensitive strains were identical to those in M. tuberculosis standard strain H37Rv. Of 44 streptomycin-resistant strains, 33 strains had AAG→AGG mutation at the codon 43 of rpsL gene, 6 strains had A→C mutation at the 513 site of rrs gene, 1 strain had A→T mutation at the 513 site of rrs gene, and the detection rate of the target genes mutation was 90 9%. In 53 M.tuberculosis clinical isolates, 40 resistant strains and 9 sensitive strains to streptomycin could be detected using dot blot hybridization and the consistent rate with the in vitro susceptibility test was 92 6%(49/53). Conclusion The reverse dot blot hybridization technique showed high sensitivity and specificity to detect Mycobacterium tuberculosis resistance to streptomycin. It possessed the simple and rapid characteristics, and could be used to detecte streptomycin-resistant M.tuberculosis clinical strains.

Human Papillomavirus Typing by Polymerase Chain Reaction-Reverse Dot Blot Hybridization and Comparison with Hybrid Capture SystemTM / 대한임상병리학회지

BACKGROUND: Infection with certain Human Papillomavirus (HPV) type is strongly associated with the development of dysplasia and cancer of the cervix uteri. About 70 HPV types have been identified and some 25 of these have been found in the genital tract. HPV typing has diagnostic and prognostic importance to discriminate between 'low', 'intermediate', 'high' risk types. A Polymerase Chain Reaction-Reverse Dot Blot Hybridization (PCR-RDBH) method was developed for typing of HPV with consensus biotinylated primer generated PCR products in a single test. We attempted to know the clinical usefulness of PCR-RDBH and also compared PCR-RDBH with Hybrid captureTM system (HCS) method in same specimens. METHODS: HPV typing was performed on cervical swab samples obtained from 100 women with abnormal cervical cytology: 37 with Atypical Squamous Cells of Undetermined Significance (ASCUS), 14 with Low Grade Squamous Intraepithelial Lesions (LGSIL), 44 with High Grade Squamous Intraepithelial Lesions (HGSIL) and 5 with invasive carcinoma of the uterine cervix. HPV PCR screening was tested with consensus biotinylated primer. If HPV PCR screening was positive, RDBH was done for the typing of HPV. In RDBH, biotinylated PCR product was used in hybridization with a membrane on which 12 different oligonucleotide probes (type 6/11/16/18/ 31/33/35/39/45/51/52/56) of genital HPV types had been immobilized. Hybrid captureTM system (HCS, Digene Diagnostics) was used for screening of HPV. RESULTS: Of 100 abnormal cervical cytology specimens, the positivity of HPV PCR screening was 67%. In 52 specimens, HPV could be typed by RDBH. Type 16 was the most frequent and mixed infection was found in 6 cases, all combined with type 16. Among the 13 cervical cancer specimens confirmed by biopsy, 12 specimens was found to be infected high and intermediate risk types of HPV. In cervical swab, there was signifincant discrepancy in positivity of HPV infection between PCR-RDBH and HCS method. In 51 cases, negative for PCR-RDBH, 16 cases (31%) were positive by HCS. In 49 cases, positive for PCR-RDBH, 15 cases (31%) were negative by HCS. CONCLUSIONS: PCR-RDBH method can do HPV typing fast and easy with non-radioactive biotinylated primer in cervical swab specimens. It is shown to be useful method for HPV typing and have a high clinical applicability. The results between PCR-RDBH and HCS methods show a significant discrepancy, so further investigation is needed.

Detection of rpoB mutations in rifampin resistant Mycobacterium tuberculosis strains by the reverse dot blot hybridization method / 中华传染病杂志

Objective To develop new method for rapid detection of mutations in rpoB gene related with resistance to rifampin of M. tuberculosis . Methods According to the sequence of wild type M. tuberculosis, five oligonucleotide probes covering the 69 bp hyper variable region of rpoB gene were designed and immobilized on nylon membrane strips. Thereafter, the target rpoB gene fragment was obtained by PCR using biotin labeled primers and thereafter the PCR product was denatured and hybridized with probes on membrane. The results of reverse dot blot hybridization were compared with the results of drug sensitivity test and sequencing. Results PCR products from 36 RFP resistant and 22 RFP susceptible isolates were detected by the assay of reverse dot blot hybridization, showing that the susceptibility and the specificity rate were 88.9% and 86.4% respectively,and the coincidence with biochemical method and sequencing is 87.9% and 89.7% respectively. Conclusions Reverse dot blot hybridization is a rapid and sensitive method to detect the rpoB gene mutations,which may be used in early detection of the resistance of M. tuberculosis to rifampin.