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.

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.

Mutations in pre-core and basic core promoter regions of hepatitis B virus in chronic hepatitis B patients

AIM: To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related indexes.METHODS: One hundred chronic hepatitis B patients treated at Shanxi Province Hospital of Traditional Chinese Medicine were included in this study. PCR-reverse dot blot hybridization and mismatch amplification mutation assay (MAMA)-PCR were used to detect the mutations in the HBV pre-C and BCP regions. HBV DNA content and liver function were compared between patients with mutant HBV pre-C and BCP loci and those with wild-type loci. The consistency between PCR-reverse dot blot hybridization and MAMA-PCR for detecting mutations in the HBV pre-C and BCP regions was assessed.RESULTS: Of the 100 serum samples detected, 9.38% had single mutations in the pre-C region, 29.17% had single mutations in the BCP region, 41.67% had mutations in both BCP and pre-C regions, and 19.79% had wild-type loci. The rates of BCP and pre-C mutations were 65.7% and 34.3%, respectively, in hepatitis B e antigen (HBeAg) positive patients, and 84.6% and 96.2%, respectively, in HBeAg negative patients. The rate of pre-C mutations was significantly higher in HBeAg negative patients than in HBeAg positive patients (χ2 = 26.62, P = 0.00), but there was no significant difference in the distribution of mutations in the BCP region between HBeAg positive and negative patients (χ2 = 2.43, P = 0.12). The presence of mutations in the pre-C (Wilcoxon W = 1802.5, P = 0.00) and BCP regions (Wilcoxon W = 2906.5, P = 0.00) was more common in patients with low HBV DNA content. Both AST and GGT were significantly higher in patients with mutant pre-C and BCP loci than in those with wild-type loci (P < 0.05). PCR-reverse dot blot hybridization and MAMA-PCR for detection of mutations in the BCP and pre-C regions had good consistency, and the Kappa values obtained were 0.91 and 0.58, respectively.CONCLUSION: HBeAg negative patients tend to have HBV pre-C mutations. However, these mutations do not cause increased DNA copies, but associate with damage of liver function.

Cloning and Sequence Analysis of Genome from the Inner Mongolia Strain of the Endogenous Betaretroviruses (enJSRV)

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia (enJSRV-NM), we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19-T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame (orf-x) that overlaps the 3' end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain (Accession No. AF153615) and USA JSRV21 strain (Accession No. AF105220). The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.

Differential gene expression between asymptomatic HBV carriers and normal adults

BACKGROUND: There are more than 100 million wa symptomatic HBV carriers (ASCs) in China and they are at a high risk of developing liver disease, which creates a serious health problem. But more than 90% of normal adults clear virus from primary HBV infection, so the difference of gene expression between ASCs and normal adults determines the differential outcomes. To identify differentially expressed genes in ASCs compared to normal adults, we used suppression subtractive hybridization to compare gene expression. METHODS: cDNA subtracted libraries were constructed by suppression subtracted hybridization from peripheral blood monocytes of ASCs and normal adults, the subtracted clones were prescreened by dot blot hybridization, and the levels of genes of interest were confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: One hundred and two and 96 positive clones were acquired from the A-N and N-A libraries, respectively, and 14 and 11 clones were identified by dot blot hybridization in the A-N and N-A libraries. Two genes were confirmed as differentially expressed between a set of ASC and normal adult samples by real-time RT-PCR. CONCLUSIONS: Differentially expressed genes in peripheral blood mononuclear cells between ASCs and normal adults were isolated by suppression subtractive hybridization, and included some new genes. Of the upregulated genes in ASCs, checkpoint suppressor I is associated with DNA damage-induced cell cycle arrest. Perforin I is associated with inflammation. The information about such alterations in gene expression could be useful for elucidating the mechanisms of ASC pathogenesis.

Expression and Cellular Localization of Interleukin-6 mRNA in Ovariectomized Rats

In order to observe the expression and cellular localization of interleukin-6 (IL-6) mRNA in bone tissue, ovaria of the rats were excised to develop osteoporosis model. The expression of IL-6 mRNA in bone tissues was detected by using dot blot hybridization assay and the cells producing IL6 identified and localized by using in situ hybridization respectively. The results showed that the expression of IL-6 mRNA was significantly increased in the ovariectomized rats as compared with that in normal control rats and strong IL-6 mRNA hybridization signals were detected in lining cells, osteoblasts and osteocytes. It was suggested that loss of ovarian function induced in vivo osteoblast lineage increased IL-6 mRNA expression. IL-6 might play important roles in the development of bone loss following ovariectomy.

Temporal analysis of mRNA expression of endogenous TGFβ And its typeⅠ,typeⅡreceptor on burn wounds

Recent studies have shown that application of exogenous transforming growth factor?(TGF?)to a wound has a beneficial effect. However,little is known about the roles of endogenous TGF? and its receptors in the course of wound healing. In the present study, mRNA expression of endogenous TGF? and its type Ⅰ,type Ⅱreceptors on wounds of burned rats was observed by using dot blot hybridization.Thermal injury could induce expression of TGF ?, and its type Ⅰ,type Ⅱ receptor genes, and their expression appeared to be regulated. The expression was the strongest at 5 and 7 d postscalding when the repair of wounds was most active.The study suggests that TGF? plays an important role in epidermis regeneration, and that expression of TGF ? receptors is one of factors regulating wound healing. The expression discrepancy between TGF ? and its type Ⅰ,type Ⅱ receptor genes at the later stages of wound healing implies the presence of a well-controlled mechanism to limit excessive effect of endogenous TGF ? on repair cells.

Screening of Species-specific DNA Probes for Identification of Fallopia multiflora

To screen species-specific DNA probes for identification of Fallopia muhiflora, the genomic DNA （gDNA） suppression subtraction hybridization （SSH） between F. muhiflora and F. muhiflora var. ciliinervis was firstly performed. The obtained differential gDNA fragments by SSH were then hybridized with gDNA ar- rays consisting of multiple whole genomes of several species （adulterants and/or closely related species of F. muhiflora） and four differential fragments were screened uniquely representing F. muhiflora, which could be used as F. muhiflora species-specific probes. The screened DNA probes were tested by reverse dot blot hybridization and the results demonstrated that these probes could be used reliably to identify F, muhiflora. The species-specific DNA probes obtained in this study exhibited broad application prospects in the preparation of gene chips for identifying Chinese traditional medicines and the authentication of germplasm re- sources and crude drugs of F. muhiflora.

Development of a Low-cost Polymerase Chain Reaction-based Method for Studying Differentially Expressed Genes in Developing Rice Leaves

Gene expression studies are important for revealing gene functions putatively involved in biological processes. We were interested in identifying differentially expressed genes during leaf development in rice. We combined the RNA arbitrarily primed-polymerase chain reaction （RAP.PCR） and dot blot hybridization methods to screen a rice leaf primordium cDNA library. Three developmental stages during vegetative growth were examined. The cDNA clones showing different hybridization patterns were further analyzed and verified. Here we demonstrate that the combination of RAP-PCR and dot blot hybridization could provide an efficient and relatively low-cost cDNA library screening approach to discover genes not previously known to be associated with leaf development in rice, We believe that the findings described here will help to elucidate the molecular mechanism（s） underlying the developmental processes of rice leaf