Evaluation of the diagnostic value of loop-mediated isothermal amplification assays targeting three different Mycobacterium tuberculosis genes.

BACKGROUND: The aim of the study was to evaluate a loop-mediated isothermal amplification (LAMP) assay for the ability to diagnose tuberculosis directly from clinical samples rapidly. METHODS: LAMP assays were performed using previously reported primer sets to amplify three specific Mycobacterium tuberculosis (MTB) gene targets, hspX, gyrB, and IS6110. Quantitated DNA from strain H37Rv were detected for assessment of analytical sensitivity; specificity was evaluated by testing eight species of non-tuberculosis Mycobacterium (NTM) and four unrelated bacterial species. Sputum samples from 68 pulmonary tuberculosis patients and a control group consisting of 45 lung cancer patients and 20 healthy controls were analyzed using LAMP assays, and then compared with smear, culture and quantitative real-time PCR (qRT-PCR) methods. RESULTS: All three LAMP assays showed 100% specificity for MTB when tested against NTM and other bacterial species. The gyrB-LAMP assay was able to detect 60 cfu/ml of H37Rv suspension within 1 h, similar to qRT-PCR, but 10 times more sensitive than the hspX-LAMP and IS6110-LAMP assays. In clinical samples, when qRT-PCR was used as the reference method, the sensitivity of the three LAMP assays targeting hspX, gyrB, and IS6110 genes was 94.6, 98.2 and 92.9%, respectively. CONCLUSIONS: LAMP is more sensitive than smear microscopy and close to qRT-PCR in sensitivity for the detection of MTB. LAMP has comparable specificity to qRT-PCR but was more rapid and convenient.

Association of mannose-binding lectin gene polymorphisms with the development of pulmonary tuberculosis in China.

BACKGROUND: Mannose-binding lectin (MBL) is an important protein in the lectin pathway of the immune system. This study explores the association between MBL polymorphism and the susceptibility to tuberculosis (TB). The association between the MBL2 polymorphisms and serum MBL levels is also analyzed in the present study. METHODS: A total of 112 inpatients with pulmonary TB and 120 healthy controls were recruited to participate in this case-control study. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism(PCR-RFLP) technology was used to genotype MBL gene (variants in -221Y/X and exon l codons 54 A/B). Serum MBL level was assayed by human MBL ELISA kit. Demographic data and exposure information were also obtained from the study participants. RESULTS: Genotypes YA/YA of MBL gene were more prevalent in the healthy control group than in the TB patient (P =0.038, OR, 0.57; 95% CI, 0.34-0.97) and genotypes XA/XA were less frequent in the healthy control group (P =0.007, OR, 6.42; 95% CI, 1.39-29.67). The resistant diplotype was more frequently found in the younger patients and retreatment cases with TB in MBL gene sites -221Y/X or codon 54 A/B. X/Y and A/B polymorphisms were strong determinants of serum MBL levels. CONCLUSION: The polymorphisms of MBL gene may be associated with susceptibility to TB and the recurrence of TB. The YA/YA may be a protected diplotype against TB.

[Hydrocamptothecin promotes the mRNA expression of Wnt signaling inhibitor DKK-1].

OBJECTIVE: To investigate the effects of hydrocamptothecin on the expression of Wnt signaling pathway inhibitor DKK-1 in tumor cells. METHODS: Human HepG2, Hep3B, LoVo and U251 cells were treated with the antitumor drug Hydrocamptothecin. DKK-1 mRNA expression in the cells was detected with RT-PCR, and beta-catenin expression was measured by fluorescence-activated cell sorting (FACS). RESULTS: DKK mRNA in Hep3B, HepG2, LoVo and U251 cells was significantly increased after hydrocamptothecin treatment for 24 h, and the percentage of beta-catenin-positive cells and fluorescence intensity for beta-catenin expression was lowered in the cells after the treatment. CONCLUSION: Hydrocamptothecin promotes mRNA expression of Wnt signaling pathway inhibitor DKK-1 in Hep3B, HepG2, LoVo and U251 cells.