Detection of Mycobacterium tuberculosis from sputum specimens using one-tube nested PCR.

One-tube nested PCR was developed for diagnosis of pulmonary tuberculosis using sequences based on thel6SrRNA gene. The usage of primers 16SOL, 16SOR, 16SIL and 16SIR with optimized conditions could detect 555 bp DNA band from 21 species, 41 strains of mycobacteria and one isolate of Nocardia asteroides. It also revealed a specific 306 bp DNA band from 59 strains of M. tuberculosis complex. Cross amplification was observed in M. marinum, M. ulcerans and a few isolates of M. fortuitum complex. The developed method could detect as little as 100 fg of M. tuberculosis DNA. The PCR mixtures could be stored at 0 degrees C for 2 months or at -20 degrees C for at least 20 months without decrease in sensitivity. Using one-tube nested PCR for detection of M. tuberculosis compared with acid fast staining and culture results from 153 sputum specimens revealed 88.6% sensitivity and 89.2% specificity in smear positive specimens and 93.2% sensitivity and 85.0% specificity in culture positive specimens.

Can serial qualitative polymerase chain reaction monitoring predict outcome of pulmonary tuberculosis treatment?

BACKGROUND: The aim of this study was to assess the use of qualitative one-tube nested polymerase chain reaction (PCR) for monitoring the treatment response in smear-positive pulmonary tuberculosis, and the factors determining the negative conversion of sputum smear, culture, and PCR during treatment. METHODOLOGY: A total of 53 patients receiving a standard short course of chemotherapy with 24 months follow-up period after treatment cessation were included in the study. Sputum specimens were collected serially for smear, culture, and PCR until the treatment was complete. RESULTS: The conversion rate for sputum culture, smear, and PCR at 8 weeks after treatment were 84.9, 58.5, and 47.1%, and at 16 weeks of treatment were 100, 88.7, and 79.2%, respectively. At the end of the treatment period, there were four PCR persisters, one of whom had disease relapse. Only cavitary disease had an influence over the negative conversion of the smear and PCR at 8 weeks (RR 3.5, 95% CI 1.04-11.95, P=0.04 for smear; RR 5.06, 95% CI 1.196-21.42, P=0.03 for PCR). CONCLUSION: Qualitative PCR was not useful for monitoring therapy in smear-positive pulmonary tuberculosis. Mycobacterium DNA was cleared slowly in cavitary disease. The PCR may be performed at the time of treatment cessation to identify those with potential for disease relapse.

Rapid detection and identification of dengue viruses by polymerase chain reaction (PCR).

A polymerase chain reaction (PCR) method using sets of newly designed primers for rapid detection and simultaneous identification of dengue virus serotypes was developed and tested. The test is based on two sets of primers specific within the envelope (E) and non-structural (NS1) regions of the dengue-virus genome. Two sets of universal primers that bind to two target sequences which are shared by all the four serotypes of the virus within the E and NS1 regions are used. The resulting products are further amplified by another pair of inner or nested universal primers, which also bind to another set of shared sequences within the E and NS1 regions, respectively. The nested PCR of both the E and NS1 regions can detect dengue virus of all the four serotypes at a sensitivity of 1 plaque forming unit (pfu) or less. For the identification of serotypes, a mixture of four pairs of serotype-specific primers, specific to the E region, was used. The primers have been designed to bind to serotype specific sequences within the regions flanked by the outer universal primers, and giving the amplified products of different sizes, each corresponds to one particular serotype (405 bp for Den1, 346 bp for Den2, 196 bp for Den3, and 143 bp for Den4). A protocol has been developed and successfully applied to detect dengue virus in cell-culture supernatants and patients sera. The technique is simple and rapid, capable of not only detecting the dengue virus but also identifying the serotypes of the virus in clinical specimens.

Recombination of two homologous MHC class III genes of the mouse (C4 and Slp) that accounts for the loss of testosterone dependence of sex-limited protein expression.

In most mouse strains, expression of a gene encoding sex-limited protein (Slp), an isotype of the fourth component of complement (C4), is induced by testosterone, or the gene is not expressed at all; however, in some wild-derived strains carrying H-2w7, H-2w16, or H-2w19 haplotype, Slp is expressed constitutively in the same way as C4. To examine the structural basis for the testosterone-independent expression of Slp, 41 overlapping clones together encoding the S region were isolated from C3H.W7 mouse (H-2w7) cosmid library. Five C4-related genes each spanning approximately 16 kb were identified among the cluster of cosmid clones and were isolated for structural study. One of the genes (C4w7) hybridized with the C4-specific oligonucleotide probe but not with the Slp-specific oligonucleotide probe, whereas the other genes (Slpw7a, Slpw7b, Slpw7c, and Slpw7d) hybridized only with the Slp-specific probe. Restriction mapping of these genes and sequencing of the selected regions of 5'-flanking regions of the genes were performed, and the results were compared with the data obtained with the C4 and Slp genes of FM (H-2d) and B10.BR (H-2k). These studies showed that three of the C4-related genes of C3H.W7 (Slpw7b, Slpw7c, and Slpw7d) are C4-Slp recombinant genes comprising a 5'-region derived from C4 gene and a 3'-region derived from Slp gene. It is suggested that 5'-flanking region derived from C4 in these C4-Slp recombinant genes accounts for testosterone-independent expression of Slp in C3H.W7 mouse.

Comparative studies on thymidylate synthetase from P. berghei and mouse reticulocytes.

Partially purified thymidylate synthetase from Plasmodium berghei and mouse reticulocytes was characterized. The mol. wt of the enzyme from P. berghei was about twice that from mouse reticulocytes. The optimum pH of the enzyme from P. berghei was found to be 6.5-7.5 while that from the host was 7.0-8.0. The enzyme from P. berghei was more susceptible to pH denaturation than the enzyme from reticulocytes. The enzyme from both sources differed in their Km values for substrates. The enzyme from reticulocytes was less sensitive to inhibition by substrate analogs than that from P. berghei.