Assessment of mycobacterial viability by RNA amplification.

We investigated whether the presence of intact RNA is a valuable indicator of viability of mycobacteria with Mycobacterium smegmatis. M. smegmatis was exposed to various concentrations of rifampin and ofloxacin suspended in broth for different periods of time. The NASBA nucleic acid amplification system was used because of its rapid, sensitive, and specific detection of 16S rRNA. During drug exposure, the viability of the mycobacteria, expressed by the number of CFU, was compared with the presence of 16S rRNA as determined by NASBA and with the presence of DNA coding for 16S rRNA as determined by PCR. Both NASBA and PCR were shown to have a detection limit of approximately 5 x 10(2) CFU/ml. The intensity of the NASBA signal corresponded well with the number of CFU, and the lack of NASBA signal coincided with a loss of viability, which was reached after 3 days of exposure to bactericidal concentrations of both drugs. The presence of mycobacterial DNA, as determined by the intensity of the PCR signal, and the viability of M. smegmatis were not related, but an increase in the number of cells and intensity of PCR signal correlated well. Bacterial viability may thus be assessed by a rapid, sensitive, and specific, and semiquantitative technique by using NASBA. This system of viability testing provides the potential for rapid evaluation of drug susceptibility testing.     

Identification of mycobacteria by nonradioisotopic single-strand conformation polymorphism analysis

Clinical isolates of mycobacteria were identified to species levels using nonradioisotopic single-strand conformation polymorphism (non-RI SSCP) analysis of 16s rRNA gene fragments amplified by polymerase chain reaction with primers common to all of mycobacterial species. The method is based on a hypervariable region within the 16s rRNA in mycobacteria, which is characterized by species-specific nucleotide sequences. A total of 92 mycobacterial strains (Mycobacterium tuberculosis, M. avium, M. gordonae, M. intracellulare, M. kansasii, M. chelonae, M. nonchromogenicum, M. xenopi, and unidentified strain) were studied. They were classified into nine types of pattern showing single-strand DNA bands having different mobilities. Each strain was shown in the species-specific mobility by non-RI SSCP analysis. The results of non-RI SSCP analysis were identical to those of standard biochemical methods and 16S rRNA sequencing.     

Lung infection caused by Mycobacterium riyadhense confused with Mycobacterium tuberculosis: the first case in Korea

A slowly growing, non-chromogenic mycobacterial strain was isolated from sputum and bronchial lavage fluid samples of a patient presenting with productive cough, blood-tinged sputum, low-grade fever, and weakness. A positive acid-fast bacilli sputum smear result prompted the initiation of an anti-tuberculosis regimen. Multiplex real-time PCR showed a negative result for Mycobacterium tuberculosis complex and a positive result for nontuberculous mycobacteria. The DNA chip test confirmed this organism as a member of the genus Mycobacterium, but could not specify the species. Interestingly, the mycolic acid patterns obtained by HPLC nearly overlapped with those of M. simulans. The sequences of the Mycobacterium 16S rRNA gene and 16S-23S internal transcribed spacer region were unique and were found to have 100% similarity with those of M. riyadhense. After a review of the literature, we report this case as the first Korean case of M. riyadhense lung infection.     

Genetic basis for clarithromycin resistance among isolates of Mycobacterium chelonae and Mycobacterium abscessus.

Resistance to clarithromycin among isolates of Mycobacterium chelonae and M. abscessus was observed in 18 of 800 (2.3%) patients tested between 1990 and 1995. Patients whose isolates were resistant had either disseminated disease or chronic lung disease, and the resistant isolates were recovered after clarithromycin monotherapy. Sequencing of the gene coding for the 23S rRNA peptidyltransferase region revealed a point mutation involving adenine at position 2058 (38%) or adenine at position 2059 (62%) in 20 of 20 relapse isolates from the first 13 patients identified. By pulsed-field gel electrophoresis or random amplified polymorphic DNA PCR, initial and relapse isolates were shown to have identical DNA patterns. M. chelonae and M. abscessus isolates were found to have only a single chromosomal copy of the rRNA operon, thus making them susceptible to single-step mutations. Thus, clarithromycin resistance in these species of rapidly growing mycobacteria relates to a point mutation in the gene coding for 23S rRNA and occurs in limited clinical situations, but was identified in almost 5% of isolates tested in 1995.     

Rapid differentiation between clinically relevant mycobacteria in microscopy positive clinical specimens and mycobacterial isolates by line probe assay

The Inno LiPA Mycobacteria assay, based on PCR amplification of the 16-23S rRNA spacer region of Mycobacterium species, has been designed for identification of mycobacteria grown in culture media and discrimination between Mycobacterium tuberculosis complex, M. avium, M. intracellulare, M. kansasii, M. gordonae, M. xenopi, scrofulaceum and M. chelonae group including M. abscessus. In order to evaluate the system as a fast diagnostic tool, the assay was for the first time used directly on 14 microscopy positive clinical specimens and 71 isolates and the results were compared to those of conventional identification using 16S rDNA analysis and biochemical properties. The assay only misidentified one strain, which was found to be M. avium complex instead of M. intracellulare as found by the conventional tests. The assay allows rapid discrimination of the eight most clinically relevant mycobacteria in microscopy positive clinical specimens and isolates within 6 h in the same procedural run.     

Mycobacteria distenct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS900-like sequences detectable IS900 polymerase chain reaction: implications for diagnosis

PCR targeting the 5' end of IS 900 has been considered specific for identification of Mycobacterium avium subsp. paratuberculosis and is frequently applied to confirm the presence of this organism in the diagnosis of Johne's disease. IS 900 PCR has also been applied to studies of the aetiology of Crohn's disease. Mycobacterium spp. isolated from the faeces of 3 clinically normal animals in 2 Australian states appeared not to be M. paratuberculosis but were positive by IS 900 PCR. The isolates were characterized using mycobactin dependency, biochemical tests, IS 900 and 16 S rRNA sequencing and restriction fragment length polymorphism (RFLP) using IS 900 as probe. DNA sequencing confirmed that the isolates had between 71% and 79% homology with M. paratuberculosis in the region of IS 900 amplified, were most closely related to Mycobacterium scrofulaceum, and confirmed the usefulness of restriction enzyme analysis of amplified product to identify the false positive results. RFLP analysis with Bst Ell detected three to five copies of the IS 900 -like element in the isolates. These were located in molecular weight fragments that were clearly different to IS 900 in previously characterized strains of M. paratuberculosis. It is likely that these isolates are environmental mycobacteria. Southern blotting with an internal probe is unlikely to provide differentiation of M. paratuberculosis from these organisms. We recommend the adoption of restriction endonuclease analysis of IS 900 PCR product as a routine precaution to prevent the reporting of false positive IS 900 PCR results.     

小儿常见感染菌16S—23S rRNA基因区间的分子生物学研究

目的：探讨聚合酶链反应（PCR）加限制性内切酶片段长度多态性（RFLP）分析在细菌rDNA区间检测中的应用。方法：以16S－23S RNA基因区间为靶序列,设计引物,选择合适的内切酶,采用PCR法加RFLP法检测标准菌株及临床菌株的rDNA区间。结果：26株不同的标准菌株经PCR扩增后,分别出现一条带,两条带,三条带及多条带的不同DNA图谱,敏感性试验可检出2．5CFU的细菌,与人类基因组DNA,真菌及病毒无交叉反应,其中14种菌经一步PCR扩增即可区分,另12种菌除肺炎克雷伯氏菌与坚韧肠球菌经Hinf I或Alu I酶切后仍不能区分外,其余经其中一内切酶酶切后均能区分,32例血培养阳性标本均扩增出与相应标准菌株相一致的图谱。结论：16S－22S rRNA区间基因PCR．扩增加RFLP技术检测细菌rDNA区间,具有特异,敏感,快速,准确的特点,为细菌感染的病原诊断提供新的科学依据。     

Capillary electrophoresis-based heteroduplex analysis with a universal heteroduplex generator for detection of point mutations associated with rifampin resistance in tuberculosis.

BACKGROUND: Slab gel heteroduplex analysis (HDA), a popular scanning method for genetic mutations, uses DNA fragments typically generated by PCR to create homo- and heteroduplex molecules with conformational differences and sequence-dependent electrophoretic profiles. Use of a universal heteroduplex generator (UHG) enhances the subtle variations caused by single-base substitutions. METHODS: The HDA-UHG slab gel format was modified for an efficient capillary-based method. The effect of staining dyes TOPRO5 and YOPRO1 on the analysis of heteroduplexes was studied, as well as ultraviolet absorbance and laser-induced fluorescence (LIF) detection methods. In addition, the entangled polymers hydroxyethyl cellulose, methyl cellulose, and linear polyacrylamide were evaluated as separation matrices. RESULTS: This assay was able to detect the presence of Mycobacterium tuberculosis and its rifampin susceptibility directly from clinical specimens in dramatically reduced analysis time (30 min vs 2.5 h). Optimized conditions included 0.3% methyl cellulose as the separation matrix, on-line staining using 1 micromol/L YOPRO1, and LIF detection for quantitative and reproducible analysis of single-base substitutions in the rifampin resistance-determining region of rpoB that give rise to the rifampin-resistant phenotype of M. tuberculosis. We generated 95% confidence limits using the wild-type sequence and used these limits to determine rifampin susceptibility in samples. CONCLUSIONS: Capillary electrophoresis, combined with the HDA-UHG technique, may be of value for rapid and efficient clinical diagnosis of rifampin-resistant tuberculosis strains.     

Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR

BACKGROUND: Broad-range PCR provides valuable information for detecting bacterial infections. This study assesses the combined use of broad-range real-time PCR and high-resolution melting analysis for rapid detection and identification of clinically important bacteria. METHODS: We subjected 46 bacterial culture colonies representing 25 clinically important bacterial species to LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye. We performed high-resolution melting analysis of the PCR products with the HR-1 instrument and used melting profiles as molecular fingerprints for bacterial species identification. We validated this method via assessment of 54 consecutive bacteria culture colonies obtained from a clinical microbiology laboratory. RESULTS: The 16S rRNA gene of all 25 bacterial species was amplifiable by this method, with PCR product lengths of 216 or 217 bp. Of the 25 bacterial species, we identified 11 via a 1-step post-PCR high-resolution melting analysis. The remaining bacterial species were identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species or by a 2nd real-time PCR targeting a different region of the 16S rRNA gene. A high-resolution melting database and a working protocol were established for identifying these 25 bacterial species. In the validation assay, a 94% accuracy rate was achieved when the bacterial species were in the high-resolution melting database. CONCLUSIONS: This assay requires no multiplexing or hybridization probes and provides a new approach for bacterial species identification in a molecular diagnostic laboratory.     

Correct heteroduplex formation for mutation detection analysis.

BACKGROUND: The majority of mutation detection methods for unknown mutations are polymerase chain reaction (PCR)-based methods dependent on the formation of heteroduplexes between wild-type and mutant strands of DNA. METHODS AND RESULTS: This report discusses the difficulties associated with forming heteroduplexes with a large DNA fragment and the implications for subsequent mutation detection by the chemical cleavage of mismatch technique and other methods reliant on heteroduplex formation. It was found that the size and sequence context of the fragment being investigated inhibited correct heteroduplex formation. The problem was overcome by dividing the sequence into two overlapping fragments. CONCLUSIONS: Early identification of this problem in other fragments will help with the rapid optimization of PCR-based mutation detection methods.     

HIV-1 detection and subtyping by PCR and heteroduplex mobility assay in blood donors: can these tests help to elucidate conflicting serological results?

Testing blood donors for the human immunodeficiency viruses (HIV-1 and 2), requires serological tests that are frequently inconclusive. Peripheral blood mononuclear cells of 50 blood donors with the screening test (ELISA) reactive to HIV-1, but with indeterminate results in the first Western Blot (WB) performed, were submitted to the polymerase chain reaction (PCR) and heteroduplex mobility assay (HMA), a nonsequencing method that can distinguish between HIV-1 subtypes (A to I). PCR amplification of HIV-1 env gene regions has been obtained in 12 (24.0%) samples. HMA of the amplified DNA showed that 12 belonged to HIV-1 B subtype and one to F subtype. PCR testing of the amplified DNA helped to elucidate doubtful serological results and HMA proved to be a relatively simple and rapid way of subtyping HIV-1 for epidemiological purposes.     

A multiplex polymerase chain reaction assay for genus-, group- and species-specific detection of mycobacteria

We developed and evaluated a single-step, multiplex polymerase chain reaction (PCR) assay for distinguishing (1) between the Mycobacterium tuberculosis complex (MTBC) and mycobacteria other than tuberculosis (MOTT) and (2) between M. tuberculosis and M. bovis species. The assay targeted the 16S and the 23S rDNA to distinguish between MTBC and MOTT species, and the oxyR gene to distinguish between M. tuberculosis and M. bovis strains. Clinical samples and reference strains (N = 156) comprised 93 strains of M. tuberculosis, 44 of M. bovis, 1 M. africanum strain, and 18 strains representing 9 different species of MOTTs. MOTTs generated only a single PCR product of about 2.5 kilobase; however, all of the MTBC strains produced a 118 base pair (bp) fragment and an additional 270 bp fragment was obtained for M. tuberculosis and M. africanum when the primer pair oxyRTB-2.1/oxyRMT-1 was used. When oxyRTB-2.1/oxyRMB-1 primers were used, the 270 bp fragment was obtained for only M. bovis. The assay needed as little as 1 pg of purified genomic DNA to make a positive identification.     

血液细菌16S rRNA基因检测的诊断价值

目的探索快速可靠的检测细菌感染的新方法。方法用PCR技术扩增实验室保留株10株的16SrRNA基因,以乙型肝炎病毒-DNA、白假丝酵母菌和人类基因组DNA为对照,检测该方法的特异性;采用10倍比稀释法进行该方法的灵敏度检测。结果对所测细菌株均获得475bp扩增产物,而与乙型肝炎病毒-DNA、白假丝酵母菌和人基因组DNA无交叉反应;PCR最低能检测1.5×104/L大肠埃希氏菌。结论16SrRN基因PCR检测细菌感染的方法具有特异性、快速性和敏感性高等特点。     

Combination of multiplex PCR with denaturing high-performance liquid chromatography for rapid detection of Mycobacterium genus and simultaneous identification of the Mycobacterium tuberculosis complex

A new assay with the combination of multiplex polymerase chain reaction and denaturing high-performance liquid chromatography analysis was developed for simultaneous detection of Mycobacterium genus and identification of the Mycobacterium tuberculosis complex (MTC). Targeting at genus-specific 16S rRNA sequence of Mycobacterium and specific insertion elements IS6110 and IS1081 of MTC, the assay was validated with 84 strains covering 23 mycobacteria species and 30 strains of non-mycobacteria species. No cross reactivity was observed. Clinical application was carried out on 198 specimens (155 human sputum and 43 bovine tissue samples) and compared with culture. The multiplex assay detected all culture-positive (36 in number) and 35.2% (57/162) culture-negative specimens. The molecular assay was fast that could be completed within 1 h on purified DNA, with the limit of detection as 0.8–1.6 pg per reaction on DNA template. This work provided a useful laboratory tool for rapid identification of Mycobacterium and differentiation of MTC and nontuberculous mycobacteria.     

Genus- and species-specific DNA probes to identify mycobacteria using the polymerase chain reaction

Differential diagnosis of Mycobacterium tuberculosis, M. avium, and other mycobacteria remains a lengthy process. Recently, the use of DNA probes has been proposed as a new approach for a more specific and rapid diagnosis. Here, we report the cloning and sequencing of a genus-specific probe for Mycobacterium and a species-specific M. avium probe. The genus-specific probe hybridizes with DNA from nine ATCC type strains and 13 isolates of mycobacteria but not to non-mycobacterial DNA. In addition, the cloned fragment could also be amplified by polymerase chain reaction (PCR) in DNa of ten different mycobacterial type strains. The M. avium specific probe hybridizes strongly to sequences amplified in M. avium but not other mycobacterial or non-mycobacterial DNA. Amplification of the target sequence by PCR allowed the detection of 1 fg of all mycobacterial DNA tested for the genus-specific probe and 1 fg of M. avium DNA for the species-specific probe.     

54株分枝杆菌国际标准株16S-23S rRNA转录间隔区序列分析

目的分析54株分枝杆菌国际标准株16S-23SrRNA转录间隔区(ITS)序列,为临床分离株的鉴定提供参考。方法用16S-23SrRNA ITS序列分析法对54株分枝杆菌国际标准株进行分析,构建系统发育树,计算相似性百分比。结果除灰尘与微黄分枝杆菌;田野与千田分枝杆菌;抗热与副偶然分枝杆菌,奥布与母牛结核分枝杆菌;杜氏与猪分枝杆菌;金色与东海分枝杆菌,海与溃疡分枝杆菌、科莫斯分枝杆菌;2株结核分枝杆菌与田鼠分枝杆菌、非洲分枝杆菌的16S-23SrRNA ITS序列完全相同无法鉴别外,其他各分枝杆菌菌种间16S-23SrRNAITS序列均不相同,可以得到很好的鉴别。结论 16S-23SrRNA ITS序列分析是一种很好的鉴定分枝杆菌的方法,国际标准株16S-23SrRNA ITS序列的研究弥补了基因数据库的不足。     

Description of a novel Mycobacterium simiae allelic variant isolated from Caribbean AIDS patients by PCR-restriction enzyme analysis and sequencing of hsp65 gene

A sudden upsurge in the isolation of Mycobacterium simiae from terminally ill AIDS patients was recently reported on the Caribbean island of Guadeloupe. Identification of these M. simiae isolates was achieved using biochemical tests and further confirmed by PCR-restriction analysis (PRA) of a 439-bp fragment of hsp65. A novel PRA profile III (three Bst EII fragments of 240/125/80 bp and four Hae III fragments of 145/125/40/25 bp) was observed in four blood isolates from two patients. The 16 S rRNA gene sequencing of the hypervariable A region confirmed that all the pattern III isolates were indeed M. simiae species, and the hsp65 sequencing confirmed the existence of a new hsp65 allele in these caribbean isolates. A hsp65 sequence-based phylogenetic tree was also created for 39 species including M. simiae and related mycobacterial species as well as other rapid and slow growing mycobacteria, and may serve as an useful tool for identification of mycobacteria to species level.     

Mycobacterium wolinskyi Infection Confirmed by rpoB Gene Sequencing

Identification of rapidly growing mycobacteria (RGM) is problematic because there are many taxonomic changes. 16S rRNA gene is commonly used to identify Mycobacterium species, but alternative gene targets have been introduced for more accurate identification. We report a rare case of a prosthetic knee infection due to Mycobacterium wolinskyi. The isolate was not identified by 16S rRNA gene sequencing alone and substantially confirmed by rpoB gene sequencing. The identification was delayed because our laboratory did not routinely identify RGM to the species level. Simultaneous sequencing of both 16S rRNA and rpoB genes will allow rapid and accurate identification of M. wolinskyi isolates. J. Clin. Lab. Anal. 26:325‐327, 2012. © 2012 Wiley Periodicals, Inc.     

Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes andreal-time PCR

A rapid and sensitive strategy for the specific identification of Mycobacterium tuberculosis (TB) was designed and evaluated using crude mycobacterial lysates. The speed of real-time polymerase chain reaction (PCR) was combined with the sensitivity of fluorogenic probes to confirm the presence of mycobacteria as well as specifically identify the presence of members of the mycobacteria tuberculosis complex (MTC) in a single-tube assay. Oligonucleotides were designed to amplify the internal transcribed spacer (ITS) from several mycobacterial species. Specific fluorogenic probes were included in the PCR reaction for the identification of TB as well as Mycobacterium bovia and Mycobacterium africanum in bacterial lysates. The combination of TB-specific fluorogenic probes with real-time PCR formed an approach determined to be fast (less than 40 min), sensitive (less than 800 copies of DNA) and reliable for the specific detection of the MTC. Our data demonstrate the use of real-time PCR and fluorogenic probes in a rapid and sensitive assay to distinguish members of the MTC from other mycobacterial species.     

54株分枝杆菌国际参考株16S rRNA序列分析

目的分析54株分枝杆菌国际参考株16S rRNA序列,为临床分离株的鉴定提供参考。方法用16S rRNA序列分析法对54株分枝杆菌国际参考株进行分析,构建系统发育树,计算相似性百分比。结果除11株（共9种4组）分枝杆菌,即产鼻疽分枝杆菌与塞内加尔分枝杆菌;溃疡分枝杆菌与海分枝杆菌;堪萨斯与胃分枝杆菌;3株结核分枝杆菌与田鼠分枝杆菌、非洲分枝杆菌16S rRNA基因序列完全相同无法相互鉴别,其它41株（共41种）分枝杆菌菌种间16S rRNA均不相同可以得到很好的鉴别。结论 16S rRNA基因序列分析是一种很好的鉴定分枝杆菌的方法,国际参考株16S rRNA基因序列的研究弥补了基因数据库的不足。