Detection and identification of Mycobacterium species

2021 ◽  
Vol 30 (1) ◽  
pp. 79-86
Author(s):  
Asaad Gaber ◽  
Hazem Hamed
Keyword(s):  
Mycobacterium Tuberculosis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Mycobacterium Species ◽  
Mycobacterial Infections ◽  
Rapid Pcr ◽  
Detection And Identification ◽  
Pcr Method ◽  
Dna Fragment

Background: Successful diagnosis and effective treatment for mycobacterial infections are mainly depending on a rapid and sensitive identification method. Objective: To detect and identify the Mycobacterium species. Methodology: PCR and LCD-microarry techniques were compared with the classical methods of Ziehl-Neelsen staining (ZN) and culturing. Two primers based on two conservative regions within the mycobacterium 16S rRNA gene were designed and amplified a DNA fragment of about 1350 bp for both complex of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM). Results: Regarding to the standard method of culture, 57 positive individuals were identified out of 100 urine samples. The PCR showed 96.30 % sensitivity and 96.70% specificity, while ZN gave Se = 67.50 % and Sp = 100 %. The LCD-microarray analysis exhibited 100 % sensitivity and specificity. One species of MTB was determined as M. tuberculosis and positively represented by 12.3% (n=7). Five species of NTM were determined and represented as M. kansasii 36.8 % (n=21), M. celatum 21 % (n=12), M. gordonae 12.2% (n=7), M. chelonae 10.5 % (n=6), and M. phlei 7% (n=4). Conclusion: The results recommended utilizing the simple and rapid PCR method for early mycobacteria detection. Also, the fast LCD-microarray protocol is very beneficial for identification and differentiation between MTB and of NTM species.

scholarly journals Utility of a PCR-based method for rapid and specific detection of toxigenic Microcystis spp. in farm ponds

2020 ◽  
Vol 32 (3) ◽  
pp. 369-381 ◽  
Author(s):  
Jian Yuan ◽  
Hyun-Joong Kim ◽  
Christopher T. Filstrup ◽  
Baoqing Guo ◽  
Paula Imerman ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Low Cost ◽  
Farm Animals ◽  
Rrna Gene ◽  
Analytical Sensitivity ◽  
Water Safety ◽  
Farm Ponds ◽  
Pcr Method

Microcystis is a widespread freshwater cyanobacterium that can produce microcystin, a potent hepatotoxin harmful to animals and humans. Therefore, it is crucial to monitor for the presence of toxigenic Microcystis spp. to provide early warning of potential microcystin contamination. Microscopy, which has been used traditionally to identify Microcystis spp., cannot differentiate toxigenic from non-toxigenic Microcystis. We developed a PCR-based method to detect toxigenic Microcystis spp. based on detection of the microcystin synthetase C ( mcyC) gene and 16S rRNA gene. Specificity was validated against toxic and nontoxic M. aeruginosa strains, as well as 4 intergeneric freshwater cyanobacterial strains. Analytical sensitivity was as low as 747 fg/µL genomic DNA (or 3 cells/µL) for toxic M. aeruginosa. Furthermore, we tested 60 water samples from 4 farm ponds providing drinking water to swine facilities in the midwestern United States using this method. Although all water samples were positive for Microcystis spp. (i.e., 16S rRNA gene), toxigenic Microcystis spp. were detected in only 34 samples (57%). Seventeen water samples contained microcystin (0.1–9.1 μg/L) determined with liquid chromatography–mass spectrometry, of which 14 samples (82%) were positive for mcyC. A significant correlation was found between the presence of toxigenic Microcystis spp. and microcystin in water samples ( p = 0.0004). Our PCR method can be a low-cost molecular tool for rapid and specific identification of toxigenic Microcystis spp. in farm ponds, improving detection of microcystin contamination, and ensuring water safety for farm animals.

scholarly journals Molecular Identification of Mycobacterium Species of Public Health Importance in Cattle in Zimbabwe by 16S rRNA Gene Sequencing

2015 ◽  
Vol 9 (1) ◽  
pp. 38-42 ◽  
Author(s):  
Leah Padya ◽  
Nyasha Chin'ombe ◽  
Marcelyn Magwenzi ◽  
Joshua Mbanga ◽  
Vurayai Ruhanya ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequences ◽  
Close Relative ◽  
Cow Dung ◽  
Rrna Gene ◽  
Mycobacterium Fortuitum ◽  
Sequencing Analysis ◽  
Public Health Importance ◽  
Detection And Identification

Mycobacteriumspecies are naturally found in the environment as well as in domestic animals such as cattle. So far, more than 150 species ofMycobacterium, some of which are pathogenic, have been identified. Laboratory isolation, detection and identification ofMycobacteriumspecies are therefore critical if human and animal infections are to be controlled. The objective of this study was to identifyMycobacteriumspecies isolated in cattle in Zimbabwe using 16S ribosomal RNA gene amplification and sequencing. A total of 134 cow dung samples were collected throughout Zimbabwe and mycobacteria were isolated by culture. Only 49 culture isolates that were found to be acid-fast bacilli positive by Ziehl-Neelsen staining. The 16S rRNA gene was successfully amplified by PCR in 41 (84%) of the samples. There was no amplification in 8 (16%) of the samples. Out of the 41 samples that showed amplification, 26 (63%) had strong PCR bands and were selected for DNA sequencing. Analysis of the DNA sequences showed that 7 (27%) belonged toMycobacterium neoaurum, 6 (23%) belonged toMycobacterium fortuitum, 3 (12%) toMycobacterium goodii, 2 (1%) toMycobacterium arupense, 2 (1%) toMycobacterium peregrinumorM. septicumand 1 isolate (0.04%) toMycobacterium elephantis. There were 5 (19%) isolates that were non-mycobacteria and identified as Gordonia terrae, a close relative ofMycobacterium. The study therefore provided a molecular basis for detection and identification ofMycobacteriumspecies in animals and humans.

A DNA fragment hybridizing to a nif probe in Rhodobacter capsulatus is homologous to a 16S rRNA gene

Gene ◽  
1986 ◽  
Vol 48 (1) ◽  
pp. 81-92 ◽  
Author(s):  
Jacoba P. Schumann ◽  
Gayle M. Waitches ◽  
Pablo A. Scolnik
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Rhodobacter Capsulatus ◽  
Rrna Gene ◽  
Dna Fragment

scholarly journals Miniprimer PCR, a New Lens for Viewing the Microbial World

2007 ◽  
Vol 74 (3) ◽  
pp. 840-849 ◽  
Author(s):  
Thomas A. Isenbarger ◽  
Michael Finney ◽  
Carlos Ríos-Velázquez ◽  
Jo Handelsman ◽  
Gary Ruvkun
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Microbial Populations ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Microbial World ◽  
Pcr Method ◽  
Environmental Sequences ◽  
The 16S Rrna Gene

ABSTRACT Molecular methods based on the 16S rRNA gene sequence are used widely in microbial ecology to reveal the diversity of microbial populations in environmental samples. Here we show that a new PCR method using an engineered polymerase and 10-nucleotide “miniprimers” expands the scope of detectable sequences beyond those detected by standard methods using longer primers and Taq polymerase. After testing the method in silico to identify divergent ribosomal genes in previously cloned environmental sequences, we applied the method to soil and microbial mat samples, which revealed novel 16S rRNA gene sequences that would not have been detected with standard primers. Deeply divergent sequences were discovered with high frequency and included representatives that define two new division-level taxa, designated CR1 and CR2, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity.

scholarly journals Detection and Identification of Banana-associated Phytoplasma Using Nested-PCR Method

2019 ◽  
Vol 23 (1) ◽  
pp. 148
Author(s):  
Saurma Mona Astrid Sibarani ◽  
Tri Joko ◽  
Siti Subandiyah
Keyword(s):  
16S Rrna ◽  
Nested Pcr ◽  
Plant Diseases ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Aster Yellows ◽  
Detection And Identification ◽  
Pcr Method ◽  
The 16S Rrna Gene

Phytoplasma is known to be associated with plant diseases in about 300 plant species from various families. Information on the presence of phytoplasma in bananas as one of the pathogens that can cause disease in bananas in Indonesia has never been reported. This research was conducted with the aim to detect the presence of banana phytoplasma by the nested-PCR method and to identify phytoplasma obtained based on the sequence analysis of the 16S rRNA gene. Standard PCR was carried out using P1/P7 primary pairs, followed by nested-PCR using a pair of R16F2n/R16R2m23SR primers separately that could amplify the target 16S rRNA genes in a row at 1600 bp. BLAST analysis shows that the results of phylogenetic analysis of banana phytoplasmic nucleotide cv. manggala from Tasikmalaya and cv. Raja nangka from Banjar has a genetic relationship that is closer to lethal wilt oil palm Phytoplasma (Candidatus Phytoplasma asteris). This phytoplasma belongs to the 16SrI-B group (aster yellows).

scholarly journals Occurrence and Genetic Diversity of Uncultured Legionella spp. in Drinking Water Treated at Temperatures below 15°C

2006 ◽  
Vol 72 (1) ◽  
pp. 157-166 ◽  
Author(s):  
Bart A. Wullings ◽  
Dick van der Kooij
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Culture Method ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Treated Water ◽  
Pcr Method ◽  
Unit Reduction ◽  
Selection Of

ABSTRACT Representatives of the genus Legionella were detected by use of a real-time PCR method in all water samples collected directly after treatment from 16 surface water (SW) supplies prior to postdisinfection and from 81 groundwater (GW) supplies. Legionella concentrations ranged from 1.1 × 103 to 7.8 × 105 cells liter−1 and were significantly higher in SW treated with multiple barriers at 4°C than in GW treated at 9 to 12°C with aeration and filtration but without chemical disinfection. No Legionellae (<50 CFU liter−1) were detected in treated water by the culture method. Legionella was also observed in untreated SW and in untreated aerobic and anaerobic GW. Filtration processes in SW and GW treatment had little effect or increased the Legionella concentration, but ozonation in SW treatment caused about 1-log-unit reduction. A phylogenetic analysis of 16S rRNA gene sequences of 202 clones, obtained from a selection of samples, showed a high similarity (>91%) with Legionella sequences in the GenBank database. A total of 40 (33%) of the 16S rRNA gene sequences obtained from treated water were identified as described Legionella species and types, including L. bozemanii, L. worsleiensis, Legionella-like amoebal pathogen types, L. quateirensis, L. waltersii, and L. pneumophila. 16S rRNA gene sequences with a similarity of below 97% from described species were positioned all over the phylogenetic tree of Legionella. Hence, a large diversity of yet-uncultured Legionellae are common members of the microbial communities in SW and GW treated at water temperatures of below 15°C.

scholarly journals Development of 16S rRNA-Gene-Targeted Group-Specific Primers for the Detection and Identification of Predominant Bacteria in Human Feces

2002 ◽  
Vol 68 (11) ◽  
pp. 5445-5451 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Junji Fujimoto ◽  
Yukiko Miyamoto ◽  
Toshihiko Takada ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Intestinal Microflora ◽  
Quantitative Detection ◽  
Rrna Gene ◽  
Human Feces ◽  
Specific Primers ◽  
Specific Pcr ◽  
Detection And Identification ◽  
Bacterial Groups

ABSTRACT For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol. 68:2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.

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