Identification of mycobacterial species by PCR restriction enzyme analysis of thehsp65gene — an Indian experience

2015 ◽  
Vol 61 (4) ◽  
pp. 293-296 ◽  
Author(s):  
Ajoy Kumar Verma ◽  
Gavish Kumar ◽  
Jyoti Arora ◽  
Paras Singh ◽  
Vijay Kumar Arora ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Nontuberculous Mycobacteria ◽  
Diagnostic Methods ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Smear Microscopy ◽  
Biochemical Tests ◽  
Mycobacterium Chelonae ◽  
Biochemical Identification ◽  
Pcr Targeting

Nowadays, nontuberculous mycobacteria (NTM) often cause pulmonary and extrapulmonary disease. Species identification of NTM determines the line of treatment and management of the disease. The routine diagnostic methods, i.e., smear microscopy and biochemical identification, of nontuberculous mycobacteria are tedious and time consuming and not all laboratories can perform these tests on a routine basis. A PCR targeting the hsp65 gene was implemented using standard strains and was applied to 109 clinical isolates. The PCR-amplified product was subjected to restriction enzyme analysis using BstEII and HaeIII. The results obtained were compared with that of biochemical tests. Of 109 NTM, 107 were identified to species level. PCR plus restriction enzyme analysis (PRA) identified 12 types of NTM. Common species identified were Mycobacterium chelonae (32), a rapid growing NTM, and Mycobacterium avium complex (21), among the slow growing NTM. PRA and biochemical identification showed 95.32% (102/107) concordant results. PRA is fast, cheap, and accurate for identification of potentially pathogenic NTM.

scholarly journals hsp65 PCR-restriction enzyme analysis (PRA) for identification of mycobacteria in the clinical laboratory

2001 ◽  
Vol 43 (1) ◽  
pp. 25-28 ◽  
Author(s):  
Carolina Feher da SILVA ◽  
Suely Yoko Mizuka UEKI ◽  
Débora de Cássia Pires GEIGER ◽  
Sylvia Cardoso LEÃO
Keyword(s):  
Restriction Enzyme ◽  
Clinical Laboratory ◽  
Specific Treatment ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Internet Site ◽  
Phenotypic Characteristics ◽  
Biochemical Tests ◽  
Hsp65 Gene ◽  
Biochemical Identification

More than 70 species of mycobacteria have been defined, and some can cause disease in humans, especially in immunocompromised patients. Species identification in most clinical laboratories is based on phenotypic characteristics and biochemical tests and final results are obtained only after two to four weeks. Quick identification methods, by reducing time for diagnosis, could expedite institution of specific treatment, increasing chances of success. PCR restriction-enzyme analysis (PRA) of the hsp65 gene was used as a rapid method for identification of 103 clinical isolates. Band patterns were interpreted by comparison with published tables and patterns available at an Internet site (http://www.hospvd.ch:8005). Concordant results of PRA and biochemical identification were obtained in 76 out of 83 isolates (91.5%). Results from 20 isolates could not be compared due to inconclusive PRA or biochemical identification. The results of this work showed that PRA could improve identification of mycobacteria in a routine setting because it is accurate, fast, and cheaper than conventional phenotypic identification.

scholarly journals Identification of Two Novel Mycobacterium avium Allelic Variants in Pig and Human Isolates from Brazil by PCR-Restriction Enzyme Analysis

1999 ◽  
Vol 37 (8) ◽  
pp. 2592-2597 ◽  
Author(s):  
Sylvia Cardoso Leão ◽  
Marcelo R. S. Briones ◽  
Marcelo Palma Sircili ◽  
Simone Carvalho Balian ◽  
Nelson Mores ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Mycobacterium Avium ◽  
Restriction Enzyme ◽  
Clinical Laboratory ◽  
Epidemiological Studies ◽  
Nucleic Acid Hybridization ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Biochemical Tests ◽  
Diagnostic Significance

Mycobacterium avium complex (MAC) is composed of environmental mycobacteria found widely in soil, water, and aerosols that can cause disease in animals and humans, especially disseminated infections in AIDS patients. MAC consists of two closely related species, M. avium and M. intracellulare, and may also include other, less-defined groups. The precise differentiation of MAC species is a fundamental step in epidemiological studies and for the evaluation of possible reservoirs for MAC infection in humans and animals. In this study, which included 111 pig and 26 clinical MAC isolates, two novel allelic M. aviumPCR-restriction enzyme analysis (PRA) variants were identified, differing from the M. avium PRA prototype in theHaeIII digestion pattern. Mutations in HaeIII sites were confirmed by DNA sequencing. Identification of these isolates as M. avium was confirmed by PCR with DT1-DT6 and IS1245 primers, nucleic acid hybridization with the AccuProbe system, 16S ribosomal DNA sequencing, and biochemical tests. The characterization of M. avium PRA variants can be useful in the elucidation of factors involved in mycobacterial virulence and routes of infection and also has diagnostic significance, since they can be misidentified as M. simiae II and M. kansasii I if the PRA method is used in the clinical laboratory for identification of mycobacteria.

scholarly journals Short Communication: Subtyping ofMycobacterium kansasiiby PCR-Restriction Enzyme Analysis of thehsp65Gene

2013 ◽  
Vol 2013 ◽  
pp. 1-4 ◽  
Author(s):  
Zofia Bakuła ◽  
Aleksandra Safianowska ◽  
Magdalena Nowacka-Mazurek ◽  
Jacek Bielecki ◽  
Tomasz Jagielski
Keyword(s):  
Lung Disease ◽  
Pulmonary Disease ◽  
Restriction Enzyme ◽  
Short Communication ◽  
Nontuberculous Mycobacteria ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Mycobacterium Kansasii ◽  
Clinical Specimens ◽  
Common Causes

Mycobacterium kansasiiis one of the most common causes of pulmonary disease resulting from nontuberculous mycobacteria (NTM). It is also the most frequently isolated NTM species from clinical specimens in Poland. The aim of this study was to investigate the distribution ofM. kansasiisubtypes among patients suspected of having pulmonary NTM disease. Fifty clinical isolates ofM. kansasiirecovered from as many patients with suspected mycobacterial lung disease between 2000 and 2010 in Poland were genotyped by PCR-restriction enzyme analysis (PCR-REA) of partialhsp65gene.Mycobacterium kansasiisubtype I was the only genotype to be identified among the isolates, both disease-associated and non-disease-associated. Isolation ofM. kansasiisubtype I from clinical specimens may be indicative of infection but may also merely represent colonization.

scholarly journals Mycobacterium franklinii sp. nov., a species closely related to members of the Mycobacterium chelonae–Mycobacterium abscessus group

2015 ◽  
Vol 65 (Pt_7) ◽  
pp. 2148-2153 ◽  
Author(s):  
Christiane Lourenço Nogueira ◽  
Keith E. Simmon ◽  
Erica Chimara ◽  
Margo Cnockaert ◽  
Juan Carlos Palomino ◽  
...  
Keyword(s):  
Housekeeping Genes ◽  
Single Species ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Mycobacterium Abscessus ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Phenotypic Analysis ◽  
Biochemical Tests ◽  
Mycobacterium Chelonae

Two isolates from water, D16Q19 and D16R27, were shown to be highly similar in their 16S rRNA, 16S–23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences to ‘Mycobacterium franklinii’ DSM 45524, described in 2011 but with the name not validly published. They are all nonpigmented rapid growers and are related phenotypically and genetically to the Mycobacterium chelonae–Mycobacterium abscessus group. Extensive characterization by phenotypic analysis, biochemical tests, drug susceptibility testing, PCR restriction enzyme analysis of the hsp65 gene and ITS, DNA sequencing of housekeeping genes and DNA–DNA hybridization demonstrated that ‘M. franklinii’ DSM 45524, D16Q19 and D16R27 belong to a single species that is separated from other members of the M. chelonae–M. abscessus group. On the basis of these results we propose the formal recognition of Mycobacterium franklinii sp. nov. Strain DSM 45524T ( = ATCC BAA-2149T) is the type strain.

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus

1985 ◽  
Vol 5 (9) ◽  
pp. 2197-2203
Author(s):  
M S Lakshmikumaran ◽  
E D'Ambrosio ◽  
L A Laimins ◽  
D T Lin ◽  
A V Furano
Keyword(s):  
Dna Sequence ◽  
Restriction Enzyme ◽  
Allelic Variation ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Repeated Dna ◽  
Coding Region ◽  
Sequence Determination ◽  
Polymorphic Region ◽  
Or Gene

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.

Restriction Enzyme Analysis (REA) of Group A Streptococcal (GAS) M-Serotypes 1, 3, and 28

1997 ◽  
pp. 221-223 ◽  
Author(s):  
Dwight R. Johnson ◽  
Cheryl L. Romana ◽  
Carey D. Rehder ◽  
Joanne Dehnbostel ◽  
Edward L. Kaplan
Keyword(s):  
Restriction Enzyme ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Group A

scholarly journals Identificação de micobactérias não tuberculosas isoladas de sítios estéreis em pacientes em um hospital universitário na cidade do Rio de Janeiro

2011 ◽  
Vol 37 (4) ◽  
pp. 521-526 ◽  
Author(s):  
Simone Gonçalves Senna ◽  
Ana Grazia Marsico ◽  
Gisele Betzler de Oliveira Vieira ◽  
Luciana Fonseca Sobral ◽  
Philip Noel Suffys ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Restriction Enzyme ◽  
Rio De Janeiro ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis

OBJETIVO: Identificar micobactérias não tuberculosas (MNT) isoladas de sítios estéreis em pacientes internados no Hospital Universitário Clementino Fraga Filho, Rio de Janeiro (RJ) entre 2001 e 2006. MÉTODOS: Durante o período do estudo, 34 isolados de MNT de sítios estéreis de 14 pacientes, a maioria HIV positivos, foram submetidos a identificação fenotípica e hsp65 PCR-restriction enzyme analysis (PRA, análise por enzimas de restrição por PCR do gene hsp65). RESULTADOS: A maioria dos isolados foi identificada como Mycobacterium avium, seguida por M. monacense, M. kansasii e M. abscessus em menores proporções. CONCLUSÕES: A combinação de PRA, um método relativamente simples e de baixo custo, com algumas características fenotípicas pode fornecer a identificação correta de MNT na rotina de laboratórios clínicos.

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