Comparison of two molecular diagnostic methods for identifying Beijing genotype of Mycobacterium tuberculosis

Background and Objectives: The Beijing family of Mycobacterium tuberculosis has been identified as a severe pathogen among this species and found in many clinical isolates during the last decade. Early identification of such genotype is import- ant for better prevention and treatment of tuberculosis. The present study performed to compare the efficiency of Real-Time PCR and IS6110-Based Inverse PCR methods to identify the Beijing family. Materials and Methods: This study was carried out on 173 clinical isolates of Mycobacterium tuberculosis complex in Golestan Province, northern Iran. DNA extraction performed by boiling and determining the Beijing and non-Beijing strains carried out using Real-Time PCR and IS6110-Based Inverse PCR. Results: In both Real-Time PCR and IS6110-Based Inverse PCR method, 24 specimens (13.9%) of the Beijing family were identified and the result of the IS6110-Based Inverse PCR method showed that all the Beijing strains in this region belonged to the Ancient Beijing sub-lineage. Conclusion: Although the efficacy of the two methods in the diagnosis of the Beijing family is similar, the IS6110-Based Inverse PCR is more applicable to the ability to detect new and old Beijing family.

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Identification and Discrimination of Mycobacterium tuberculosis Complex with Traditional and Real-Time PCR in Different Specimens in Iraq

Journal of the Faculty of Medicine Baghdad ◽

10.32007/jfacmedbagdad.6231787 ◽

2020 ◽

Vol 62 (3) ◽

Author(s):

Shaymaa Mahmood Ali ◽

Mohammad A. Al-Faham ◽

Ahmed A. Mankhi

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Real Time Pcr ◽

Mycobacterium Tuberculosis Complex ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Tuberculosis Complex ◽

Direct Smear ◽

Smear Examination ◽

Lowenstein Jensen

Background: Tuberculosis (TB) is a major public health issue and a main cause of global morbidity and mortality. TB is the world's ninth leading cause of death despite the numerous treatment strategies for managing the disease.Objective: To assess the traditional method (direct smear examination and culture) against real-time PCR as pulmonary and extrapulmonary tuberculosis laboratory diagnostic techniques.Cases and methods: Samples were collected from (612) TB cases, (409) of whom were pulmonary tuberculosis (PTB) and (203) were extrapolmonary tuberculosis (EPTB). The cases were seeking care at the Specialized Chest and Respiratory Disease Center/ National Reference Laboratory for Tuberculosis (NRL) in Baghdad, during the period 1st of May -1st of October 2019. Direct smear examination, Lowenstein-Jensen culture and Real Time PCR were used to diagnose TB.Results: Out of 612 samples received, 82(13.4%) were positive by smear microscopy, while 90(14.7%) were able to grow on Lowenstein-Jensen (LJ) media. Ninety DNA extracts from the samples which were positive on LJ media and 25 control specimens, were diagnosed with molecular analysis by using real time PCR to determine the species of Mycobacterium tuberculosis complex. The results revealed that the 71 samples (78.9%) were M. tuberculosis, three specimens (3.3%) were combined M. bovis and M. tuberculosis, and one M. tuberculosis, M. bovis, and M. bovis BCG, while15 (16.7%) were negative and subsequently excluded from study.Conclusion: The comparison between molecular diagnostic methods by using Real time PCR with conventional diagnostic methods, provides a new promising technique and is potentially a practical and rapid alternative to the slower traditional pulmonary and EPTB diagnostic culture. The results show M. bovis overall contribution on human TB in comparison to M. tuberculosis is minor among PTB and EPTB cases in the sample studied.

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Rapid Detection of Mycobacterium tuberculosis Beijing Genotype Strains by Real-Time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.01491-06 ◽

2006 ◽

Vol 44 (9) ◽

pp. 3472-3472

Author(s):

D. Hillemann ◽

R. Warren ◽

T. Kubica ◽

S. Rusch-Gerdes ◽

S. Niemann

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Beijing Genotype

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A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

PLoS ONE ◽

10.1371/journal.pone.0089395 ◽

2014 ◽

Vol 9 (2) ◽

pp. e89395 ◽

Cited By ~ 8

Author(s):

Jiyoun Yeo ◽

Erin L. Crawford ◽

Thomas M. Blomquist ◽

Lauren M. Stanoszek ◽

Rachel E. Dannemiller ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Diagnostic Testing ◽

Molecular Diagnostic ◽

Molecular Diagnostic Testing ◽

Ffpe Samples ◽

Pcr Method

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Improved real-time PCR for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates

Diagnostic Microbiology and Infectious Disease ◽

10.1016/s0732-8893(02)00521-7 ◽

2003 ◽

Vol 45 (3) ◽

pp. 207-212 ◽

Cited By ~ 24

Author(s):

Maria J Torres ◽

Antonio Criado ◽

Maite Ruiz ◽

Ana C Llanos ◽

Jose C Palomares ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Clinical Isolates ◽

Isoniazid Resistance

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Use of Real-Time PCR and Fluorimetry for Rapid Detection of Rifampin and Isoniazid Resistance-Associated Mutations in Mycobacterium tuberculosis

Journal of Clinical Microbiology ◽

10.1128/jcm.38.9.3194-3199.2000 ◽

2000 ◽

Vol 38 (9) ◽

pp. 3194-3199 ◽

Cited By ~ 69

Author(s):

Maria J. Torres ◽

Antonio Criado ◽

Jose C. Palomares ◽

Javier Aznar

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Real Time Pcr ◽

Antimicrobial Agents ◽

Dna Amplification ◽

Single Strand Conformation Polymorphism ◽

Clinical Isolates ◽

Polymorphism Analysis ◽

Antibiotic Resistant ◽

Hybridization Probes

Very fast amplification of DNA in small volumes can be continuously monitored with a rapid cycler that incorporates fluorimetric detection. Primers were designed to amplify a 157-bp fragment of therpoB gene spanning codons 526 and 531 and a 209-bp fragment of the katG gene spanning codon 315 of Mycobacterium tuberculosis. Most mutations associated with resistance to rifampin (RMP) and isoniazid (INH) in clinical isolates occur in these codons. Two pairs of hybridization probes were synthesized; one in each pair was 3′ labeled with fluorescein and hybridized upstream of the codon with the mutation; the other two probes were 5′ labeled with LightCycler-Red 640. Each pair of probes recognized adjacent sequences in the amplicon. After DNA amplification was finished by using a LightCycler, the temperature at which the Red 640 probe melted from the product was determined in a 3-min melt program. Twenty M. tuberculosis clinical isolates susceptible to streptomycin, INH, RMP, and ethambutol and 36 antibiotic-resistant clinical M. tuberculosis isolates (16 resistant to RMP, 16 to INH, and 4 to both antimicrobial agents) were amplified, and the presence of mutations was determined using single-strand conformation polymorphism analysis, the LiQor automated sequencer, and the LightCycler system. Concordant results were obtained in all cases. Within 30 min, the LightCycler method correctly genotyped all the strains without the need of any post-PCR sample manipulation. Overall, this pilot study demonstrated that real-time PCR coupled to fluorescence detection is the fastest available method for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.

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Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2014000100005 ◽

2014 ◽

Vol 34 (1) ◽

pp. 29-33 ◽

Cited By ~ 4

Author(s):

Gisele M. Bacanelli ◽

Carlos A. N. Ramos ◽

Flábio R. Araújo

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Anaplasma Marginale ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Chain Reaction ◽

The Real ◽

End Point ◽

Polymerase Chain

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

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