scholarly journals Evaluation of PCR pncA-restriction fragment length polymorphism and PCR amplification of genomic regions of difference for the identification of M. bovis strains in lymph nodes cultures

2021 ◽  
Vol 21 (3) ◽  
pp. 985-989
Author(s):  
Imen Bouzouita ◽  
Henda Draoui ◽  
Samia Mahdhi ◽  
Leila Essalah ◽  
Leila Slim Saidi
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Lymph Nodes ◽  
Mycobacterium Bovis ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Biochemical Tests ◽  
Accurate Identification ◽  
Restriction Fragment Length

Background: A rapid accurate identification of Mycobacterium bovis is essential for surveillance purposes. Objectives: A PCR pncA-Restriction Fragment Length Polymorphism (RFLP) and a multiplex PCR based on the detection of 3 regions of difference (RD-PCR): RD9, RD4 and RD1 were evaluated for the identification of M. bovis in lymph nodes cultures, in Tunisia, during 2013-2015. Methods: Eighty-two M. tuberculosis complex strains were identified using the biochemical tests, GenoType MTBC assay, PCR pncA-RFLP and RD-PCR. Results: The PCR pncA-RFLP showed that 54 M. bovis strains, identified by GenoType MTBC, had a mutation at position 169 of pncA gene. Twenty-eight strains did not show any mutation at this position 27 M. tuberculosis isolates and one M. caprae. The PCR pncA-RFLP had a sensitivity of 100.0% (95%CI: 93.3 -100.0) and a specificity of 100.0% (95%CI: 87.9- 100.0) for identifying M. bovis. The RD-PCR showed that all M. bovis strains had the RD9 and RD4 deleted but presented RD1. RD-PCR also presented high sensitivity and specificity in detecting M. bovis strains (100.0%). Conclusions: PCR pncA-RFLP and RD-PCR represent very accurate and rapid tools to identify M. bovis. They can be easily implemented in each laboratory due to their low cost and easy use. Keywords: GenoType MTBC; lymph nodes; Mycobacterium bovis; PCR pncA-RFLP; RD-PCR.

scholarly journals Characterization of the Mycobacterium bovis Restriction Fragment Length Polymorphism DNA Probe pUCD and Performance Comparison with Standard Methods

2000 ◽  
Vol 38 (9) ◽  
pp. 3362-3369 ◽  
Author(s):  
Rory O'Brien ◽  
Bret S. Danilowicz ◽  
Louise Bailey ◽  
Orla Flynn ◽  
Eamon Costello ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Mycobacterium Bovis ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Performance Comparison ◽  
High Level ◽  
Restriction Fragment Length

In this study, the newly described Mycobacterium bovisrestriction fragment length polymorphism (RFLP) typing probe pUCD was characterized by sequence analysis and the previously observed polymorphic banding pattern was reproduced with a combination of three oligonucleotide probes in a single, mixed hybridization. In addition, the ability of pUCD to distinguish between 299 M. bovisisolates from the Republic of Ireland was assessed in relation to established methods and a statistical function for objective comparison of RFLP probes was derived. It was found that typing with pUCD alone produced greater discrimination between M. bovis isolates than typing with the commonly used mycobacterial DNA probes IS6110, PGRS, and DR and also by the spoligotyping technique. pUCD and DR in combination produced the highest level of discrimination while maintaining a high level of concordance with known epidemiological data relating to the samples. The reduction of pUCD to the level of oligonucleotides should in future allow pUCD and DR to be included together in a mixed hybridization, thus producing a high level of M. bovis strain type discrimination from a single round of RFLP analysis.

scholarly journals Restriction Fragment Length Polymorphism Analysis of Mycobacterium Bovis Isolates from Captive and Free-Ranging Animals

1997 ◽  
Vol 9 (4) ◽  
pp. 381-386 ◽  
Author(s):  
Diana L. Whipple ◽  
P. Ryan Clarke ◽  
Jerald L. Jarnagin ◽  
Janet B. Payeur
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Mycobacterium Bovis ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Mule Deer ◽  
Rflp Analysis ◽  
Free Ranging ◽  
Restriction Fragment Length

Mycobacterium bovis isolates from cattle, captive elk, and free-ranging mule deer and coyotes were examined by restriction fragment length polymorphism (RFLP) analysis. DNA extracted from each isolate was digested with restriction endonucleases AluI and PvuII. DNA probes used for Southern hybridizations were a 37-base oligonucleotide and a 123-base–pair sequence specific for the insertion sequence IS 6110 and a plasmid, pTBN12, which contains a polymorphic GC-rich repetitive sequence present in several species of mycobacteria. Generally, M. bovis isolates originating from a single herd of either cattle or captive elk had identical RFLP patterns, whereas isolates from unrelated sources had distinct patterns. The RFLP patterns for M. bovis isolates from free-ranging mule deer and coyotes were identical to patterns observed for isolates from a captive elk herd that was located in the area where the free-ranging animals were found. These results indicate that the captive elk herd may have been the source of M. bovis that infected the free-ranging animals. Results of this study show that RFLP analysis is a useful tool for differentiation of M. bovis isolates and for molecular epidemiology studies to determine possible sources of infection in outbreaks of tuberculosis in animals.

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