A Simple and Efficient Multiplex PCR Assay for the Identification of Mycobacterium Genus and Mycobacterium Tuberculosis Complex to the Species Level

Overview

Journal Yonsei Med J

Specialty General Medicine

Date 2013 Aug 7

PMID 23918573

Citations 14

Authors

Yeun Kim

Yeonim Choi

Bo-Young Jeon

Hyunwoo Jin

Sang-Nae Cho

Hyeyoung Lee

Affiliations

Soon will be listed here.

Abstract

Purpose: The Mycobacterium tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis bacillus Calmette-Guérin (BCG) and M. africanum, and causes tuberculosis in humans and animals. Identification of Mycobacterium spp. and M. tuberculosis complex to the species level is important for practical use in microbiological laboratories, in addition to optimal treatment and public health.

Materials And Methods: A novel multiplex PCR assay targeting a conserved rpoB sequence in Mycobacteria spp., as well as regions of difference (RD) 1 and RD8, was developed and evaluated using 37 reference strains and 178 clinical isolates.

Results: All mycobacterial strains produced a 518-bp product (rpoB), while other bacteria produced no product. Virulent M. tuberculosis complex strains, M. tuberculosis, M. bovis and M. africanum, produced a 254-bp product (RD1), while M. bovis BCG, M. microti and nontuberculous mycobacteria produced no RD1 region product. Additionally, M. tuberculosis and M. africanum produced a 150-bp product (RD8), while M. bovis and M. bovis BCG produced a 360-bp product (deleted form of RD8). M. microti and nontuberculous mycobacteria produced no RD8 region product. This assay identified all Mycobacterium spp. and all M. tuberculosis complex strains to the species level.

Conclusion: The multiplex PCR assay of the present study could be implemented as a routine test in microbiology laboratories, and may contribute to more effective treatment and surveillance of tuberculosis stemming from the M. tuberculosis complex.

Citing Articles

Study of Zoonotic Pathogens in Alien Population of Veiled Chameleons () in the Canary Islands (Spain).

Pino-Vera R, Abreu-Acosta N, Foronda P Animals (Basel). 2023; 13(14).

PMID: 37508064 PMC: 10376624. DOI: 10.3390/ani13142288.

Zoonotic Bacteria in sp., an Invasive Species Introduced to the Canary Islands (Spain).

Abreu-Acosta N, Pino-Vera R, Izquierdo-Rodriguez E, Afonso O, Foronda P Animals (Basel). 2023; 13(3).

PMID: 36766304 PMC: 9913382. DOI: 10.3390/ani13030414.

Isolation of multidrug-resistant (MDR) Mycobacterium bovis from a dog in Korea.

Cho H, Choi U, Oh Y J Vet Med Sci. 2022; 84(10):1358-1362.

PMID: 35922936 PMC: 9586030. DOI: 10.1292/jvms.21-0347.

Molecular surveillance of tuberculosis-causing mycobacteria in wastewater.

Mtetwa H, Amoah I, Kumari S, Bux F, Reddy P Heliyon. 2022; 8(2):e08910.

PMID: 35198775 PMC: 8842018. DOI: 10.1016/j.heliyon.2022.e08910.

Detection of native interferon-γ in nyala (Tragelaphus angasii): Towards diagnosing tuberculosis.

Roux L, McCall A, Michel A Onderstepoort J Vet Res. 2019; 86(1):e1-e3.

PMID: 31714142 PMC: 6852497. DOI: 10.4102/ojvr.v86i1.1796.

References

Streicher E, Victor T, van der Spuy G, Sola C, Rastogi N, Van Helden P . Spoligotype signatures in the Mycobacterium tuberculosis complex. J Clin Microbiol. 2006; 45(1):237-40. PMC: 1828946. DOI: 10.1128/JCM.01429-06. View

Gordon S, Eiglmeier K, Garnier T, Brosch R, Parkhill J, Barrell B . Genomics of Mycobacterium bovis. Tuberculosis (Edinb). 2001; 81(1-2):157-63. DOI: 10.1054/tube.2000.0269. View

Frothingham R . Differentiation of strains in Mycobacterium tuberculosis complex by DNA sequence polymorphisms, including rapid identification of M. bovis BCG. J Clin Microbiol. 1995; 33(4):840-4. PMC: 228052. DOI: 10.1128/jcm.33.4.840-844.1995. View

Dobner P, Feldmann K, Rifai M, Loscher T, Rinder H . Rapid identification of mycobacterial species by PCR amplification of hypervariable 16S rRNA gene promoter region. J Clin Microbiol. 1996; 34(4):866-9. PMC: 228907. DOI: 10.1128/jcm.34.4.866-869.1996. View

Lee H, Park H, Cho S, Bai G, Kim S . Species identification of mycobacteria by PCR-restriction fragment length polymorphism of the rpoB gene. J Clin Microbiol. 2000; 38(8):2966-71. PMC: 87161. DOI: 10.1128/JCM.38.8.2966-2971.2000. View

Halse T, Escuyer V, Musser K . Evaluation of a single-tube multiplex real-time PCR for differentiation of members of the Mycobacterium tuberculosis complex in clinical specimens. J Clin Microbiol. 2011; 49(7):2562-7. PMC: 3147876. DOI: 10.1128/JCM.00467-11. View

Raviglione M, Marais B, Floyd K, Lonnroth K, Getahun H, Migliori G . Scaling up interventions to achieve global tuberculosis control: progress and new developments. Lancet. 2012; 379(9829):1902-13. DOI: 10.1016/S0140-6736(12)60727-2. View

Vasconcellos S, Huard R, Niemann S, Kremer K, Santos A, Suffys P . Distinct genotypic profiles of the two major clades of Mycobacterium africanum. BMC Infect Dis. 2010; 10:80. PMC: 2859774. DOI: 10.1186/1471-2334-10-80. View

Kamphuis J, Buiting A, Misere J, van Berge Henegouwen D, van Soolingen D, Rensma P . BCG immunotherapy: be cautious of granulomas. Disseminated BCG infection and mycotic aneurysm as late complications of intravesical BCG instillations. Neth J Med. 2001; 58(2):71-5. DOI: 10.1016/s0300-2977(00)00098-x. View

10.

Ernst J, Trevejo-Nunez G, Banaiee N . Genomics and the evolution, pathogenesis, and diagnosis of tuberculosis. J Clin Invest. 2007; 117(7):1738-45. PMC: 1904327. DOI: 10.1172/JCI31810. View

11.

Parsons L, Brosch R, Cole S, Somoskovi A, Loder A, Bretzel G . Rapid and simple approach for identification of Mycobacterium tuberculosis complex isolates by PCR-based genomic deletion analysis. J Clin Microbiol. 2002; 40(7):2339-45. PMC: 120548. DOI: 10.1128/JCM.40.7.2339-2345.2002. View

12.

Sankar S, Ramamurthy M, Nandagopal B, Sridharan G . An appraisal of PCR-based technology in the detection of Mycobacterium tuberculosis. Mol Diagn Ther. 2011; 15(1):1-11. DOI: 10.1007/BF03257188. View

13.

Butler W, Jost Jr K, Kilburn J . Identification of mycobacteria by high-performance liquid chromatography. J Clin Microbiol. 1991; 29(11):2468-72. PMC: 270356. DOI: 10.1128/jcm.29.11.2468-2472.1991. View

14.

Kjeldsen M, Bek D, Rasmussen E, Prieme A, Thomsen V . Line probe assay for differentiation within Mycobacterium tuberculosis complex. Evaluation on clinical specimens and isolates including Mycobacterium pinnipedii. Scand J Infect Dis. 2009; 41(9):635-41. DOI: 10.1080/00365540903127425. View

15.

Frota C, Hunt D, Buxton R, Rickman L, Hinds J, Kremer K . Genome structure in the vole bacillus, Mycobacterium microti, a member of the Mycobacterium tuberculosis complex with a low virulence for humans. Microbiology (Reading). 2004; 150(Pt 5):1519-1527. PMC: 2964484. DOI: 10.1099/mic.0.26660-0. View

16.

Lamm D, Stogdill V, Stogdill B, Crispen R . Complications of bacillus Calmette-Guerin immunotherapy in 1,278 patients with bladder cancer. J Urol. 1986; 135(2):272-4. DOI: 10.1016/s0022-5347(17)45606-0. View

17.

Pinsky B, Banaei N . Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level. J Clin Microbiol. 2008; 46(7):2241-6. PMC: 2446918. DOI: 10.1128/JCM.00347-08. View

18.

OReilly L, Daborn C . The epidemiology of Mycobacterium bovis infections in animals and man: a review. Tuber Lung Dis. 1995; 76 Suppl 1:1-46. DOI: 10.1016/0962-8479(95)90591-x. View

19.

de la Rua-Domenech R . Human Mycobacterium bovis infection in the United Kingdom: Incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis. Tuberculosis (Edinb). 2005; 86(2):77-109. DOI: 10.1016/j.tube.2005.05.002. View