New Microbiol. 2013 Oct;36(4):413-8.

RAPD discloses high molecular diversity of Mycobacterium tuberculosis from Michoacan, Mexico.

Guill N-Nepita AL, Vazquez-Marrufo G, Blanco-Guillot FT, Figueroa-Aguilar GA, Vazquez-Garciduenas MS.

Centro Multidisciplinario de Estudios en Biotecnologia, Facultad de Medicina;Veterinaria y Zootecnia, Universidad Michoacana de San Nicolas de Hidalgo.

 

Abstract

Random amplified polymorphism DNA (RAPD) is an easy, inexpensive technique for the characterization of pathogens in low-income countries. In this study we used RAPD to assess the genetic diversity of a small collection of isolates of mycobacteria from the Mexican state of Michoacan. In contrast with the low annual tuberculosis incidence in Michoacan relative to the national average, we found a high molecular diversity value suggesting high population diversity of M. tuberculosis in the studied region. Our findings justify further typing efforts with other molecular tools such as MIRU-VNTR and spoligotyping.

PMID: 24177304

 

SUPPLEMENT:

Mycobacterium tuberculosis is the causal agent of tuberculosis. When the 16SrRNA gene became a common way for typing bacteria, it proved to be useless for typing M. tuberculosis because it appeared to be very similar to other Mycobacterium species (1,2).  Afterwards, RAPD (Random amplified polymorphic DNA) was developed and widely used to obtain the molecular fingerprint of a variety of bacteria. Spoligotyping and IS1661-RFLP were later introduced for identifying different strains of M. tuberculosis and closely related mycobacteria, which although retrieving good typing information are time consuming and technically demanding. Thus, RAPD remained as a fast, easy technique for evaluating the diversity of bacterial populations through individual fingerprints that could be compared between each other.

The results of RAPD fingerprints appear as multiple bands in a lane (Figure 1). These bands are produced by PCR reactions that are carried out with a single short primer (3). Because of its short length, the primer is able to align with multiple sequences along both DNA strands.  When the primer aligns in plus and minus DNA strands in a close enough position, the segment between the sites is amplified generating a detectable band, something that usually occurs more than once.  This happens along the entire chromosome. Point mutations in the alignment sites and insertions or deletions in the amplified segments change the number and size of the bands obtained. These differences allow for distinguishing closely related bacteria (i.e. two strains of the same species). The resulting banding patterns are used to build a branching diagram (dendrogram) reflecting the genetic relatedness between the typed isolates. Additionally, a mathematical index such as the Shannon index is calculated in order to have a numerical magnitude of the displayed diversity.  A higher value denotes higher diversity.

Banding patterns obtained by RAPD in different laboratories are difficult to be compared with each other, because of which the method was not adopted as a standard typing technique.  However, differences between the obtained values of the Shannon index are a valid comparison. In this way, RAPD is highly valuable as a first line tool for assessing molecular diversity in bacterial populations of interest.

We are interested in the mycobacterial population that causes tuberculosis in the Mexican state of Michoacán, where it is claimed that the incidence of tuberculosis is of about 7 cases for every 100,000 inhabitants. In order to have an idea of how diverse this population is, we typed by RAPD a small sample of clinical isolates of M. tuberculosis. We discovered that the sample was highly diverse. We also noticed that three isolates from meningeal tuberculosis cases had different fingerprints, meaning that different strains were causing this severe form of tuberculosis.

Thanks to these results, we now have a precedent for the ongoing M. tuberculosis typing with gold standard techniques and were able to identify an interesting question: Which is the genetic background of the M. tuberculosis strains that are able to reach the central nervous system? We are working to answer this question, which will have important public health implications both for Michoacán as throughout the world (4).

FIGURE 1Figure 1. A single short primer aligns in more than one sequence within bacterial chromosome, generating more than one PCR products. Changes in the bacterial chromosome within amplified regions are reflected in the resulting banding pattern. The presence or absence of each possible band is recorded as a 1 o 0, respectively, in a binary matrix. This matrix is used to calculate the genetic distances between the strains and a branching diagram is constructed to visualize such distances. In the example, the strain B appears in a different branch, separated from the strains A and C. This happens because the strain B has accumulated more genetic changes than A or B, as is shown in the top of the figure.

 

REFERENCE:

1. Kirschner P, Springer B, Vogel U, Meier A, Wrede A, Kiekenbeck M, Bange FC, Bötteger EC. 1993. Genotypic Identification of Mycobacteria by Nucleic Acid Sequence Determination: Report of a 2-Year Experience in a Clinical Laboratory. J. Clin. Microbiol. 31(11):2882-2889

2. Frothingham R, Hills HG, Wilson KH. 1994. Extensive DNA Sequence Conservation throughout the Mycobacterium tuberculosis Complex. J. Clin. Microbiol. 32(7):1639-1643

3. Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18(22):6531–6535.

4. Thwaites GE, van Toorn R, Schoeman J. 2013. Tuberculous meningitis: more questions, still too few answers. Lancet Neurol. 12(10):999-1010.

 

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