Solid-Phase Hybridization Capture of Low-Abundance Target DNA Sequences: Application to the Polymerase Chain Reaction Detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum

doi:10.1006/abio.1995.1232

Analytical Biochemistry

Volume 226, Issue 2, April 1995, Pages 325-330

https://doi.org/10.1006/abio.1995.1232 Get rights and content

Abstract

Polymerase chain reaction (PCR) has been widely applied to the detection of microorganisms. Overall sensitivity of PCR tests may be substantially reduced due to a large excess of nontarget DNA and inhibitory substances in the sample. We used a 5′-biotinylated 513-bp probe from the 3′ region of the IS 900 element specific for Mycobacterium paratuberculosis (Mptb) to capture target Mptb DNA from crude sample DNA extracts. Captured target DNA was separated using streptavidin-coated magnetic particles (Dynal). Since the IS 900 element shares homology over this region with IS 902 in Mycobacterium avium subsp. silvaticum (Mavs), target DNA from this other pathogen was also retained. Highly specific PCR for the detection of either organism directed to the 5′ regions of IS 900 or IS 902 was then performed directly on the solid phase. Hybridization capture of target DNA using sequence adjacent to the desired specific PCR site applied to Mptb increased overall sensitivity of detection in tissue and fecal extracts 10- to 100-fold. False positives due to contamination artifact were substantially excluded since the capture probe did not retain amplicons from the detection PCR. Development of the method to involve covalent 5′ immobilization of capture probes on heat-resistant polymers should, in the future, provide a simple system with broad potential applications.

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The PCR assay was compared with serological tests such as ELISA and AGID. The results of the present study corroborated very well with those reported earlier on naturally (Gwozdz et al., 1997; Singh et al., 2007) and experimentally infected sheep (Millar et al., 1995). “Indigenous serum ELISA” has been reported to be most sensitive, fast and inexpensive test for screening of goats as compared to ZN staining of impression smears and tissue section as well as tissue PCR (Raguvanshi et al., 2010).
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Several targets have been found to increase the specificity and sensitivity of PCR. IS900 is generally used as a representative target in PCR diagnosis of MAP because it is a MAP specific marker with high sensitivity because there are 12–18 copies of it in the MAP genome (Englund et al., 1999; Millar et al., 1995; Moss et al., 1991; Vary et al., 1990). However, the IS900-like sequence has been found in non-MAP mycobacteria, posing a problem of specificity (Cousins et al., 1999; Englund et al., 2002; Godfroid et al., 2005; Motiwala et al., 2004; Rajeev et al., 2005).
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Citation Excerpt :
Moreover, as biosecurity measures are not practiced at gaushalas in India, wandering of these animals or use of their dung in the nearby farms or grasslands creates a continuous source of exposure for susceptible animals. The PCR-based detection of Map is limited by the low number of Map bacilli in feces [8], poor recovery of DNA due to the lysis-resistant mycobacterial cell wall and by the presence of high concentrations of inhibitors in fecal material [22,23]. The dilution of samples with distilled water and concentration steps prior to DNA extraction were employed in the present study to overcome these limitations.
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In this study, the isolation of 52 mycobactin-independent fast growing mycobacteria from 631 bulk milk samples (8.2%), is reported. These strains, isolated during a bulk milk survey for Mycobacterium avium subsp. paratuberculosis (Map), strongly affected Map detection both by PCR and by culture, as they gave a positive IS900 PCR signal and resulted to totally inhibit the growth of Map when spotted on HEYM slants already inoculated with 200 μl of 10-fold dilutions containing from 5 × 10 to 5 × 10³ Map cells/ml. 16S rRNA gene sequencing, using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems), was performed on a subset of six strains, identifying Mycobacterium porcinum with 100% homology in all six cases. The 52 strains were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene, which confirmed the identification of M. porcinum for all the isolates. Using specific primers designed on the Map-IS900 sequence and on the M. porcinum sequence determined in this study, a 1385 bp sequence from the M. porcinum genome was characterized. This IS900-like sequence showed 82% homology with Map IS900. From our findings the following results emerged: (a) any culture showing one or more M. porcinum colonies represents a potential “false negative” result and should therefore be considered as contaminated; (b) IS900-like elements could be more widespread than was previously thought; (c) IS900 PCR positive results should be interpreted cautiously, as confirmed by the evidence that the primer pair used in this study resulted not to be specific.

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Research ReportSolid-Phase Hybridization Capture of Low-Abundance Target DNA Sequences: Application to the Polymerase Chain Reaction Detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum

Abstract

Research Report
Solid-Phase Hybridization Capture of Low-Abundance Target DNA Sequences: Application to the Polymerase Chain Reaction Detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum