Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases
Year : 2003, Volume : 24, Issue : 2
First page : ( 153) Last page : ( 154)
Print ISSN : 0970-9320.

Diagnosis of Mycoplasma mycoides subsp. mycoides type lc infection by polymerase chain reaction

Kumar Manoj, M., Singh V.P.*, Srivastava N.C., Sharma Bhaskar

National Referral Laboratory on Mycoplasma, Division of Bacteriology & Mycology Indian Veterinary Research Institute, Izatnagar-243122 (U.P.)

*Corresponding authors

Received:  21  February,  2004.

Abstract

PCR assay was undertaken for rapid diagnosis of caprine pleuropneumonnia caused by Mycoplasma mycoides subsp . mycoides type LC directly from the experimental clinical materials without prior isolation of the organisms. Specific amplifications were observed with M. mycoides cluster specific, M. mycoides group specific and M. mycoides subsp . mycoides LC specific primers confirming the suitability of PCR for rapid diagnosis of mycoplasma infections by overcoming the problems associated with their isolation.

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Material and methods

Organism

Mycoplasma mycoides subsp. mycoides LC strain (Y-Goat) obtained from the National Referral Laboratory on Mycoplasma, Division of Bacteriology and Mycology, IVRI, Izatnagar, was used in the present study.

Experimental infection

Five healthy goats of age 6-12 months, whose sera were found to be negative for mycoplasmal antibodies as well as nasal passage was free of mycoplasmal organisms, were taken into the experiment. These were inoculated each with 1 ml (7.3 x 10⁹ cfu) of overnight grown virulent culture of M. mycoides subsp. mycoides LC by intratracheal route. The goats were observed for the development of clinical symptoms for 7 to 10 days. Later on, the clinical materials viz. lungs, lymphnodes, spleen and liver were collected from dead or sacrificed goats and then stored at −20°C till further use.

Extraction of Genomic DNA

DNA was extracted from the above clinical materials as per the protocol of Bashriuddin et al. (1994) with slight modifications. Approximately 1gm of the tissue was homogenized in 1 ml of TE (Tris ethylene diamine tetraacetic acid) buffer (pH 8.0) in a tissue homogenizing tube. A 500 µl of the supernatant was removed from the settled homogenate and the cell pellet was separated by centrifugation at 13,000 rpm for 10 minutes.

The cell pellet was lysed in a mixture of 200 µl of T.E., 20 µl of 10% SDS (Sodium dodecyl sulphate) and 5 µl of proteinase K (20 mg/ml) at 37°C for 1 hour and then it was sequentially extracted with phenol followed by phenol : chloroform : isoamylalcohol. Finally, the DNA was precipitated with 20 µl of 3 M sodium acetate (pH 5.2) and 400 µl of chilled ethanol and left overnight at −20°C. The DNA pellet was washed in 80% ethanol, dried and then re-suspended in 10 µl of TE buffer.

Pcr amplification

Three sets of primers were used for amplification of DNA extracted from the clinical materials. viz. M. mycoides cluster specific primers MC 323 (5′-TAGAGGTACTTTAGATACTCAAGG-3′) and MC 358 (5′-GATATCTAAAGGTGATGGT-3′), M. mycoides group specific primers MM450 (5′-GTATTTTCCTTTCTAATTTG-3′) and MM 451 (5′-AAATCAAATTAATAAGTTTG-3′) (Bashiruddin et al., 1994) and the M. mycoides subsp. mycoides LC specific primers P4 (5′-ACTGAGCAATTCCTCTT-3′) and P5 (5′-TTAAATAAGTTTGTATATGAAT-3′) (Hotzel et al., 1996). Each reaction mixture consisted of 5 µl of 10 x PCR buffer (Sigma), 200 µm of each dNTP (Sigma), 50 p mol of each primer (Genei), 1.5 units of Taq DNA polymerase (Genei), 1 µl of the extracted DNA and distilled water added to make up the volume ot 50 µl and overlaid with 340 µl of mineral oil (Sigma).

The DNA amplifications were carried out in a thermocycler (Techne, U.K.) at cycling conditions of initial denaturation at 94°C for 1 minute and final extension at 72°C for 5 minutes with 30 intermediary cycles of denaturation at 94°C for 1 minute, annealing at 50°C for 1 minute and extension at 72°C for 2 minutes. For the primer pair of P4/P5, a slightly different programme was used, consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 1 minute and extension at 72°C for 1^½ minutes. A 10 µl aliquot of each PCR product was characterized on 1% agarose gel alongwith 100 bp ladder marker (Gibco-BRL). The gels were stained with ethidium bromide and photographed under UV transilluminator.

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Results and discussion

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Figure

Fig. 1: PCR using DNA extracted from M. mycoides subsp. mycoides LC infected materials. ? ? ? ? ?Lane M : 100bp ladder; Lane 1: M. mycoides cluster specific PCR using primers MC323/MC358; Lane 2: M. mycoides group specific PCR using primers MM450/MM451; Lane 3: M. mycoides subsp. mycoides type LC specific PCR using primers TopBack

Acknowledgements

The authors are thankful to the Head, Division of Bacteriology & Myclogy and Director, IVRI, Izatnagar for providing the necessary facilities for carrying out this work.

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