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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  References

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ORIGINAL ARTICLE
Year : 2002  |  Volume : 20  |  Issue : 1  |  Page : 25-28
 

Arbitrarily primed PCR- A rapid and simple method for typing of leptospiral serovars


Department of Animal Biotechnology, Madras Veterinary College, Chennai - 600 007, India

Correspondence Address:
Department of Animal Biotechnology, Madras Veterinary College, Chennai - 600 007, India

 ~ Abstract 

PURPOSE: To investigate the use of arbitrarily primed polymerase chain reaction (AP-PCR) for typing of leptospiral serovars. METHODS: AP-PCR was adopted for identification of laboratory strains of leptospires and leptospiral cultures at serovar level. A primer of 12 bp was used for amplifying DNA of 13 laboratory strains of leptospires as well as culture pellets of leptospires. RESULTS: Each serovar produced distinct DNA fingerprint which was characteristic for each serovar. These patterns were used for typing of 81 serum culture samples obtained from human leptospiral cases. Of these samples, 39 could be typed based on AP-PCR fingerprints belonging to serovars autumnalis, pomona, canicola, javanica, icterohaemorrhagiae, patoc and pyrogenes. These results were confirmed by RAPD fingerprinting of the DNA samples of the respective leptospiral serovars after culturing -*them in EMJH media. One of the important findings of this work was that straight culture sample could be used for AP-PCR assay, without purification of DNA. By having more number of AP-PCR reference fingerprints, more serovars could be typed. CONCLUSIONS: AP-PCR technique provides great potential for simple and rapid identification of leptospires at serovar level, which could be useful in molecular epidemiological studies of leptospirosis.

How to cite this article:
Ramadass P, Latha D, Senthilkumar A, Srinivasan P, Saranya N. Arbitrarily primed PCR- A rapid and simple method for typing of leptospiral serovars. Indian J Med Microbiol 2002;20:25-8


How to cite this URL:
Ramadass P, Latha D, Senthilkumar A, Srinivasan P, Saranya N. Arbitrarily primed PCR- A rapid and simple method for typing of leptospiral serovars. Indian J Med Microbiol [serial online] 2002 [cited 2019 Oct 19];20:25-8. Available from: http://www.ijmm.org/text.asp?2002/20/1/25/8334


Leptospirosis, which is caused by Leptospira interrogans, affects men and animals and has a worldwide distribution. Diagnosis of leptospirosis is important for early treatment of infected individuals and for better prognosis. Currently, there are over 212 recognized serovars of leptospires, divided into 23 serogroups.[1] Traditionally, leptospires are classified and identified by cross absorption test.[2] However, this technique is laborious and shows poor inter-laboratory reproducibility. Monoclonal antibody methods are more rapid and specific,[3] but cannot distinguish all serovars. DNA-based methods for identification of leptospires include: DNA restriction endonuclease analysis (REA),[4],[5] DNA-DNA hybridization,[6],[7] polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP)[8],[9] and random amplified polymorphic DNA (RAPD) fingerprinting.[10],[11]
REA, though highly discriminatory, requires DNA from purified cultures for analysis and the number of DNA bands produced are more, making the comparison difficult. Slot blot hybridization method is used for typing different serovars. However, the procedure is too lengthy and laborious. PCR-RFLP method differentiates few serovars. RAPD methods have shown some promise for differentiating individual serovars, however, purified DNA samples are required for RAPD fingerprinting.
The present study investigated the use of arbitrarily primed PCR method (AP-PCR) for typing of leptospiral serovars from direct serum culture and was found to be a good and sensitive method.

 ~ Materials and Methods Top

Bacterial strains
A total of 13 commonly prevalent laboratory strains of leptospiral serovars were grown in EMJH media (Difco Lab, USA) along with 5-fluorouracil which prevented the growth of other bacteria. Following serovars were taken for the study: autumnalis (strain Akiyami A), australis (strain Ballico), bataviae (strain Swart), canicola (strain Hond Utrecht IV), hardjo (strain Hardjoprajitno), hebdomadis (strain Hebdomadis), icterohaemorrhagiae (strain RGA), javanica (strain Veldrat Bat.46), pomona (strain Pomona), pyrogenes (strain Salinem), carlos (strain C3), ramisi (strain Musa) and weerasinghe (strain Weerasinghe).
DNA extraction

DNA was extracted from the laboratory strains by treatment with lysozyme, sodium dodecyl sulphate and protease.[12] After overnight incubation at 550C in a waterbath, the DNA was purified by repeated extraction with phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol/vol), followed by a final extraction with chloroform. The DNA was concentrated by precipitation with 99% ethanol.
Serum samples
Blood samples were collected from human patients who were suspected to be suffering from leptospirosis and the serum was separated and tested for the presence of leptospires under Dark field microscopy (DFM). The DFM positive serum samples were stored in freezer until used.
Serum Culture
One loopful of serum was used for the inoculation into semisolid EMJH media (Difco) for culturing of leptospires. It was cultured for 7-21 days at 210C until turbidity of cells could be seen.
Processing of serum culture
One loopful of serum culture samples were taken in 100 無 of phosphate buffered saline and centrifuged at 10,000 rpm for 20 minutes to pellet the leptospires. The pellets were suspended in 20無 of autoclaved triple distilled water. From this, 5 無 was used for PCR reaction after boiling for 10 minutes in a boiling water bath.

Arbitrarily-primed PCR

Primer PB-1, 5'-GCG CTG GCT CAG -3' was used for AP-PCR analysis[13] of laboratory strains of leptospires as well as of serum culture samples. PCR mixture (final volume 50 mL) contained about 50 ng of purified DNA (1 無) or 5 無 of processed serum culture sample, 0.3 然 primer, 250 然 of each dNTP, 3 mM MgCl2, 0.5 U of Taq DNA polymerase (Finnzymes) in 10 mM Tris.HCl (pH 9.0) and 50 mM KCl. Healthy human serum samples were used as negative controls.
The PCR amplification was carried out in a thermal cycler (DNA Engine, MJ Research, USA) using 0.5 mL thick microfuge tubes. The temperature for amplification consisted of one cycle of 7 minutes at 940蚓, 1 minute at 40蚓 and 1 minute at 72蚓; four cycles of 1 minute at 94蚓, 1 minute at 40蚓 and 1 minute at 720C; 24 cycles of 1 minute at 94蚓, 1 minute at 55蚓, 1 minute at 72蚓 and one cycle of 1 minute at 94蚓, 1 minute at 55蚓 and 7 minutes at 720C. Reaction products (about 20 無) were electrophoresed on a 2% agarose gel using Tris borate buffer (pH 8.3) containing ethidium bromide (0.5 痢/mL).
Standard AP-PCR patterns were produced first using the purified DNA of standard laboratory leptospiral strains. It was used for the comparative analysis of AP-PCR patterns obtained from direct serum culture and serum.
RAPD Fingerprinting
Random amplified polymorphic fingerprinting of DNA of leptospiral isolates were carried out as per the methods of Gerritsen et al.[14] and Ramadass et al.[11] A pair of primers of following sequences were used for amplification.
Primer 1: 5' CCGGA AGAAG GGGCG CCAT 3'
Primer 2: 5' CGATT TAGAA GGACT TGCAC AC 3'

 ~ Results Top

The purified DNA of commonly prevalent laboratory strains were subjected to AP-PCR and different patterns were obtained. The bands were spread out between the size range of 100 bp to 3000 bp and representative patterns for few of the laboratory strains are given in [Figure - 1] Serovars like bataviae and icterohaemorrhagiae produced lesser number of bands, while serovars like patoc, autumnalis and hardjo produced more number of bands.
A total of 146 human serum samples were screened by DFM and 126 serum samples were found positive by DFM. All DFM positive serum samples were inoculated into EMJH media for culturing. Of these, 81 samples showed positive culture and all these were examined by AP-PCR method. Of these samples, 39 could be typed to belong to one or other serovar. Since we did AP-PCR with direct serum culture samples without purifying the DNA, the patterns obtained were similar to that of the standard patterns obtained with the purified DNA. Only differences in some minor bands were found. Few samples did not produce any amplified products and few of the patterns did not match with the DNA fingerprints available in the laboratory. Of the 39 samples typed, serovar autumnalis was identified in 14 of the samples, serovar pomona in nine samples, serovar canicola and javanica were identified in 5 samples each, serovar icterohaemorrhagiae in 4 samples and serovar patoc and pyrogenes in one each case. This typing pattern agreed with the typing carried out with RAPD fingerprinting method.
[Figure:2] shows representative patterns obtained from serum culture samples. We could identify serovars pyrogenes (lane 1), javanica (lane 4) and pomona (lane 6). Other samples (lanes 2, 3 and 5) could not be identified, as it did not match any of the patterns we have Figure:2a. Similarly, in Figure:2b, we could type six samples as belonging to serovars javanica (lane 1) and autumnalis (lanes 2, 3, 4, 5 and 6). Sample in lane 7 could not be identified since the bands were not
Though more number of bands were obtained when using DNA in contrast to serum culture samples, few serovar specific bands could be identified for each serovar. The purity of DNA sample definitely improves the RAPD fingerprints obtained. However, the fingerprints obtained with serum culture samples are quite clear enough to identify the serovar.

 ~ Discussion Top

AP-PCR method has been found to be an useful and reasonably simple and quick method for typing of leptospires, when compared to cross absorption test, nucleic acid probe technology and RAPD fingerprinting method. Brown and Levett13 reported similar results for AP-PCR in differentiating different serovars but in their study few of the closely related serovars produced similar patterns like pomona and georgia, icterohaemorrhagiae and copenhageni and pomona and tarassovi. These workers used DNA samples in their study. However, in the present study, each of the serovars tested produced different patterns. Our earlier study11 showed the usefulness of RAPD fingerprints for typing leptospires. But, that procedure required purified DNA after leptospiral culturing. The present study has proved that even straight culture samples could be used directly for AP-PCR amplification and the resultant DNA pattern produced could be used for the typing of the leptospires.
In an earlier work,[15] AP-PCR method differentiated hardjo isolates of the Hardjoprajitno and Hardjobovis genotypes. These workers also used purified DNA samples for AP-PCR analysis. Similarly, Ralph et al [8] categorized leptospiral serovars from various serogroups by AP-PCR method.
In this study the patterns obtained with the direct serum culture samples were confirmed by purifying their DNA and their AP-PCR pattern which confirmed the direct serum culture typing and also the whole experiment was further confirmed by RAPD technique using the purified DNA of serum culture. The results from this study show that AP-PCR is a simple and rapid method for typing leptospiral serovars. The main advantage of this method is that the culture samples could be directly used for typing, thus characterization of leptospires at serovar level without the need for DNA preparation. 

 ~ References Top

1.Kmety E and Dikken H. Revised list of Leptospira serovars. University of Groningen, Groningen, The Netherlands 1988.  Back to cited text no. 1    
2.Cole JR, Sulzer CR and Pursell AR. Improved microtechnique for the leptospiral microscopic agglutination test. Appl Microbiol 1973; 25: 976-980.  Back to cited text no. 2    
3.Kobayashi Y, Tamai T, Sada E, et al. Antigenic analysis of serogroup Icterohaemorrhagiae using monoclonal antibodies. Isr J Vet Med 1987; 43: 277-287.  Back to cited text no. 3    
4.Marshall RB, Wilton BE and Robinson AJ. Identification of Leptospira serovars by restriction endonuclease analysis. J Med Microbiol 1981; 14: 163-166.  Back to cited text no. 4    
5.Senthilkumar A, Ramadass P and Nachimuthu K. Typing of leptospiral serovars by DNA restriction enzyme analysis. Ind J Ani Sci 1997; 67: 457-459.  Back to cited text no. 5    
6.Yasuda PH, Steigerwalt AG, Sulzer KR, Kaufmann AF, Rogers F and Brenner DJ. Deoxyribonucleic acid relatedness between serogroups and serovars in the family Leptospiraceae with proposals for seven new Leptospira species. Int J Syst Bacteriol 1987; 37: 407-415.  Back to cited text no. 6    
7.Ramadass P, Jarvis BDW, Corner RJ, Penny D and Marshall RB. Genetic characterization of pathogenic Leptospira species by DNA hybridization. Int J Syst Bacteriol 1992; 42: 215-219.   Back to cited text no. 7    
8.Ralph D, McClelland M, Welsh J, Baranton G and Perolat P. Pathogenic Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCR-amplified rRNA genes. J Bacteriol 1993; 175: 973-981.  Back to cited text no. 8    
9.Venkatesha MD. Molecular characterization of Leptospiral serovars. Ph.D Thesis submitted to the Tamil Nadu Veterinary and Animal Sciences University, Chennai (1997.  Back to cited text no. 9    
10.Corney BG, Colley J, Djordjevic SP, Whittington R and Graham GC. Rapid identification of some Leptospira isolates from cattle by random amplified polymorphic DNA fingerprinting. J Clin Microbiol 1993; 31: 2927-2932.  Back to cited text no. 10    
11.Ramadass P, Meerarani S, Venkatesha MD, Senthilkumar A and Nachimuthu K. Characterization of Leptispiral serovars by Random amplified polymorphic DNA fingerprinting. Int J Syst Bacteriol 1997; 47: 575-576.  Back to cited text no. 11    
12.Van Eys GJJM, Gravekamp C, Gerritsen MJ, Quint W, Cornelissen MTE, Ter Schegget J and Terpstra WJ. Detection of leptospira in urine by polymerase chain reaction. J Clin Microbiol 1989; 27: 2258-2262.  Back to cited text no. 12    
13.Brown PD and Levett PN. Differentiation of Leptospira species and serovars by PCR-restriction endonuclease analysis, arbitrarily primed PCR and low-stringency PCR. J Med Microbiol 1997; 46: 173-181.   Back to cited text no. 13    
14.Gerritsen MA, Smits MA and Olyhoek T. RAPD fingerprinting for rapid identification of leptospires of serogroup Sejore. J med microbiol 1995;42: 336-339.  Back to cited text no. 14    
15.Perolat P, Merien F, Ellis WA and Baranton G. Characterization of Leptospira isolates from serovar Hardjo by Ribotyping, Arbitrarily primed PCR and Mapped restriction site polymorphisms. J Clin Microbiol 1994; 32: 1949-1957.   Back to cited text no. 15    
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