STANDARDIZATION OF LOOP MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) FOR IDENTIFICATION OF CLOSTRIDIUM PERFRINGENS
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Date
2014-10
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT:
The Loop Mediated Isothermal Amplification (LAMP) was standardized for rapid detection of Clostridium perfringens toxin types. Different combinations of outer and inner primers were tried and the outer primers concentration of 10p mol/μl and 160p mol/μl of inner primers (FIP and BIP) were found to be optimum for positive LAMP reaction. The enzyme concentration ranging from 0.5 to 1.5μl were tried and the concentration of 1.0μl was found to be optimum for positive LAMP reaction. The temperature of 55 °C and 60 min time was found to be optimum for positive LAMP reaction. The LAMP reaction was tried with and without betaine at 5M concentration and no difference was observed in both the methods. The specificity of the LAMP amplified products were tested by digesting with restriction enzyme XmnI for alpha toxin gene. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in single band in gel electrophoresis Attempts were made to standardize LAMP for amplification of epsilon toxin gene. Amplification of epsilon toxin was not observed with all possible combination of time, temperature and reagents. A total of 120 faecal samples were collected from enterotoxaemia suspected lambs from different regions of chittoor district viz., Peruru, Pudipatla, Srikalahasthi and K.V. Palli. The bacterial lysate of 24h broth culture from clinical samples were used for screening of C. perfringens alpha toxin gene by LAMP. Out of 120 samples screened 38 (31.66%) samples were positive for alpha toxin gene. All 120 samples were further tested by multiplex PCR and found amplification of only alpha toxin gene in 29 (24.16%) samples and amplification of both alpha and epsilon toxin genes in 09 (07.50%) samples, all together 38 (31.66%) positives for C. perfringens which indicated equal sensitivity for both LAMP and multiplex PCR. Out of 120 samples attempted for isolation by culturing only 21 (17.50%) isolates were obtained which indicates low sensitivity of the culturing for isolation when compared LAMP and multiplex PCR. Out of 25 samples tested from Peruru, 10 (40%) isolates were obtained. A total of 20 samples tested from Pudipatla yielded 8 (40%) isolates. Out of 25 samples tested from Srikalahasthi 6 (24%) isolates were obtained. Fifty samples tested from K.V. Palli yielded 14 (28%) isolates. All the isolates were found to be from LAMP and multiplex PCR positive samples.
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