Abstract
Aim:The loop mediated isothermal amplification (LAMP) was standardized for rapid detection of Clostridium perfringens.Materials and Methods:A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene.Results:Out of 120 samples screened 112 (93.3%) samples were positive by both LAMP and polymerase chain reaction (PCR) for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis.Conclusion:Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases.
Highlights
The Clostridium perfringens is a Gram-positive anaerobic, spore forming, rod-shaped bacteria that commonly found in the gastrointestinal tract of both humans and animals [1]
Same concentration of the Bst DNA polymerase enzyme was used by earlier workers for successful Loop mediated isothermal amplification (LAMP) reaction for amplification of Bordetella pertussis, Capri poxviruses viral genome [6,12,13]
The double strandard DNA in LAMP reaction mixture will be in dynamic equilibrium at the temperature around 65°C and one of the LAMP primers could anneal to the complimentary sequence to initiate LAMP reaction
Summary
The Clostridium perfringens is a Gram-positive anaerobic, spore forming, rod-shaped bacteria that commonly found in the gastrointestinal tract of both humans and animals [1]. C. perfringens Type A is implicated in ovine and caprine enterotoxemia in some parts of the world. The cpb producing C. perfringens Type A has been linked to disease in several animal species, including sheep and goats and poultry [2,3]. Various polymerase chain reaction (PCR) protocols including multiplex PCR assays have been established to genotype C. perfringens isolates [4,5]. PCR got several intrinsic disadvantages, such as requirement of thermal cycling, time-consuming, post-PCR analysis, and high-risk for cross-contamination [6]. Loop mediated isothermal amplification (LAMP) is a novel nucleic acid amplification which relies on auto-cycling strand displacement DNA
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