Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

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.


Introduction
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]. The organism is classified into five toxinotypes (A to E) based on the production of four major toxins, namely alpha (cpa), beta (cpb), epsilon (etx), and iota (itx). C. perfringens Type A is implicated in ovine and caprine enterotoxemia in some parts of the world. The cpb2 producing C. perfringens Type A has also 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 synthesis performed by the BstDNA polymerase large fragment [7][8][9][10].
Taking above factors into consideration, attempts were made in the present study to develop LAMP with ease of operation for amplification of cpa gene of C. perfringens.

Reference strain
C. perfringens Type D reference strain (Microbial Type Culture Collection [MTCC]No.49) was obtained from MTCC, Chandigarh was used for standardization of LAMP and as positive control for multiplex PCR reactions. The DNA was extracted from 24 h culture of C. perfringens reference strains by phenol-chloroform-isoamylalcohol method [11].

Collection and processing of samples
A total of 120 dung samples were collected from enterotoxemia suspected lambs of 1-3 months age group. The samples were collected aseptically with sterile swabs and transferred to the lab under ice within 1-2 h. Samples were suspended in sterile phosphate buffer saline and kept in the water bath at 80°C for 5-10 min to eliminate enteric contaminant. The supernatant was inoculated into Robertson's cooked meat medium and incubated anaerobically with anaerobic gas pack (BD, USA) in anaerobic gas jar at 37°C for 24 h. The DNA was extracted from the 24 h broth culture by boiling method and the sample was preserved at −20°C. Available at www.veterinaryworld.org/Vol.9/January-2016/11.pdf

Primer design
Toxin gene specific LAMP primers were designed on the basis of the published sequence of cpa gene of C. perfringens (GenBank accession number, JX091649) with the LAMP primer design software program Primer Explorer V4, from Ekin Chemical Company, Japan (http://primerexplorer.jp/elamp3.0.0/ index.html). A set of four AT-rich primers comprising two outer and two inner primers were designed. The two outer primers were known as the forward outer primer (F3) and the backward outer primer (B3), which helps in strand displacement. The inner primers were known as the forward inner primer (FIP) and the backward inner primer (BIP), respectively. FIP contains F1C (complementary to F1), a TTTT spacer and the F2 sequence. BIP contains the B1C sequence (complementary to B1), a TTTT spacer and B2 sequence. The primer details and location were given in Table-

LAMP amplification
The LAMP amplification was done in hot water bath with thermostat by the following steps with negative controls added with nuclease free water in place of template DNA.
In the initial reaction nuclease free water, Bsm buffer (×10), dNTP mix and template were added into ependorff tube with initial denaturation at 95°C for 5 min. After chilling the samples for 30 s to 1 min, outer primers, inner primers and enzyme were added. LAMP reaction was allowed for 60 min at 55°C followed by enzyme inactivation step for 10 min at 80°C. The amplified LAMP products were stored at −20°C.
LAMP amplification reaction was carried out in hot water bath at 55°C for 60 min. Enzyme inactivation was carried out after completion of reaction at 80°C for 10 min. The amplified LAMP products were stored at−20°C.

Electrophoresis of LAMP products
LAMP products were subjected to agarose gel electrophoresis with 3.0% agarose in tris-borate-ethylenediaminetetraacetic acid buffer with ethidium bromide at 5 μg/ml in genie submerged gel apparatus at 35 mA and amplified products were visualized under ultraviolet (UV) transilluminator and photographed with gel documentation system (Alpha Innotech, Alphaimager, HP).

Testing the specificity of LAMP for cpa using restriction enzyme
To confirm that the amplification product had corresponding DNA sequences, the amplified product was digested with restriction enzyme Xmnl (New  Available at www.veterinaryworld.org/Vol.9/January-2016/11.pdf England Biolabs) with suitable controls. 9 μl of the LAMP product was treated with the restriction enzyme-1μl; NE buffer (×10) -1 μl and incubated at 37°C for 15 min. The product was subjected to electrophoresis for 1 h and visualized under UV transilluminator and photographed using gel documentation system.

LAMP amplification of clinical samples
The reaction mixture was prepared, and LAMP was run as described earlier, with DNA extracted described earlier and visualized under UV-transilluminator.

Screening of clinical samples by PCR
All the 24 h broth culture, samples were screened by PCR for the presence of cpa gene as per the standard protocol [5].

Comparison of diagnostic sensitivity of LAMP with PCR
Diagnostic sensitivity of LAMP was compared with PCR for detection of C. perfringens by testing 120 clinical samples by both the methods.

Results and Discussion
The enzyme Bsm polymerase large fragment with high functional similarity to Bst DNApolymerase, the large fragment was used successfully in the present study in place of Bst DNApolymerase. The enzyme concentration of 1.0 μl was found to be optimum for LAMP reaction in the present study (Figure-2). Same concentration of the Bst DNA polymerase enzyme was used by earlier workers for successful LAMP reaction for amplification of Bordetella pertussis, Capri poxviruses viral genome [6,12,13]. The outer primers concentration of 10 pmol/μl and 160 pmol/μl of inner primers (FIP and BIP) were found to be optimum for LAMP reaction in the present study (Figure-3). Similar combinations of outer and inner primers were successfully used previously for detection of enterotoxigenic C.perfringens from meat samples [14]. 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. Hence, heat denaturation of the double strandard DNA into a single strand, like with PCR was not recommended in LAMP reaction [5]. However, initial denaturation step of template DNA was added in the present study to increase the reaction efficiency as recommended by several previous workers [6,12,13,[15][16][17]. To confirm that the amplification product had corresponding DNA sequences in the present investigation, the amplified product was digested with restriction enzyme XmnI. The enzyme produced single cut at 81 bp within specific recognition sequences in the LAMP amplified product of size 162 bp (Figure-4). The product size was visualized as single band (Figure-5), similar methods were adopted by previous authors to test the specificity of the LAMP products during the amplification of west nile virus and coronovirus genome [6,10,17]. Out of 120 samples screened by LAMP for alpha toxin specific primers 112 (93.30%) samples showed amplification of alpha toxin gene in the samples. Similarly, PCR using 24 h broth culture of fecal samples collected from lambs revealed amplification of alpha toxins gene in all LAMP positive samples indicating similar sensitivity of LAMP and PCR. A similar type of comparison between reverse transcription (RT)-LAMP and RT-PCR was made and it was shown that RT-LAMP was more sensitive than RT-PCR for detection of SARS corona virus from the clinical sample [17]. Though LAMP was said to be having advantages over PCR, the inability of multiplexing in LAMP for simultaneous amplification of more than one gene which is possible with multiplex PCR cannot be over ruled [18]. Moreover, PCR is a basic  technique of DNA amplification with several variants and potential applications in molecular biology apart from its use as a simple diagnostic tool.
Hence, comparison of LAMP with PCR should be limited only to diagnostic applications in clinical laboratories in the developing counties.
Further, standardization of LAMP reaction for amplification of etx and cpb 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 help of sophisticated machinery like thermal cycler. Considering LAMPs simplicity in operation and high sensitivity, the test can be used for clinical diagnosis and surveillance of infectious diseases. To achieve these levels in resource-poor endemic areas, specimen processing methods, and a closed amplification and detection system need to be developed. This will facilitate the provision of a same-day testing strategy in even the most remote rural health facilities.

Conclusion
Based on the present study we conclude that 24 h broth cultures of the fecal samples collected from enterotoxemia suspected lambs could be used for amplification of C. perfringenscpa gene by LAMP and PCR. Further the study reveals the efficacy of toxin genotyping from the clinical samples, compared with the conventional isolation, in vitro and in vivo toxin detection methods. Identification of the prevailing C. perfringens toxin types in the region will help to understand the epidemiology of enterotoxemia in sheep, particularly in lambs which helps in developing suitable control measures.