Dataset Information

LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples.

ABSTRACT: Animal species detection is one of the crucial steps for consumer's food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to minimum 5% level of cow components admixed in goat milk/meat samples and can be completed within 1 h 40 min starting from DNA extraction from milk/meat samples and can be performed in a water bath. Developed LAMP methodology is simple; rapid and sensitive techniques that can detect adulterant like cow components in goat milk/meat are more accurate than other existing DNA based technologies.

SUBMITTER: Deb R

PROVIDER: S-EPMC5534258 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples.

Deb R R Sengar G S GS Singh U U Kumar S S Raja T V TV Alex R R Alyethodi R R RR Prakash B B

Iranian journal of veterinary research 20170101 2

Animal species detection is one of the crucial steps for consumer's food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to ...[more]

PMID: 28775755

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Project description:BackgroundAmphimeriasis is a fish-borne disease caused by the liver fluke Amphimerus spp. that has recently been reported as endemic in the tropical Pacific side of Ecuador with a high prevalence in humans and domestic animals. The diagnosis is based on the stool examination to identify parasite eggs, but it lacks sensitivity. Additionally, the morphology of the eggs may be confounded with other liver and intestinal flukes. No immunological or molecular methods have been developed to date. New diagnostic techniques for specific and sensitive detection of Amphimerus spp. DNA in clinical samples are needed.Methodology/principal findingsA LAMP targeting a sequence of the Amphimerus sp. internal transcribed spacer 2 region was designed. Amphimerus sp. DNA was obtained from adult worms recovered from animals and used to optimize the molecular assays. Conventional PCR was performed using outer primers F3-B3 to verify the proper amplification of the Amphimerus sp. DNA target sequence. LAMP was optimized using different reaction mixtures and temperatures, and it was finally set up as LAMPhimerus. The specificity and sensitivity of both PCR and LAMP were evaluated. The detection limit was 1 pg of genomic DNA. Field testing was done using 44 human stool samples collected from localities where fluke is endemic. Twenty-five samples were microscopy positive for Amphimerus sp. eggs detection. In molecular testing, PCR F3-B3 was ineffective when DNA from fecal samples was used. When testing all human stool samples included in our study, the diagnostic parameters for the sensitivity and specificity were calculated for our LAMPhimerus assay, which were 76.67% and 80.77%, respectively.Conclusions/significanceWe have developed and evaluated, for the first time, a specific and sensitive LAMP assay for detecting Amphimerus sp. in human stool samples. The procedure has been named LAMPhimerus method and has the potential to be adapted for field diagnosis and disease surveillance in amphimeriasis-endemic areas. Future large-scale studies will assess the applicability of this novel LAMP assay.

Project description:BackgroundStrongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples.Methodology/principal findingsStool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests.Conclusions/significanceOur Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially applied for effective diagnosis of strongyloidiasis in a clinical setting.

Project description:Milk fatty acids are essential for many dairy product productions, while intramuscular fat (IMF) is associated with the quality of meat. The triacylglycerols (TAGs) are the major components of IMF and milk fat. Therefore, understanding the polymorphisms and genes linked to fat synthesis is important for animal production. Identifying quantitative trait loci (QTLs) and genes associated with milk and meat production traits has been the objective of various mapping studies in the last decade. Consistently, the QTLs on chromosomes 14, 15, and 9 have been found to be associated with milk and meat production traits in cattle, goat, and buffalo and sheep, respectively. Diacylglycerol O-acyltransferase 1 (DGAT1) gene has been reported on chromosomes 14, 15, and 9 in cattle, goat, and buffalo and sheep, respectively. Being a key role in fat metabolism and TAG synthesis, the DGAT1 has obtained considerable attention especially in animal milk production. In addition to milk production, DGAT1 has also been a subject of interest in animal meat production. Several polymorphisms have been documented in DGAT1 in various animal species including cattle, buffalo, goat, and sheep for their association with milk production traits. In addition, the DGAT1 has also been studied for their role in meat production traits in cattle, sheep, and goat. However, very limited studies have been conducted in cattle for association of DGAT1 with meat production traits in cattle. Moreover, not a single study reported the association of DGAT1 with meat production traits in buffalo; thus, further studies are warranted to fulfill this huge gap. Keeping in view the important role of DGAT1 in animal production, the current review article was designed to highlight the major development and new insights on DGAT1 effect on milk and meat production traits in cattle, buffalo, sheep, and goat. Moreover, we have also highlighted the possible future contributions of DGAT1 for the studied species.

Dataset Information

LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples.

Publications

LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples.

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