• 대한수의학회지
• /
• 제54권2호
• /
• pp.113-115
• /
• 2014

Salmonella are causative agents of gastroenteritis and systemic disease in animals. The invA gene was selected as a target sequence of loop-mediated isothermal amplification (LAMP) assay for diagnosis of Salmonella infection. The detection limits for broth dilution, spiked feces and enrichment were $10^4$, $10^5$ and $10^2$ CFUs/mL, respectively. The LAMP assay developed in the present study may be a reliable method for detection of Salmonella spp. in pig feces.

• Animal Bioscience
• /
• 제36권4호
• /
• pp.654-670
• /
• 2023

Objective: This study aimed to develop loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) and compare it with LAMP-AGE, polymerase chain reaction (PCR), and standard Salmonella culture as reference methods for detecting Salmonella contamination in animal products and animal production environmental samples. Methods: The SalInvA01 primer, derived from the InvA gene and designed as a new probe for LFD detection, was used in developing this study. Adjusting for optimal conditions by temperature, time, and reagent concentration includes evaluating the specificity and limit of detection. The sampling of 120 animal product samples and 350 animal production environmental samples was determined by LAMP-LFD, comparing LAMP-AGE, PCR, and the culture method. Results: Salmonella was amplified using optimal conditions for the LAMP reaction and a DNA probe for LFD at 63℃ for 60 minutes. The specificity test revealed no cross-reactivity with other microorganisms. The limit of detection of LAMP-LFD in pure culture was 3×10² CFU/mL (6 CFU/reaction) and 9.01 pg/μL in genomic DNA. The limit of detection of the LAMP-LFD using artificially inoculated in minced chicken samples with 5 hours of pre-enrichment was 3.4×10⁴ CFU/mL (680 CFU/reaction). For 120 animal product samples, Salmonella was detected by the culture method, LAMP-LFD, LAMP-AGE, and PCR in 10/120 (8.3%). In three hundred fifty animal production environmental samples, Salmonella was detected in 91/350 (26%) by the culture method, equivalent to the detection rates of LAMP-LFD and LAMP-AGE, while PCR achieved 86/350 (24.6%). When comparing sensitivity, specificity, positive predictive value, and accuracy, LAMP-LFD showed the best results at 100%, 95.7%, 86.3%, and 96.6%, respectively. For Kappa index of LAMP-LFD, indicated nearly perfect agreement with culture method. Conclusion: The LAMP-LFD Salmonella detection, which used InvA gene, was highly specific, sensitive, and convenient for identifying Salmonella. Furthermore, this method could be used for Salmonella monitoring and primary screening in animal products and animal production environmental samples.

• 한국축산식품학회지
• /
• 제32권2호
• /
• pp.241-246
• /
• 2012

This report outlines the development of a rapid, simple, and sensitive detection system for pathogenic bacteria using a capillary electrophoresis-based, single strand conformation polymorphism (CE-SSCP) combined with PCR. We demonstrate that this method, used with primers targeting the V4 region of the16S rRNA gene, is capable of the simultaneous detection of 10 microbes that could be associated with foodborne illness, caused by animal-derived foods: Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7, Campylobacter jejuni, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Yersinia enterocolitica, Vibrio parahaemolyticus, and Enterobacter sakazakii. The traditional detection techniques are time-consuming and labor-intensive, due to the necessary task of separate cultivation of each target species. As such, the CE-SSCP-PCR method, that we have developed, has the potential to diagnose pathogens rapidly, unlike the traditional technique, in order to prevent foodborne illness in a much more efficient manner.

• Journal of Animal Science and Technology
• /
• 제65권4호
• /
• pp.748-758
• /
• 2023

This study was conducted to investigate the change in activity and mounting behavior in Hanwoo (Korean Native Cattle) during the peri-estrus period and its application to estrus detection. A total of 20 Hanwoo cows were fitted with a neck-collar accelerometer device, which measured the location and acceleration of cow movements and recorded the number of instances of mounting behavior by the altitude data. The data were analyzed in three periods (24-, 6-, and 2-h periods). Blood samples were collected for 5 days after the prostaglandin F2α (PGF2α) injection, and the concentrations of estradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone were determined by enzyme-linked immunosorbent assays. Activity and mounting behavior recorded over 2-h periods significantly increased as estrus approached and were more efficient at detecting estrus than over 24- and 6-h periods (p < 0.05). Endocrine patterns did not differ with the variation of individual cows during the peri-estrus period (p > 0.05). Activity was selected as the best predictor through stepwise discriminant analysis. However, activity alone is not enough to detect estrus. We suggest that a combination of activity and mounting behavior may improve estrus detection efficiency in Hanwoo. Further research is necessary to validate the findings on a larger sample size.

• Journal of Veterinary Science
• /
• 제19권6호
• /
• pp.855-857
• /
• 2018

Porcine parvovirus 7 (PPV7) was first detected in Korean pig farms in 2017. The detection rate of PPV7 DNA was 24.0% (30/125) in aborted pig fetuses and 74.9% (262/350) in finishing pigs, suggesting that PPV7 has circulated among Korean domestic pig farms. Phylogenetic analysis based on capsid protein amino acid sequences demonstrated that the nine isolated Korean strains (PPV-KA1-3 and PPV-KF1-6) were closely related to the previously reported USA and Chinese PPV7 strains. In addition, the Korean strains exhibit genetic diversity with both insertion and deletion mutations. This study contributes to the understanding of the molecular epidemiology of PPV7 in Korea.

• Journal of Dairy Science and Biotechnology
• /
• 제29권2호
• /
• pp.51-57
• /
• 2011

Milk and dairy products are nutritionally one of the most important food in human health and the quality of raw milk is significantly important to ensure safety of dairy products. However, milk and dairy products are commonly related with chemical and microbial contaminations. Therefore, rapid and reliable detection of hazardous (e.g. pathogenic bacteria, pesticides, antibiotics, microbial toxins) in milk and dairy products is essential to ensure human health and food safety. Conventional methods for detection of food hazardous are mostly time-consuming to yield a results. Recently, biosensors have been focused as its rapidity and high sensitivity to analyse chemical and microbial hazardous from a variety of foods and environments. This study reviewed the recent trends and applications of biosensors as rapid detection method of hazardous in milk and dairy products.

• 한국축산식품학회지
• /
• 제34권5호
• /
• pp.717-723
• /
• 2014

A rapid and specific PCR assay for the simultaneous detection of Listeria monocytogenes, Escherichia coli O157:H7, Bacillus cereus, Salmonella spp., and Staphylococcus aureus in foods was developed to reduce the detection time and to increase sensitivity. Multiplex PCR developed in this study produced only actA, fliC, hbl, invA, ileS amplicons, but did not produce any non-specific amplicon. The primer sets successfully amplified the target genes in the multiplex PCR without any non-specific or additional bands on the other strains. The multiplex PCR assays also amplified some target genes from five pathogens, and multiplex amplification was obtained from as little as 1 pg of DNA. According to the results from the sensitivity evaluation, the multiplex PCR developed in this study detected 10 cells/mL of the pathogens inoculated in milk samples, respectively. The results suggested that multiplex PCR was an effective assay demonstrating high specificity for the simultaneous detection of five target pathogens in food system.

• Journal of Dairy Science and Biotechnology
• /
• 제31권1호
• /
• pp.59-65
• /
• 2013

Antibiotic residues are undesirable in milk and milk products for a number of reasons. In particular, they can have harmful effects on public health and harm to the manufacturer of the cultured milk products, e.g. MRSA etc. Although government regulatory agencies and the dairy industry have been successful in decreasing the presence of high concentrations of antibiotic residues, violations still occur and lead to contaminated products. As a result, several rapid and reliable methods for the detection of antibiotic residues have been developed, including microbiological and instrumental analysis methods. The conventional methods are time consuming, but recent improvements have allowed for better detection time, sensitivity, and accuracy. An example of an advanced detection instrument is the biosensor, which has several applications in food and environmental science, e.g. food-born pathogen detection, antimicrobial residues etc. In the present review, the recent trends in the methods used to test for antibiotic residues in milk and dairy products, as well as their specific applications, have been discussed.

• 한국축산식품학회지
• /
• 제32권6호
• /
• pp.706-712
• /
• 2012

The present study was aimed to produce a chicken polyclonal antibody against Cronobacter muytjensii and to develop an immunoassay for its detection. Purification of anti-C. muytjensii IgY from egg yolk was accomplished using various methods such as water dilution and salt precipitation. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 30 and 66 kDa, corresponding to a light and a heavy chain, respectively. Indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was performed to determine the effectiveness of the chicken IgY against C. muytjensii. The optimum conditions for detecting C. muytjensii by indirect ELISA and checkerboard titration of the antigen revealed an optimum average absorbance at the concentration of 18 ${\mu}g/mL$, having ca. $10^8$ coated cells per well. The anti-C. muytjensii IgY antibody had high specificity for C. muytjensii and low cross-reactivity with other tested pathogens. In this assay, no cross-reactivity was observed with the other genera of pathogenic bacteria including Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, Bacillus cereus, Enterobacter aerogenes, Salmonella Enteritidis and Listeria monocytogenes. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of IC-ELISA for C. muytjensii, with similar detection limit of $10^5$ CFU/mL as shown in standard curve. These findings demonstrate that the developed method is able to detect C. muytjensii in infant formula powder. Due to the stable antibody supply without sacrificing animals, this IgY can have wide applications for the rapid and accurate detection of C. muytjensii in dairy foods samples.

• 한국동물위생학회지
• /
• 제45권1호
• /
• pp.19-30
• /
• 2022

In this study, a simple loop-mediated isothermal amplification (LAMP) combined with visual detection method (vLAMP) assay was developed for the rapid and specific detection of African swine fever virus (ASFV), overcoming the shortcomings of previously described LAMP assays that require additional detection steps or pose a cross-contamination risk. The assay results can be directly detected by the naked eye using hydroxynaphthol blue after incubation for 40 min at 62℃. The assay specifically amplified ASFV DNA and no other viral nucleic acids. The limit of detection of the assay was <50 DNA copies/reaction, which was ten times more sensitive than conventional polymerase chain reaction (cPCR) and comparable to real-time PCR (qPCR). For clinical evaluation, the ASFV detection rate of vLAMP was higher than cPCR and comparable to OIE-recommended qPCR, showing 100% concordance, with a κ value (95% confidence interval) of 1 (1.00~1.00). Considering the advantages of high sensitivity and specificity, no possibility for cross-contamination, and being able to be used as low-cost equipment, the developed vLAMP assay will be a valuable tool for detecting ASFV from clinical samples, even in resource-limited laboratories.