A Polymerase Chain Reaction Procedure for the Detection of Salmonella spp. within 24 Hours

1998 ◽  
Vol 61 (8) ◽  
pp. 1039-1042 ◽  
Author(s):  
PIETER A. GOUWS ◽  
MARINDA VISSER ◽  
VOLKER S. BRÖZEL
Keyword(s):  
Detection Method ◽  
Detection Methods ◽  
Salmonella Spp ◽  
Culture Methods ◽  
Conventional Culture ◽  
Food Borne ◽  
Pcr Product ◽  
Peptone Water ◽  
Polymerase Chain ◽  
Phosphate Buffered Saline

Salmonella spp. are one of the most important groups of food-borne pathogens worldwide. Conventional methods for the detection of Salmonella spp. in foodstuffs are generally cumbersome and time consuming. Whereas various more rapid detection methods have been developed over the past few years, there is currently no reliable true 24-hour detection method available. We report here a reliable Salmonella PCR detection method yielding results within 24 h. Chicken samples were preenriched in buffered peptone water (BPW) for 6 h. The DNA was extracted using phosphate-buffered saline (PBS) and then heated at 95°C for 10 min. The Salmonella-specific primers ST11 and ST15 were used to amplify a 429-bp region specific to all Salmonella spp. This approach proved to be sufficient for the reliable detection of Salmonella spp. from both artificially and naturally contaminated poultry samples. The characteristic 429-bp PCR product was obtained in artificially contaminated samples with a detection limit of 50 CFU. A variety of chicken samples confirmed to harbor Salmonella spp. by conventional culture methods tested positive by our 24-h procedure, whereas no detectable amplification product was detected in those samples testing negative by culture methods. This method proved to be an excellent tool for the rapid and sensitive detection of Salmonella spp. from poultry samples using a specific primer set (ST11 and ST15) after only 6 h of preenrichment.

scholarly journals Yersinia enterocoliticaandYersinia pseudotuberculosisDetection in Foods

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
H. Fukushima ◽  
S. Shimizu ◽  
Y. Inatsu
Keyword(s):  
Food Safety ◽  
Detection Methods ◽  
Culture Methods ◽  
Selective Media ◽  
Conventional Culture ◽  
Food Borne Pathogens ◽  
Food Borne ◽  
Isolation Methods ◽  
Pcr Assays ◽  
Virulence Properties

Yersinia enterocoliticaandY. pseudotuberculosiswhich can cause yersiniosis in humans and animals are thought to be significant food-borne pathogens and be important as hygiene indicator in food safety. The pathogenicY. enterocoliticaserotypes/biotypes are O:3/4 and 3 variant VP negative, O:5, 27/2, O:8/1b, and O:9/2, have been reported worldwide.Y. pseudotuberculosisis distributed less widely thanY. enterocolitica. Isolation methods usually involve selective and recovery enrichment of the food sample followed by plating onto selective media, confirmation of typical colonies and testing for virulence properties of isolated strains. Recently, DNA-based methods, such as PCR assays, have been developed to detect pathogenicY. enterocoliticaandY. pseudotuberculosisin foods more rapidly, and sensitivity than can be achieved by conventional culture methods. This paper reviews commercially available conventional and PCR-based procedures for the detection of pathogenicYersiniain food. These methods are effective as the isolation and detection methods to target pathogenicY. enterocoliticaandY. pseudotuberculosisin foods.

scholarly journals DETEKSI BAKTERI PENYEBAB PENYAKIT TYPHUS PADA MANUSIA DENGAN POLYMERASE CHAIN REACTION

2011 ◽  
Vol 1 (1) ◽  
pp. 45
Author(s):  
Muktiningsih Nurjayadi ◽  
Fera Kurnia Dewi ◽  
Dahlia Dahlia ◽  
S, Restu.N S ◽  
Fitri W
Keyword(s):  
Polymerase Chain Reaction ◽  
Detection Method ◽  
Serological Test ◽  
Research Result ◽  
Salmonella Typhi ◽  
Detection Methods ◽  
Chain Reaction ◽  
Specificity And Sensitivity ◽  
Dna Fragment ◽  
Polymerase Chain

Salmonella typhi is bacteria that cause typhoid disease in humans. In Indonesia, the morbidity number of typhoid disease tends to be increase. Thus, it has been requiring the alternative for handling or preventing that disease. Recently, the detection method commonly uses for S. typhi detection is Serological test. The weakness of this method is often producing less accurate and not specific detection. The previous research was successfully discovered S. typhi gene that codes protein which is contributed at adherents or colonization those bacteria in epithelial human cell. That result was base to develop detection on S. typhi method by Polymerase chain reaction (PCR). The aim of this research is developing a specific and accurate detection method for S. typhi bacteria by PCR. The research result is performed successfully to amplify the fimbrial-C S. typhi gene using pairs of primer FW-INT 2- REV-1A NEW which was designed and synthesized in previous step. That success showed by the finding of the DNA fragment of 0.2 kilobase (kb) proffers to size of DNA fragment which is hopefully in using S. typhi genome as a template. Specificity and sensitivity test for those primers are still conducting to reproducibility results. Base on the results can be concluded that the research have successfully conducted in developing S. typhi detection method using pairs of S. typhi fimbrial-C primer. Hopefully, the studied of developing detection methods was conducted better compare with former detection methods.Keywords: S. typhi detection method, fim-C S. typhi gene, PCRAbstrakSalmonella typhi merupakan bakteri penyebab penyakit tifus pada manusia. Di Indonesia, angka morbiditas penderita penyakit typhus cenderung meningkat, sehingga diperlukan suatu alternatif untuk penanganan atau pencegahan penyakit tersebut. Sampai saat ini metode deteksi S. typhi yang banyak digunakan adalah uji serologi. Kelemahan metode ini adalah sering menghasilkan deteksi yang kurang akurat dan tidak spesifik. Pada penelitian yang dilakukan sebelumnya, telah berhasil ditemukan gen fimbrial-C S. typhi pengkode protein yang berperan dalam penempelan S. typhi pada usus manusia, hasil ini dijadikan landasan untuk pengembangan metode deteksi menggunakan teknik PCR. Tujuan penelitian ini mengembangkan metode deteksi yang akurat dan spesifik untuk bakteri penyebab penyakit typhus pada manusia. Hasil penelitian menunjukkan bahwa telah berhasil dilakukan amplifikasi gen fimbrial-C S. typhi menggunakan pasangan primer hasil perancangan yaitu FW-INT 2- REV-1A NEW. Keberhasilan tersebut ditunjukkan dengan diperolehnya pita DNA berukuran 0.2 kilo basa (kb) sesuai dengan ukuran pita DNA yang diharapkan dengan menggunakan template DNA genom bakteri S. typhi. Uji sensitivitas dan spesifisitas terhadap primer hasil rancangan sedang di kaji lebih lanjut untuk memperoleh reprodusibiltas hasil pengujian. Berdasarkan hasil yang diperoleh dapat disimpulkan bahwa telah berhasil dilakukan pengembangan metode deteksi S. typhi menggunakan pasangan primer fimbrial-C S. typhi. Pengkajian pengembangan metode deteksi yang dihasilkan ini diharapkan dapat lebih baik dibanding beberapa metode deteksi yang sudah ada.Kata Kunci: Metode Deteksi Bakteri typhus, fim-C S. typhi, PCR

scholarly journals An outbreak of viral gastroenteritis associated with consumption of sandwiches: implications for the control of transmission by food handlers

1998 ◽  
Vol 121 (3) ◽  
pp. 615-621 ◽  
Author(s):  
U. D. PARASHAR ◽  
L. DOW ◽  
R. L. FANKHAUSER ◽  
C. D. HUMPHREY ◽  
J. MILLER ◽  
...  
Keyword(s):  
Food Handler ◽  
Common Source ◽  
Viral Gastroenteritis ◽  
Food Handlers ◽  
Rt Pcr ◽  
Source Of Infection ◽  
Food Borne ◽  
Pcr Product ◽  
Stool Specimens ◽  
Polymerase Chain

Although food handlers are often implicated as the source of infection in outbreaks of food-borne viral gastroenteritis, little is known about the timing of infectivity in relation to illness. We investigated a gastroenteritis outbreak among employees of a manufacturing company and found an association (RR=14·1, 95% CI=2·0–97·3) between disease and eating sandwiches prepared by 6 food handlers, 1 of whom reported gastroenteritis which had subsided 4 days earlier. Norwalk-like viruses were detected by electron microscopy or reverse transcriptase-polymerase chain reaction (RT-PCR) in stool specimens from several company employees, the sick food handler whose specimen was obtained 10 days after resolution of illness, and an asymptomatic food handler. All RT-PCR product sequences were identical, suggesting a common source of infection. These data support observations from recent volunteer studies that current recommendations to exclude food handlers from work for 48–72 h after recovery from illness may not always prevent transmission of Norwalk-like viruses because virus can be shed up to 10 days after illness or while exhibiting no symptoms.

scholarly journals Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Salmonella spp. in Terms of Sensitivity and Applicability

2020 ◽  
pp. 1-5
Author(s):  
Yanhong Liu ◽  
Deguo Wang ◽  
Meng Zhang ◽  
Yanhong Liu ◽  
Yongzhen Wang
Keyword(s):  
Genomic Dna ◽  
Pathogenic Bacteria ◽  
Detection Limits ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
23S Rrna ◽  
Lamp Method ◽  
Salmonella Spp ◽  
Loop Mediated Isothermal Amplification ◽  
Food Borne

Salmonella spp. are important food-borne pathogens that can cause diseases in humans. Many detection methods have been established in Salmonella spp. using loop-mediated isothermal amplification (LAMP) or reverse transcription loop-mediated isothermal amplification (RT-LAMP). The detection limits of these assays varied from 1 CFU/reaction to 104 CFU/reaction, from 100 fg genomic DNA/reaction to 10 pg genomic DNA/reaction, or from 2.0×101 CFU/mL to 107 CFU/mL for food samples. In this study, LAMP assays were developed using genomic DNA for the detection of Salmonella spp. Two sets of LAMP primers were designed using the invA gene and the 16S-23S rRNA intergenic spacer region (ITS) of S. enterica as the target sequences for two LAMP assays. The detection limits of the two methods were respectively 20 pg S. enterica DNA/reaction and 10 pg S. enterica DNA/reaction at the optimized temperature, and the LAMP methods were of high repeatability and specificity for S. enterica detection. This study provides a baseline for the application of LAMP for the detection of food-borne pathogenic bacteria.

scholarly journals Probing the dissonance among the diagnostic outputs of multiple approaches used for detection of Methicillin-resistant Staphylococcus aureus (MRSA)

2020 ◽  
Author(s):  
Ujjwal Ranjan Dahiya ◽  
Arnab Sikidar ◽  
Priyanka Sharma ◽  
Chitra Rawat ◽  
Benu Dhawan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Culture Method ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Diagnostic Efficiency ◽  
Coagulase Negative Staphylococci ◽  
Culture Methods ◽  
Methicillin Resistant ◽  
Conventional Culture

Methicillin-resistant staphylococcus aureus (MRSA) is an extremely infectious hospital acquired bacterial pathogen often found in post-surgical patients globally. Early detection of such pathogens is a critical requirement to eliminate or reduce the incidence of antimicrobial resistance as well as for effective management of the disease. Despite the development of multiple biochemical, microbiological and nucleic acid amplification techniques (NAATs), conventional culture methods are widely used clinically owing to high variability between the methods, technical skills, and infrastructural needs. Further, multiple reports suggest a significant variation among diagnostic output for MRSA detection. This work attempts to probe the discordance among the diagnostic output of three commonly used methods while trying to understand the underlying cause of variability. MRSA detection on 217 clinical pus isolates was carried out using three different methods namely, conventional culture method, qPCR-based amplification, and a modern LAMP-based detection approach. Also, to confirm the presence of MRSA and distinguish from coagulase-negative staphylococci (CoNS), as well as to investigate the observed differences between qPCR and LAMP outputs, melt curve analysis was performed on discordant samples. LAMP-based MRSA detection was found to be the optimum method. In summary, this study evaluates the diagnostic efficiency of the different detection methods, while probing for possible explanations for the observed differences.

Evaluation of Detection Methods for Screening Meat and Poultry Products for the Presence of Foodborne Pathogens

2005 ◽  
Vol 68 (12) ◽  
pp. 2637-2647 ◽  
Author(s):  
VALERIE M. BOHAYCHUK ◽  
GARY E. GENSLER ◽  
ROBIN K. KING ◽  
JOHN T. WU ◽  
LYNN M. McMULLEN
Keyword(s):  
Foodborne Pathogens ◽  
Culture Method ◽  
Lateral Flow ◽  
Detection Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Processed Meat ◽  
Culture Methods ◽  
E Coli ◽  
Poultry Products ◽  
Conventional Culture

Rapid and molecular technologies such as enzyme-linked immunosorbent assay (ELISA), PCR, and lateral flow immunoprecipitation can reduce the time and labor involved in screening food products for the presence of pathogens. These technologies were compared with conventional culture methodology for the detection of Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7 inoculated in raw and processed meat and poultry products. Recommended protocols were modified so that the same enrichment broths used in the culture methods were also used in the ELISA, PCR, and lateral flow immunoprecipitation assays. The percent agreement between the rapid technologies and culture methods ranged from 80 to 100% depending on the pathogen detected and the method used. ELISA, PCR, and lateral flow immunoprecipitation all performed well, with no statistical difference, compared with the culture method for the detection of E. coli O157:H7. ELISA performed better for the detection of Salmonella, with sensitivity and specificity rates of 100%. PCR performed better for the detection of Campylobacter jejuni, with 100% agreement to the culture method. PCR was highly sensitive for the detection of all the foodborne pathogens tested except Listeria monocytogenes. Although the lateral flow immunoprecipitation tests were statistically different from the culture methods for Salmonella and Listeria because of false-positive results, the tests did not produce any false negatives, indicating that this method would be suitable for screening meat and poultry products for these pathogens.

Isolation, identification, molecular detection and sensitivity to antibiotics of Salmonella from cattle faeces

2021 ◽  
Vol 24 (1) ◽  
pp. 57-66
Author(s):  
M. N. K. Khan ◽  
M. R. Das ◽  
M. A. Sabur ◽  
M. M. Rahman ◽  
M. B. Uddin ◽  
...  
Keyword(s):  
Antibiotic Sensitivity ◽  
Culture Method ◽  
Sensitivity Study ◽  
Culture Methods ◽  
Isolation And Identification ◽  
Conventional Culture ◽  
Faecal Samples ◽  
Antibiotic Sensitivity Test ◽  
Cattle Faeces ◽  
Polymerase Chain

The present study was designed with the aim of isolation and identification of Salmonella by con-ventional culture method and their confirmation by polymerase chain reaction (PCR). Antibacte-rial sensitivity study of isolated Salmonella from cattle faeces was also performed. During the study period of July 2017 to June 2018, a total of 200 faecal samples were collected from different government and private farms in Sylhet district of Bangladesh. Out of 200 samples, 24 (12%) were found to be positive for Salmonella by conventional culture methods. Among the twenty four suspected colonies of Salmonella, seventeen were confirmed by biochemical test and same number was detected by PCR estimating a prevalence of 8.5% (17/200). The prevalence was high-er in calves under 1 year of age (16%) compared with older animals (11.25% of 1–2 years; 10% of above 2 years of age) but without statistically significant differences (χ2=4.835, P=0.089). Moreo-ver, in diarrhoeic animals the prevalence was significantly higher (32.14%, χ2=49.414, P<0.01) than in apparently healthy animals (8.72%). The antibiotic sensitivity test showed that highest number of Salmonella isolates were sensitive to ciprofloxacin (100%), gentamicin (100%) and neomycin (100%). On the other hand, significantly high resistance of Salmonella isolates was detected to erythromycin (100%), amoxicillin (100%), cotrimoxazole (81.48%), streptomycin (62.96%) followed by tetracycline (55.56%).

Evaluation of TA10 Broth for Recovery of Listeria monocytogenes from Ground Beef

2017 ◽  
Vol 100 (2) ◽  
pp. 470-473
Author(s):  
Naoko Kamisaki-Horikoshi ◽  
Yukio Okada ◽  
Kazuko Takeshita ◽  
Makoto Takada ◽  
Shinichi Kawamoto ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Ground Beef ◽  
Culture Method ◽  
Most Probable Number ◽  
Salmonella Spp ◽  
Probable Number ◽  
Enrichment Broth ◽  
Conventional Culture ◽  
Significant Difference ◽  
Peptone Water

Abstract In 2009, the enrichment broth TA10 was released for simultaneous recovery of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7. This medium was compared with other Salmonella enrichment broths [lactose (LAC) broth, buffered peptone water (BPW), and universal pre-enrichment (UP) broth] for the recovery of heat- and freeze-injured Salmonella spp. in beef by the conventional culture method. There was a significant difference between TA10 and LAC enrichment broths for detecting injured Salmonella spp. In this study, the International Organization for Standardization Listeria pre-enrichment broth (Half-Fraser/Fraser) was compared with TA10 broth for the recovery of L. monocytogenes from ground beef. Ground beef samples were contaminated with single Listeria serovars at levels of 0.096 to 0.001 most probable number/g. Twenty 25 g test portions of the contaminated ground beef were pre-enriched in each broth, and the ISO-11290-1 Listeria official isolation protocol was used thereafter. There was a significant difference between TA10 broth (48 h) and Half-Fraser/Fraser broth (72 h) in the recovery of L. monocytogenes. In addition, the incubation time for TA10 broth was shorter than for Half-Fraser/Fraser broth. The results indicate that TA10 broth should be used instead of Half-Fraser/Fraser broth for analysis of beef that may be contaminated with very low levels of L. monocytogenes.

scholarly journals The water environment as a source of potentially pathogenic mycobacteria

2013 ◽  
Vol 12 (2) ◽  
pp. 254-263 ◽  
Author(s):  
Jitka Makovcova ◽  
Michal Slany ◽  
Vladimir Babak ◽  
Iva Slana ◽  
Petr Kralik
Keyword(s):  
Water Environment ◽  
Polymerase Chain Reaction Analysis ◽  
Epidemiologic Studies ◽  
Fish Ponds ◽  
Culture Methods ◽  
Conventional Culture ◽  
Principal Source ◽  
Polymerase Chain ◽  
First Time ◽  
Fresh Water Environment

Nontuberculous mycobacteria (NTM) are ubiquitous organisms of a wide variety of environmental reservoirs, including natural and municipal water, soil, aerosols, protozoans, animals and humans. Several of these species are potential pathogens which affect human health. The aim of this study was to determine the occurrence of NTM in the water environment. Samples were taken from 13 water-related facilities including fish ponds, storage ponds, drinking water reservoirs and an experimental recirculation system. Altogether, 396 samples of water, sediment and aquatic plants were collected and analysed. All samples were examined using conventional culture methods. Suspected microbial isolates were subjected to polymerase chain reaction analysis and identified using partial sequence analysis of the 16S rDNA gene. The culture revealed 94/396 samples (23.7%) that contained mycobacteria. Among known NTM we identified potentially pathogenic mycobacteria isolated from the fresh water environment for the first time: Mycobacterium asiaticum, M. chimaera, M. interjectum, M. kumamotonense, M. lentiflavum, M. montefiorense, M. nebraskense, M. paraffinicum and M. simiae. Epidemiologic studies suggest that the natural water environment is the principal source of human exposure. Our results indicate that besides the well-known potentially pathogenic mycobacteria it is important to observe occurrence, proliferation and persistence of newly discovered mycobacterial species.

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