Multiplex PCR for detection of water-borne bacteria

Microbial water-borne diseases still affect developing countries and are major water quality concerns throughout the world. Routine culture-based methods of identifying bacterial pathogens in water sources are laborious and time-consuming. Recently, the use of molecular techniques such as the polymerase chain reaction (PCR) has provided rapid and highly promising detection methods. In this study, we developed two multiplex PCR assays for simultaneous detection of six water-borne bacteria. Two triplex PCR protocols were developed to detect six target genes. The first protocol targets uidA (Escherichia coli), int (Shigella spp.), and gyrB (Pseudomonas aeruginosa) genes, while invA (Salmonella spp.), ompW (Vibrio cholera), and lacZ (coliforms) were amplified by the second protocol. Specificity testing was carried out for 12 reference strains. Furthermore, the applicability of the multiplex PCR assays for detection of these bacteria was investigated for 52 surface water samples. The results indicated that all primer pairs showed specificities only for their corresponding target organisms. The detection sensitivity of both multiplex PCR assays was 3 × 102 − 3 × 103 colony forming units. The developed assays represent simple and efficient diagnostic procedures for co-detection of water-borne bacteria and have the potential to provide earlier warnings of possible public health threats and more accurate surveillance of these organisms.

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Multiplex PCR for Simultaneous Detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in Meat Samples

Journal of Food Protection ◽

10.4315/0362-028x-68.3.551 ◽

2005 ◽

Vol 68 (3) ◽

pp. 551-556 ◽

Cited By ~ 95

Author(s):

SUSUMU KAWASAKI ◽

NAOKO HORIKOSHI ◽

YUKIO OKADA ◽

KAZUKO TAKESHITA ◽

TAKASHI SAMESHIMA ◽

...

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Simultaneous Detection ◽

Detection Sensitivity ◽

Rapid Screening ◽

Escherichia Coli O157 ◽

Salmonella Spp ◽

E Coli ◽

Meat Samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 103 CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.

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Detection of Sarcocystis Parasites in Retail Beef: A Regional Survey Combining Histological and Genetic Detection Methods

Journal of Food Protection ◽

10.4315/0362-028x-71.10.2144 ◽

2008 ◽

Vol 71 (10) ◽

pp. 2144-2147 ◽

Cited By ~ 37

Author(s):

BOBBI PRITT ◽

THOMAS TRAINER ◽

LINDA SIMMONS-ARNOLD ◽

MARK EVANS ◽

DETIGER DUNAMS ◽

...

Keyword(s):

Molecular Techniques ◽

Detection Sensitivity ◽

Detection Methods ◽

Human Pathogen ◽

Regional Survey ◽

Histologic Section ◽

Sarcocystis Spp ◽

Genetic Detection ◽

Pcr Assays

Sarcocystis spp. are parasitic protists acquired when undercooked, cyst-laden meat is consumed. While both Sarcocystis hominis and S. cruzi encyst in beef, only S. hominis is pathogenic to humans. In this study, we used histological methods and novel molecular techniques to determine the regional prevalence and identity of Sarcocystis spp. in retail beef. Of 110 samples, 60 supported amplification of parasite rRNA by PCR. All 41 sequenced representatives were identified as S. cruzi. To compare detection methods, 48 samples were then examined in parallel by histology and PCR, and 16 and 26 samples, respectively, were positive. Five samples positive by initial histologic sections were not amplified by PCR. Fifteen PCR-positive samples did not contain sarcocysts on initial histologic section, but additional sections from these samples revealed sarcocysts in an additional 12 samples. When combined, histology with additional sections and PCR detected 31 positive specimens of the 48 total specimens. We found no evidence of human pathogen S. hominis and confirm that cattle pathogen S. cruzi is highly prevalent in this regional sample. PCR assays may increase the detection sensitivity of Sarcocystis spp. and contribute diagnostic precision.

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Rapid and Simultaneous Detection of Vibrio parahaemolyticus, Salmonella spp. and Escherichia coli in Fish by Multiplex PCR

SQUALEN Bulletin of Marine and Fisheries Postharvest and Biotechnology ◽

10.15578/squalen.v15i2.444 ◽

2020 ◽

Vol 15 (2) ◽

pp. 53

Author(s):

Radestya Triwibowo ◽

Novalia Rachmawati ◽

Dwiyitno Dwiyitno

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Internal Control ◽

Vibrio Parahaemolyticus ◽

Pathogenic Bacteria ◽

Target Genes ◽

Simultaneous Detection ◽

Salmonella Spp ◽

Fish Products ◽

Confirmatory Assay

Pathogenic bacteria are commonly found as natural contaminants in seafood and fish products. Globally, several countries have been imposing strict regulations on the maximum levels of pathogens and consequently require microbial testing of pathogens before the products can be marketed. A culture-based method with biochemical assay has been widely used to detect pathogenic bacteria in food, despite its long and extensive process. Meanwhile, the alternative molecular-based method to overcome this problem, cannot differentiate between viable and nonviable cells, which may lead to underestimation. This study aimed to develop a multiplex PCR (mPCR) method as a confirmatory assay for the culture-based method to detect pathogens in fish products simultaneously. This method applied a pre-enrichment step to ensure the growth of low-level pathogens and the injured cells in the sample. The target genes were ToxR, InvA, and UidA for Vibrio parahaemolyticus, Salmonella spp. and Escherichia coli, respectively. This assay also amplified the 16S rDNA gene of bacteria as an internal control for the PCR reaction. By implementing liquid-based DNA extraction during analysis, the developed-mPCR was comparable to detect the targeted bacteria in artificially-contaminated samples. The method was more sensitive in naturally-contaminated samples, where the number of E. coli, Salmonella spp. and V. parahaemolyticus detected were 28, 7, and 22, respectively. While the conventional method only detected 26, 5, and 19 of the respective pathogens. With a relatively shorter time and lower operation cost, the mPCR method is potential as an alternative for the culture-based method.

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EVALUATION OF THE ISOLATION AND DETECTION METHODS FOR SALMONELLA SPP. FROM EGG SHELL CONTAMINATION USING MULTIPLEX PCR.

Basrah Journal of veterinary Research ◽

10.33762/bvetr.2015.102445 ◽

2015 ◽

Vol 14 (1) ◽

pp. 282-288

Author(s):

Israa Adnan Ibraheam Al-Baghdady ◽

Ashwak Bassim Jassim ◽

Zainab Khudher Ahmed

Keyword(s):

Multiplex Pcr ◽

Detection Methods ◽

Salmonella Spp ◽

Egg Shell

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Primers with 5′ Flaps Improve the Efficiency and Sensitivity of Multiplex PCR Assays for the Detection of Salmonella and Escherichia coli O157:H7

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-428 ◽

2013 ◽

Vol 76 (4) ◽

pp. 668-673 ◽

Cited By ~ 9

Author(s):

CHRIS TIMMONS ◽

SHEFALI DOBHAL ◽

JACQUELINE FLETCHER ◽

LI MARIA MA

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Fold Increase ◽

Detection Sensitivity ◽

Escherichia Coli O157 ◽

Bacterial Cells ◽

Robust Detection ◽

E Coli ◽

Pcr Multiplex ◽

Pcr Assays

Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5′ end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5′ flap (5′-AATAAATCATAA-3′). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

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