Multiplex detection and identification of pathogens Escherichia coli, Bacillus cereus, Clostridioides difficile, and Vibrio parahaemolyticus using PCR high-resolution melt assays

2020 ◽  
Author(s):  
Allison C. Bender ◽  
Jessica Faulkner ◽  
Katherine Tulimieri ◽  
Kelly M. Elkins
Keyword(s):  
Escherichia Coli ◽  
High Resolution ◽  
Bacillus Cereus ◽  
Vibrio Parahaemolyticus ◽  
Multiplex Detection ◽  
High Resolution Melt ◽  
Clostridioides Difficile ◽  
Detection And Identification

scholarly journals High Resolution Melt Assays to Detect and Identify Vibrio parahaemolyticus, Bacillus cereus, Escherichia coli, and Clostridioides difficile Bacteria

2020 ◽  
Vol 8 (4) ◽  
pp. 561
Author(s):  
Allison C. Bender ◽  
Jessica A. Faulkner ◽  
Katherine Tulimieri ◽  
Thomas H. Boise ◽  
Kelly M. Elkins
Keyword(s):  
Escherichia Coli ◽  
High Resolution ◽  
Bacillus Cereus ◽  
Vibrio Parahaemolyticus ◽  
Bacterial Species ◽  
Human Pathogens ◽  
High Resolution Melt ◽  
E Coli ◽  
Clostridioides Difficile ◽  
Sensitivity Specificity

Over one hundred bacterial species have been determined to comprise the human microbiota in a healthy individual. Bacteria including Escherichia coli, Bacillus cereus, Clostridioides difficile, and Vibrio parahaemolyticus are found inside of the human body and B. cereus and E. coli are also found on the skin. These bacteria can act as human pathogens upon ingestion of contaminated food or water, if they enter an open wound, or antibiotics, and environment or stress can alter the microbiome. In this study, we present new polymerase chain reaction (PCR) high-resolution melt (HRM) assays to detect and identify the above microorganisms. Amplified DNA from C. difficile, E. coli, B. cereus, and V. parahaemolyticus melted at 80.37 ± 0.45 °C, 82.15 ± 0.37 °C, 84.43 ± 0.50 °C, and 86.74 ± 0.65 °C, respectively. A triplex PCR assay was developed to simultaneously detect and identify E. coli, B. cereus, and V. parahaemolyticus, and cultured microorganisms were successfully amplified, detected, and identified. The assays demonstrated sensitivity, specificity, reproducibility, and robustness in testing.

scholarly journals Detecting and Differentiating Phytoplasmas Belonging to Subgroups 16SrIV-A and 16SrIV-D Associated With Lethal Declines of Palms in Florida Using qPCR and High-Resolution Melt Analysis (HRMA)

Plant Disease ◽  
2017 ◽  
Vol 101 (8) ◽  
pp. 1449-1454 ◽  
Author(s):  
Brian W. Bahder ◽  
Ericka E. Helmick ◽  
Nigel A. Harrison
Keyword(s):  
High Resolution ◽  
Sequence Data ◽  
Management Strategies ◽  
Economic Losses ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis ◽  
Melting Temperatures ◽  
Detection And Identification ◽  
Lethal Yellowing ◽  
Sufficient Variation

Lethal yellowing (LY) and Texas Phoenix palm decline (TPPD) are two important phytoplasma diseases of palms in Florida. Both have been responsible for major economic losses historically and remain a constant threat to the sustainability of palm production in the landscaping and nursery industries in Florida. These two diseases cause rapid, lethal decline in afflicted palms, so rapid detection and identification is crucial to implement appropriate management strategies to reduce further spread and losses. In this study, a qPCR assay was developed to detect and identify the causal agents of LY and TPPD. Based on sequence data of the 16S gene for the 16SrIV-A phytoplasma (LY) and the 16SrIV-D phytoplasma (TPPD), two regions were identified in the gene that possessed sufficient variation to yield amplicons with measurable differences in melting temperature based on high resolution melt analysis (HRMA). One region was in the 5′ region and the other was located in the 3′ region of the gene. Products from both regions yielded amplicons with significantly different melting temperatures between the two phytoplasma strains. This research allows for the detection and identification of phytoplasmas in palms rapidly by eliminating many lengthy and post-PCR steps commonly used in phytoplasma identification.

A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food

2016 ◽  
Vol 79 (5) ◽  
pp. 810-815 ◽  
Author(s):  
FEREIDOUN FORGHANI ◽  
SHUAI WEI ◽  
DEOG-HWAN OH
Keyword(s):  
Staphylococcus Aureus ◽  
Listeria Monocytogenes ◽  
High Resolution ◽  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Cost Savings ◽  
High Resolution Melt ◽  
And Staphylococcus Aureus

ABSTRACTThree important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 103 CFU/g without an enrichment step and 3.7 × 101 CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus.

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