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M-PCR (Multiplex Polymerase Chain Reaction) primers, probes and detection methods for vibrio cholerae, vibrio parahaemolyticus and salmonella

A technology for hemolytic Vibrio and Vibrio cholerae, which is applied in the field of detection of pathogenic microorganisms in food, can solve the problems of multiple PCR technology and achieve high sensitivity and specificity

Inactive Publication Date: 2011-08-17
麻丽丹 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The multiple real-time PCR technology that uses more than one pair of primers and probes with different fluorescent markers to detect in the PCR reaction has been used in the detection of multiple pathogenic bacteria in the same sample, but it has not been seen in the prior art. Multiplex PCR available for detection of Vibrio cholerae, Vibrio parahaemolyticus and Salmonella

Method used

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  • M-PCR (Multiplex Polymerase Chain Reaction) primers, probes and detection methods for vibrio cholerae, vibrio parahaemolyticus and salmonella
  • M-PCR (Multiplex Polymerase Chain Reaction) primers, probes and detection methods for vibrio cholerae, vibrio parahaemolyticus and salmonella
  • M-PCR (Multiplex Polymerase Chain Reaction) primers, probes and detection methods for vibrio cholerae, vibrio parahaemolyticus and salmonella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, establishment of detection method

[0035] 1. Design of primers and probes

[0036] According to the results of homologous gene screening comparison, the fimY gene (SEQ ID NO.10) of Salmonella, the hlyA gene (SEQ ID NO.11) of Vibrio cholerae and the toxR of Vibrio parahaemolyticus were selected with high specificity The gene (SEQ ID NO.12) is the target gene for detecting amplification. According to the nucleic acid sequences of the above-mentioned genes published on NCBI, and through comprehensive consideration of factors such as the consistency of the annealing temperature of the detection primers and probes and the similarity of GC content, the following specific primers and probes were designed:

[0037] ① Detection primer and probe sequence of fimY gene of Salmonella:

[0038] Forward primer (SEQ ID NO.1): 5'-CCGTATGGCTGGGCGTTT-3',

[0039] Reverse primer (SEQ ID NO.2): 5'-AGTACGGCTAAAGCTTTCCGATAAG-3',

[0040] Probe (SEQ ID NO.3): 5'FAM-CAGAGGCCA...

Embodiment 2

[0070] Embodiment 2, specificity test

Embodiment 3

[0078] Embodiment 3, sensitivity test

[0079] 1. Detection sensitivity of pure culture of standard strains

[0080] Using the specific primers, probes and detection methods of Example 1, the detection sensitivity of pure cultures of Salmonella, Vibrio cholerae and Vibrio parahaemolyticus standard strains was studied.

[0081] Take the above three standard strains and culture them in ordinary broth medium for about 18 hours, measure their OD value, estimate the number of bacteria, and then carry out gradient dilution to 10 times in ten-fold increments. -7 , take the bacterial solution of the last four dilutions for plate counting, repeat 3 times for each dilution, perform colony counting and take the average value after culturing according to the corresponding method to determine the true bacterial density. Simultaneously each dilution respectively gets 1mL bacterium liquid in 2mL centrifuge tube and makes 3 tubes to repeat, is used for extracting DNA and applies the method o...

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Abstract

The invention discloses m-PCR (Multiplex Polymerase Chain Reaction) primers, probes and detection methods for vibrio cholerae, vibrio parahaemolyticus and salmonella. The primers and the probes are detection primer and probe for fimY genes of the salmonella with sequences shown as SEQ ID NO. 1 to 3 (Sequence Identity Numbers 1 to 3), detection primer and probe for hlyA genes of the vibrio cholerae with sequences shown as SEQ ID NO. 4 to 6 and detection primer and probe for toxR genes of the vibrio parahaemolyticus with sequences shown as SEQ ID NO. 7 to 9 respectively. On the basis, the invention also discloses a detection method containing the primer and probe sequences. The probes and the primers have high specificity; and detection kits and the method are simple and convenient, are easy to use, and have accurate results and extremely high specificity and sensitivity.

Description

technical field [0001] The invention relates to a method for detecting pathogenic microorganisms in food, in particular to a method for detecting Vibrio cholerae, Vibrio parahaemolyticus and Salmonella in samples by using real-time fluorescent PCR technology. It also relates to the specific primer and probe sequences used for detection. Background technique [0002] Rapid and accurate detection of pathogenic bacteria in food is an important condition for effective prevention and control of pathogenic bacteria infection. Among the existing detection methods, conventional microbial detection methods are still used for the detection of Vibrio cholerae, Vibrio parahaemolyticus, and Salmonella. These methods are time-consuming and laborious, and cannot meet the requirements of rapid and high-throughput detection. If it is positive, it will affect the analysis of the results and may cause greater economic losses. Moreover, in food testing, one sample is often used to detect seve...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/42C12R1/63
CPCY02A50/30
Inventor 麻丽丹于兵王殿夫高世光陈晓东冯颖
Owner 麻丽丹
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