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Vibrio parahaemolyticus constant-temperature detection primer set, detection kit and detection method capable of avoiding false negative detection result

A detection kit and a technology for detecting primers, which are applied in the field of molecular biology detection, can solve the problems of the harm of Vibrio parahaemolyticus and the complexity of detection samples, and achieve the effects of low primer-dimer probability, simple identification and high sensitivity.

Inactive Publication Date: 2015-06-24
苏州华麦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the detection samples are complicated, and because the current detection methods do not adopt monitoring measures for false negative detection results, there is a possibility of missed detection, but Vibrio parahaemolyticus is serious, and the damage caused by missed detection is immeasurable. In addition, PCR amplification requires expensive Many basic testing units do not have the corresponding testing conditions

Method used

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  • Vibrio parahaemolyticus constant-temperature detection primer set, detection kit and detection method capable of avoiding false negative detection result
  • Vibrio parahaemolyticus constant-temperature detection primer set, detection kit and detection method capable of avoiding false negative detection result
  • Vibrio parahaemolyticus constant-temperature detection primer set, detection kit and detection method capable of avoiding false negative detection result

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Establishment of Vibrio parahaemolyticus LAMP detection kit

[0057] LAMP-PCR detection kit for Vibrio parahaemolyticus, including detection primer set, internal standard primer set, LAMP reaction solution, Bst DNA polymerase, positive control, negative control and internal standard.

[0058] (1) Detection primer set: take the Vibrio parahaemolyticus VPtlh gene as the target gene to design LAMP primers, the detection primer set includes a pair of outer primers, a pair of inner primers and a pair of loop primers, the nucleotide sequences of which are respectively As follows:

[0059] VPtlh-F3: 5'-TGATTCGTTTGACGGACG-3' (SEQ ID NO.1);

[0060] VPtlh-B3: 5'-GAACAAGGCGTGAGTATCAA-3' (SEQ ID NO. 2);

[0061] VPtlh-FIP: 5'-ACTTAAACTGAGGCGCTTTCGTAGGTGCGAAGAACTTCATG-3' (SEQ ID NO. 3);

[0062] VPtlh-BIP: 5'-TCGTGCGAAAGTGCTTGAGATTGATGTCGTAACCTTGCG-3' (SEQ ID NO.4);

[0063] VPtlh-LF: 5'-CGTCTGGCAGTGTCATCA-3' (SEQ ID NO.5);

[0064] VPtlh-LB: 5'-GGCTCAAGCGATGTACTAC...

Embodiment 2

[0076] The LAMP detection method of embodiment 2 Vibrio parahaemolyticus

[0077] Utilize the LAMP detection kit of Vibrio parahaemolyticus of embodiment 1 to detect the sample, the steps are as follows:

[0078] (1) Extract the DNA of the sample to be tested.

[0079] (2) Using the LAMP primer composition of claim 1 to carry out LAMP constant temperature amplification of the sample DNA to be tested:

[0080] The 25μl reaction system of LAMP constant temperature amplification contains: VPtlh-F3 0.2μM, VPtlh-B3 0.2μM, VPtlh-FIP 1.6μM, VPtlh-BIP 1.6μM, VPtlh-LF 0.8μM, VPtlh-LB 0.8μM, VPgyrB-F3 0.2 μM, VPgyrB-B3 0.2μM, VPgyrB-FIP 1.6μM, VPgyrB-BIP 1.6μM, VPgyrB-LoopF ​​0.8μM, VPgyrB-LoopB 0.8μM, LAMP reaction solution 12.5μl, DNA polymerase 8U, 10×SYBR Green I 0.5μl , 2 μl of the sample to be tested, 2 μl of the internal standard, make up to 25 μl with ultrapure water;

[0081] The program of LAMP constant temperature amplification is: react at 63-65°C for 30-60min, and cont...

Embodiment 3

[0087] Embodiment 3 Sensitivity experiment

[0088] The constructed plasmid was subjected to a sensitivity experiment, and the 1 pg / μl plasmid was diluted in a 10-fold gradient into four gradients of 1 pg / μl, 100 fg / μl, 10 fg / μl, and 1 fg / μl as quality control standards. 2 for detection, the minimum detection limit of the internal standard gene was determined to be 10 fg / μl through a sensitivity experiment (such as figure 1 shown), and review the minimum detection limit ( figure 2 ), and set the internal standard concentration as the concentration of the lowest detection limit.

[0089] Cultivate the cultured Vibrio parahaemolyticus, perform 10-fold serial dilution of the cultured bacteria, extract DNA, and count the diluted bacterial solution on a plate, and compare the plate culture counting results with the lowest sensitivity of this kit, The lowest detection degree of this kit is 2.4×10 3 CFU / mL, test results see image 3 .

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Abstract

The invention discloses a vibrio parahaemolyticus constant-temperature detection primer set, detection kit and detection method. The primer kit comprises a detection primer set and an internal standard primer set. The detection primer set is designed according to the vibrio parahaemolyticus VPtlh gene, and the sequences are disclosed as SEQ ID NO.1-6. The internal standard primer set is designed according to the vibrio parahaemolyticus VPgrB gene, and the sequences are disclosed as SEQ ID NO.7-12. Besides the primer set, the Vibrio parahaemolyticus constant-temperature detection kit also comprises a DNA (deoxyribonucleic acid) polymerase, an LAMP (loop-mediated isothermal amplification) reaction / internal standard target piece, a positive control and a negative control. The detection primers, kit and detection method have the advantages of high speed, high efficiency, high specificity, high sensitivity and the like, is simple to operate and suitable for field detection, can effectively prevent false negative from generation, and is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to a constant temperature detection primer set, kit and detection method for vibrio parahaemolyticus. Background technique [0002] Vibrio parahemolyticus (Bibrio Parahemolyticus) belongs to non-cholerae Vibrio and is the most common foodborne bacterial pathogen. Vibrio parahemolyticus contamination often occurs in various foods such as aquatic products and pickled products. Clinically, the main symptoms are acute onset, abdominal pain, vomiting, diarrhea and watery stool. [0003] The traditional detection method of Vibrio parahaemolyticus is a simple microbial multiplication step, which is specific to pathogens that use food as a carrier. Although it is reliable, it is laborious and time-consuming, often requiring 4-7 days to complete. Therefore, it is usually not used when it is necessary to evaluate the safety of microorganisms in food in a timel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/63
CPCC12Q1/689C12Q1/6844C12Q2600/166C12Q2531/119C12Q2545/101
Inventor 黄曦黄裴
Owner 苏州华麦生物科技有限公司
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