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Multi-DPO-PCR primer composition for detecting vibrio parahaemolyticus and vibrio cholerae

A DPO-PCR, Vibrio cholerae technology, applied in biochemical equipment and methods, bioreactor/fermenter combination, recombinant DNA technology, etc., can solve the problems of complex system, low stability, limited use, etc., to achieve High throughput, high sensitivity, and wide applicability

Inactive Publication Date: 2016-08-17
湛江出入境检验检疫局检验检疫技术中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the addition of multiple pairs of primers in the PCR reaction system, the system is complex and the stability is not high, which limits its use

Method used

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  • Multi-DPO-PCR primer composition for detecting vibrio parahaemolyticus and vibrio cholerae
  • Multi-DPO-PCR primer composition for detecting vibrio parahaemolyticus and vibrio cholerae
  • Multi-DPO-PCR primer composition for detecting vibrio parahaemolyticus and vibrio cholerae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This example provides two sets of primer pairs and their applications.

[0058] The screening method of two sets of primer pairs is as follows: for Vibrio parahaemolyticus collagenase gene (GenBank ID: AF326572.1) and Vibrio cholerae ompW gene (GenBank ID: X51948.1), multiple multiplex DPO-PCR primers were designed and carried out A large number of screening, comprehensive its specificity, sensitivity, the interaction between primers, and the adaptability of each primer used to the multiplex DPO-PCR amplification kit, finally screened out the specificity, good repeatability and The following combination of multiple DPO-PCR primers with high sensitivity can simultaneously detect Vibrio parahaemolyticus and Vibrio cholerae (I in the primer sequence is hypoxanthine):

[0059] VP-F: 5'-AGCGCAAGTCACAGAGAAAGTTGAIIIIIIATCAGCACGA-3' (SEQ ID NO: 1);

[0060] VP-R: 5'-CTTTGCCACGTTGTACATGTGGTIIIIIITCAAACGCCG-3' (SEQ ID NO: 2);

[0061] VC-F: 5'-CGCTCCTGTATTTGCTCACCAAGIIIIIIGACTT...

Embodiment 2

[0067] This embodiment provides a kit and application thereof, the kit comprising:

[0068] (1) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 (from Example 1);

[0069] (2) dNTPs, Mg 2+ , PCR reaction buffer, and at least one of Taq DNA polymerase.

[0070] The above dNTPs, Mg 2+ , PCR reaction buffer, and Taq DNA polymerase are preferably derived from a kit with a product number of RR060A from TaKaRa Company.

[0071] It further preferably includes a DNA extraction kit, and the DNA extraction kit is preferably derived from a bacterial genome DNA extraction kit with a product number of DP302 from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0072] It is further preferred to include a positive control and a negative control.

[0073] Experiments have shown that the above-mentioned bacterial genomic DNA extraction kit, the kit with the product number RR060A of TaKaRa Company, and the combined use with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO...

Embodiment 3

[0076] This embodiment provides a method for detecting or assisting in the detection of Vibrio parahaemolyticus and Vibrio cholerae, such as detecting or assisting in detecting whether a biological sample is infected with Vibrio parahaemolyticus and Vibrio cholerae, or detecting or assisting in detecting whether the pathogenic bacteria is or is a candidate For Vibrio parahaemolyticus and Vibrio cholerae, the method uses two sets of primer pairs in Example 1 or the kit in Example 2.

[0077] Above-mentioned method comprises the steps:

[0078] Step 1: Multiplex DPO-PCR amplification

[0079] Using the DNA of biological samples or pathogenic bacteria as a template, two sets of primer pairs are used to perform multiple DPO-PCR amplification to obtain multiple DPO-PCR amplification products, and a blank control is set at the same time (the template is ultrapure water).

[0080] The multiplex DPO-PCR reaction system is shown in Table 1. Among them, Mix 2 contains dNTPs, MgCl 2 a...

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Abstract

The invention provides two primer pairs and further provides application of the two primer pairs for detecting or detecting vibrio parahaemolyticus and vibrio cholerae in an aided mode or preparing products for detecting or detecting vibrio parahaemolyticus and vibrio cholerae in an aided mode. The invention further provides a kit comprising the two primer pairs and application, and a method for detecting vibrio parahaemolyticus and vibrio cholerae on the basis of the two primer pairs or the kit. An experiment proves that the two primer pairs are good in specificity and high in sensitivity and detection efficiency, the established multi-DPO-PCR method for vibrio parahaemolyticus and vibrio cholerae on the basis of the two primer sets is high in throughput, sensitive, accurate and rapid, and the more effective method is provided for simultaneously detecting vibrio parahaemolyticus and vibrio cholera.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to methods, primers and kits for detecting Vibrio parahaemolyticus and Vibrio cholerae. Background technique [0002] Multiplex PCR refers to adding multiple pairs of primers in one reaction tube to simultaneously amplify multiple target genes, which can achieve high-throughput detection of samples, and has been widely used in the detection of genetically modified species. Due to the addition of many pairs of primers in the PCR reaction system, the system is complicated and the stability is not high, which limits its use. The main principle of DPO (Dual priming oligonucleotide) primer technology is that its primers contain two independent specific primer regions, the 5' end sequence consists of 18-25 bases and is paired with the target gene sequence, and the 3' end sequence consists of 6 -12 bases are used to guide the specific extension of the PCR reaction. These two independen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/04C12M1/00C12M1/34
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2545/113Y02A50/30
Inventor 龙阳汪天杰魏霜马新华袁俊杰张娜杨卓瑜杨劲丁秀琼刘骁
Owner 湛江出入境检验检疫局检验检疫技术中心
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