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
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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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