A primer pair and method for detecting the loss of a homologous recombination cell suicide plasmid of Vibrio parahaemolyticus
A technology for suicide plasmids and Vibrio hemolyticus, applied in the field of molecular biology, can solve the problems of difficult detection of plasmid loss, time-consuming and labor-intensive, and achieve high accuracy, simple operation, and strong specificity
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Embodiment 1
[0031] Using pDM4 as a positive control, the clinical isolate HZ of Vibrio parahaemolyticus as a negative control, and the HZ-Δtdh mutant strain of Vibrio parahaemolyticus constructed using the pDM4 plasmid as a sample, the suicide plasmid was detected directly. HZ-Δtdh is a mutant strain that has successfully deleted the tdh gene by homologous recombination technology. After confirming the successful recombination, it has been tested with primers targeting the knocked-out gene tdh. The gene cannot be detected, indicating that the plasmid has been lost.
[0032] The plasmid extraction of pDM4 used Shanghai Bioengineering Co., Ltd. plasmid mini-extraction kit, and the specific steps can be referred to the kit instructions.
[0033] The R6K fragment was amplified by PCR, and the primers were as follows:
[0034] Upstream primer (SEQ ID NO.1): 5'-ATGTCAGCCGTTAAGTGTTCC-3'
[0035] Downstream primer (SEQ ID NO.2): 5'-GAAGATCAGCAGTTCAACCTGTT-3'
[0036] The PCR reaction system is ...
Embodiment 2
[0041] Using pNQ705 as positive control, clinical isolates of Vibrio parahaemolyticus HZ and HZ-Δtdh as negative controls, and Vibrio parahaemolyticus HZ-Δtdh-tdh as samples, the suicide plasmid was detected directly. HZ-Δtdh-tdh uses pNQ705 to knock in the tdh gene at the tdh deletion position of the HZ-Δtdh mutant strain.
[0042] The plasmid extraction of pDM4 used Shanghai Bioengineering Co., Ltd. plasmid mini-extraction kit, and the specific steps can be referred to the kit instructions.
[0043] The R6K fragment was amplified by PCR, and the primers were as follows:
[0044] Upstream primer (SEQ ID NO.1): 5'-ATGTCAGCCGTTAAGTGTTCC-3'
[0045] Downstream primer (SEQ ID NO.2): 5'-GAAGATCAGCAGTTCAACCTGTT-3'
[0046] The PCR reaction system is as follows:
[0047]
[0048]The PCR reaction conditions were: 95°C for 5 minutes; 30 cycles of 94°C for 35s, 60°C for 30s, and 72°C for 30s; 72°C for 10 minutes. The amplified product was electrophoresed in 1% agarose gel, 0.5×T...
Embodiment 3
[0051] HZ-Δvpa1037 and HZ-Δvpa1044 mutant strains were prepared by homologous recombination technology, and pYAK1 plasmid loss was detected for 7 strains of HZ-Δvpa1037 and 4 strains of HZ-Δvpa1044 mutant strains after successful gene exchange.
[0052] The R6K fragment was amplified by PCR, and the primers were as follows:
[0053] Upstream primer (SEQ ID NO.1): 5'-ATGTCAGCCGTTAAGTGTTCC-3'
[0054] Downstream primer (SEQ ID NO.2): 5'-GAAGATCAGCAGTTCAACCTGTT-3'
[0055] The PCR reaction system is as follows:
[0056]
[0057] ●PCR reaction conditions are: 95°C for 5min; 30 cycles of 94°C for 35s, 45°C for 30s, 72°C for 30s; 72°C for 10min.
[0058] The amplified product was electrophoresed in 1% agarose gel, 0.5×TBE buffer, and stained by Goldview
[0059] Analyzed with a gel imaging system.
[0060] Such as image 3 As shown: channel 1 is the positive control of plasmid pYAK1, and a band of 385bp can be detected; channels 2-8 are 7 strains of HZ-Δvpa1037 with successf...
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