Kit and detection method for simultaneously detecting vibrio bacteria and edwardsiella ictaluri
The technology of a Vibrio genus and a kit, applied in the field of bacterial detection, can solve the problems of simultaneous detection of Vibrio genus bacteria and Edwardsiella spp., unfavorable mixed infection detection, heavy workload, etc., and achieve good application prospects, Inexpensive and time-consuming results
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Embodiment 1
[0027] Example 1 Primer Design
[0028] 1. Experimental method
[0029] 1. PCR primer design and synthesis
[0030] Two pairs of primers were designed according to the common rpoA gene sequence of Vibrio bacteria and the phosphoserine aminotransferase gene sequence of Edwardsiella spp. (Table 1). Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0031] Table 1PCR primer information
[0032]
[0033] 2. Template preparation
[0034] Activated Edwardsiella and Vibrio parahaemolyticus were extracted using the Bacterial Genomic DNA Extraction Kit produced by Tiangen Biochemical Technology Co., Ltd.
[0035] 3. PCR amplification
[0036] Use the DNA extracted in step 2 as a template, and the template for the negative control is ddH 2 O, get two pairs of primers in step 1 and amplify respectively according to the following system and program:
[0037] PCR reaction system (20μl):
[0038]
[0039] PCR reaction program:
[0040] A...
Embodiment 2
[0050] Embodiment 2 primer specificity test
[0051] 1. Test method
[0052] 1. PCR primers
[0053] Take two pairs of primers shown in Table 1 in Example 1.
[0054] 2. Template preparation:
[0055] Streptococcus agalactiae, Streptococcus iniae, Pseudomonas fluorescens, Aeromonas hydrophila, Flavobacterium columnar, Aeromonas caviae standard, Flavobacterium columnar, Salmonella, Bacillus, Aeromonas temperatus Genomic DNA was used as a template and extracted using a bacterial genomic DNA extraction kit produced by Tiangen Biochemical Technology Co., Ltd.
[0056] 3. PCR amplification
[0057] Take the genomic DNA obtained in step 2 as a template, and the template for the negative control is ddH 2 O, using a mixture of 2.5 μl of Edwardsiella DNA and 2.5 μl of Vibrio parahaemolyticus DNA as a positive template, amplify according to the amplification procedure in Example 1.
[0058] 4. Result detection
[0059] 1.5% agarose gel was used to perform electrophoresis at a const...
Embodiment 3
[0063] Embodiment 3 primer sensitivity test
[0064] 1. Test method
[0065] 1. PCR primers
[0066] Take two pairs of primers shown in Table 1 in Example 1.
[0067] 2. Template preparation:
[0068] Take the positive sample DNA (a mixture of 2.5 μl Edwardsiella DNA and 2.5 μl Vibrio parahaemolyticus DNA) for concentration determination, and follow the original concentration of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7、 10 -8 、10 -9 Doubling dilutions were performed.
[0069] 3. PCR amplification
[0070] Take the genomic DNA obtained in step 2 as a template, and the template for the negative control is ddH 2 O, amplify according to the amplification procedure in Example 1.
[0071] 4. Result detection
[0072] 1.5% agarose gel was used to perform electrophoresis at a constant voltage of 100V for 60 min, and the results of PCR amplification were observed under a gel imaging system.
[0073] 2. Results
[0074] Such as image 3 As shown, according to th...
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