Three-in-one seven-fold PCR detection primer set, kit and detection method for virulence genes of Streptococcus agalactiae

A technology of virulence gene and Streptococcus lactis, which is applied in the field of triplex seven-fold PCR detection primer set, can solve the problems of indetermining the source and typing of the host of Streptococcus agalactiae, as well as the variation and deletion of virulence gene spectrum, and achieve saving Cost and time, rapid pathogen detection, and the effect of reducing labor costs

Active Publication Date: 2021-12-24
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

In addition, comparisons with other S. agalactiae genomes revealed that the scpB and lmb gene sequences were missing in fish-derived S. agalactiae strains
At present, the detection of virulence genes of Streptococcus agalactiae is less involved, such as only using single, double and triple PCR methods to detect virulence genes such as sip, cpsE, hylB, ponA and cfb, these methods are only for the simple detection of some Whether the gene is present or not, the host source, typing and virulence gene spectrum variation of Streptococcus agalactiae cannot be determined

Method used

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  • Three-in-one seven-fold PCR detection primer set, kit and detection method for virulence genes of Streptococcus agalactiae
  • Three-in-one seven-fold PCR detection primer set, kit and detection method for virulence genes of Streptococcus agalactiae
  • Three-in-one seven-fold PCR detection primer set, kit and detection method for virulence genes of Streptococcus agalactiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Genomic DNA templates were extracted from the human Streptococcus agalactiae standard strain ATCC BAA-1138 and the fish-derived Streptococcus agalactiae standard strain ATCC51487, respectively.

[0057] Extract human Streptococcus agalactiae standard strain ATCC BAA-1138 genomic DNA template:

[0058] (1) Collect the cultured standard strain of Streptococcus agalactiae ATCC BAA-1138 by centrifugation and resuspend the bacteria in 1000 μL of TE buffer, take 180 μL of the bacterial resuspension, transfer it to a 1.5 mL centrifuge tube, and add 20 μL concentration 50mg / mL lysozyme, incubated at 30°C for 10min.

[0059] (2) Add 10 μL of 10% SDS and 5 μL of 20 mg / mL proteinase K to the solution in step (1), and incubate at 37° C. for 1 h.

[0060] (3) Add 50 μL of 5 mol / L NaCl solution to the solution incubated in step (2), mix well, then add 40 μL of CTAB NaCl solution, and incubate at 65°C for 20 minutes.

[0061] (4) Add a phenol-chloroform-isoamyl alcohol mixed solutio...

Embodiment 2

[0067] The three-linked seven-fold PCR reaction system of the standard strain of human Streptococcus agalactiae ATCC BAA-1138 and the standard strain of Streptococcus agalactiae ATCC51487 of fish were respectively constructed.

[0068] The first group: 25 μL Mix, 17 μL mixed primer set, 4 μL sample DNA template, supplemented with ddH 20 to 50 μL system, the mixed primer group is composed of each primer pair solution with a primer concentration of 10 μM. According to the volume ratio, it is sip primer pair: fbsA primer pair: hylB primer pair: cfb primer pair: sodA primer pair: DltR primer pair: cspA Primer pair = 3:3:2:3:2:2:2 mixed;

[0069] The second group: 25 μL Mix, 17 μL mixed primer set, 4 μL sample DNA template, supplemented with ddH 2 0 to 50 μL system, the mixed primer set is composed of each primer pair solution with a primer concentration of 10 μM. According to the volume ratio, it is ponA primer pair: bibA primer pair: srr-1 primer pair: bca primer pair: iagA prim...

Embodiment 3

[0079] Embodiment 3 sensitivity test

[0080] Take the fish-derived Streptococcus agalactiae standard strain ATCC51487 bacterium liquid, extract the bacterial genomic DNA with the method of Example 1, and dilute the genomic DNA concentration: 228ng / μL, 45.6ng / μL, 9.12ng / μL, 1.824ng / μL, 0.912 ng / μL, 0.456ng / μL, 0.228ng / μL, 0.114ng / μL. Carry out PCR amplification and electrophoresis detection respectively according to the method for embodiment 2, the result is as follows Figure 2-4 shown. It can be obtained from the figure that the sensitivity of the first group of primers is 0.912ng / μL, the sensitivity of the second group of primers is 0.912ng / μL, and the sensitivity of the third group of primers is 1.824ng / μL. It can be seen that the detection of Streptococcus agalactiae of the present invention The detection limit is relatively low and has high sensitivity.

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Abstract

The invention discloses a triplex seven-fold PCR detection primer set for virulence genes of Streptococcus agalactiae, including virulence genes sip, fbsA, hylB, cfb, sodA, DltR, cspA, ponA, bibA, srr-1, bca, PCR amplification primer pairs corresponding to iagA, scpB, fbsB, pavA, psaA, spb1, bac, cppA, lmb and cylE. The sequences of each primer are shown in the sequence table. The invention also discloses a reagent kit comprising the above primer set and a three-in-one seven-fold PCR detection method of the Streptococcus agalactiae virulence gene using the above primer set. The detection method uses sip, fbsA, hylB, cfb, sodA, DltR and cspA as the first group, ponA, bibA, srr-1, bca, iagA, scpB and fbsB as the second group, pavA, psaA, spb1, bac , cppA, lmb and cylE are the third group, and PCR reaction is carried out under the same reaction conditions at the same time, and the target sequences of 21 virulence gene target fragments are amplified at the same time. Source and typing of Streptococcus lactis host, and evaluate the variation of strain virulence gene profile.

Description

technical field [0001] The invention relates to the detection of Streptococcus agalactiae, in particular to a primer set, a kit and a detection method for three-link seven-fold PCR detection of virulence genes of Streptococcus agalactiae. Background technique [0002] Streptococcus agalactiae (Streptococcus agalactiae) belongs to Bacillus class, Lactobacillus order, Streptococcus family, Streptococcus genus, also known as group B streptococci (GBS), is a Gram-positive coccus that causes neonatal It is an important pathogenic bacterium of infantile meningitis, septicemia, pneumonia and cow mastitis, which can infect a variety of aquatic animals. At present, more than 80 kinds of farmed fishes are found to be susceptible to Streptococcus agalactiae infection, and the loss caused by Streptococcus agalactiae in the world's aquaculture industry is as high as 10 billion US dollars every year. [0003] The molecular serotypes and ST types of Streptococcus agalactiae derived from h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/46
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 苏友禄刘婵冯娟孙秀秀邓益琴郭志勋
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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