Detection primer set of Streptococcus agalactiae, detection kit and multiplex PCR detection method

A detection kit and technology for Streptococcus lactis, applied in biochemical equipment and methods, methods based on microorganisms, measurement/testing of microorganisms, etc., can solve the problem of loss of target fragments, missed or false detections, primer dimers, etc. problems, to reduce labor costs, improve quality and safety, and quickly detect pathogens

Active Publication Date: 2017-06-06
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the molecular detection methods of Streptococcus agalactiae have been reported at home and abroad, most of them use purely cultured bacteria liquid for detection. Using ordinary PCR method, only one gene can be detected at a time, which often leads to missed detection or false detection.
[0003] Multiplex PCR is a target sequence that can simultaneously amplify multiple target fragments in one reaction. This technology can be used for the detection or identification of various pathogenic microorganisms. Multiple pairs of specific primers are added to the same PCR reaction tube at the same time for PCR amplification. , the biggest problem lies in the design of primers and the optimization of reaction conditions, because the band loss phenomenon will occur after the amplification of various primers is mixed, and there is still a competitive relationship between the primers, and the strong primers may cover the weak primers, resulting in the loss of the target fragment. Even primer-primer interactions can generate severe primer-dimers
At present, there are reports in the literature on the construction of a triple PCR detection method for Streptococcus agalactiae, but the minimum detection concentration of this detection technique for DNA samples of Streptococcus agalactiae is only 3.2×10 -1 ng / μL

Method used

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  • Detection primer set of Streptococcus agalactiae, detection kit and multiplex PCR detection method
  • Detection primer set of Streptococcus agalactiae, detection kit and multiplex PCR detection method
  • Detection primer set of Streptococcus agalactiae, detection kit and multiplex PCR detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Extraction of sample genomic DNA

[0041] 1. Extraction of genomic DNA from fish (including bacteria in fish):

[0042] 1) Take 50~100 mg of fish tissue to be tested and add 1000 μL of TE buffer solution, fully homogenize with a homogenizer, take 180 μL of tissue homogenate, transfer it to a 1.5ml centrifuge tube, add 20 μL of concentration Incubate at 30°C for 10 min with 50 mg / mL lysozyme.

[0043] 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.

[0044] 3) Add 50 μL of 5 mol / L NaCl solution to the solution after incubation in step 2), mix well, then add 40 μL of CTAB NaCl solution, and incubate at 65°C for 20 min.

[0045] 4) Add a mixed solution of phenol-chloroform-isoamyl alcohol equal to the volume of the incubated solution to the incubated solution in step 3), mix well, and centrifuge at 12000 g / min for 4-5 min.

[0046] 5) Transfer the supernatant after centrifugation in step 4) to...

Embodiment 2

[0057] Example 2 Design and Effectiveness Detection of Streptococcus agalactiae Multiplex PCR Detection Primer Set

[0058] 1. Selection of three specific virulence genes of Streptococcus agalactiae hyB , pon A and cfb , using Primer Premier 6.0 software to analyze and design corresponding primer pairs, each primer pair can specifically identify the specificity of the above-mentioned bacterial virulence genes respectively, and the primer sequences are as follows:

[0059]

[0060] 2. Effectiveness testing:

[0061] (1) Synthesize the primer set designed above, and use the primers of the primer set to perform single PCR verification on the DNA of Streptococcus agalactiae extracted in Example 1, wherein the single PCR reaction system is as follows:

[0062]

[0063] (2) Simultaneously perform multiple PCR verification on the three virulence genes of Streptococcus agalactiae, using the following multiple PCR reaction system:

[0064]

[0065] (3) Use the following...

Embodiment 4

[0075] Example 4 Kit

[0076] The detection kit of this embodiment can be used for multiplex PCR to quickly detect whether the sample contains Streptococcus agalactiae, and the detection kit consists of Streptococcus agalactiae hyB , pon A and cfb Gene detection primer set, TE buffer, lysozyme, proteinase K, SDS solution, phenol-chloroform-isoamyl alcohol mixed solution, isopropanol, ethanol with a volume concentration of 70%, NaCl solution of CTAB, positive control substance and PCR DsMix consists of:

[0077] (1) Primer set for Streptococcus agalactiae detection: 1 tube containing Streptococcus agalactiae hyB Gene primers hylB-F and hylB-R, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; pon A Gene primers ponA-F and ponA-R, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively; 3 tubes contain Streptococcus agalactiae cfb The nucleotide sequences of gene primers cfb-F and cfb-R are respectiv...

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PUM

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Abstract

The invention discloses a detection primer set of Streptococcus agalactiae. The detection primer set of Streptococcus agalactiae comprises a primer pair hylB, a primer pair ponA and a primer pair cfb, wherein the primer pair hylB comprises a primer hylB-F of which the nucleotide sequence is as shown in SEQ ID NO.1 and a primer hylB-R of which the nucleotide sequence is as shown in SEQ ID NO.2; the primer pair ponA comprises a primer ponA-F of which the nucleotide sequence is as shown in SEQ ID NO.3 and a primer ponA-R of which the nucleotide sequence is as shown in SEQ ID NO.4; the primer pair cfb comprises a primer cfb-F of which the nucleotide sequence is shown in SEQ ID NO.5 and a primer cfb-R of which the nucleotide sequence is shown in SEQ ID NO.6. The invention further discloses a detection kit containing the primer set and a multiplex PCR detection method using the detection kit. The primer set and the detection method disclosed by the invention are good in specificity, high in sensitivity, simple and fast, high-efficient and accurate, suitable for rapid inspection and quarantine of Streptococcus agalactiae in pollution-free aquatic products, and can be directly applied to early monitoring and early warning of aquaculture diseases. The lowest concentration of Streptococcus agalactiae DNA detected by the detection kit is 7.74*10<-3>ng / uL, and the detection kit can achieve minimally invasive sampling without bacterial culture, and requiring small quantity of samples for detection.

Description

technical field [0001] The invention belongs to the field of detection of pathogenic bacteria in aquaculture animals, and in particular relates to a detection primer set for Streptococcus agalactiae. At the same time, the present invention also relates to a detection kit comprising the above primer set and a multiplex PCR detection method using the kit. Background technique [0002] Streptococcus agalactiae ( Streptococcus agalactiae ) belongs to Bacillus, Lactobacillus, Streptococcus, and Streptococcus. It is an important zoonotic pathogen. So far, it has been found that more than 30 kinds of farmed freshwater fish and more than 50 kinds of seawater fish are susceptible to the infection of Streptococcus agalactiae, and the annual loss caused by Streptococcus agalactiae in the world's aquaculture industry is as high as 10 billion U.S. dollars. Non-fish are most susceptible to the invasion of the pathogen. The onset of Streptococcus agalactiae is relatively fast, and ther...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12N15/11C12R1/46
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 苏友禄刘婵冯娟郭志勋王江勇
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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