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Primer and method for rapid detection of Streptococcus iniae

A technology for detection of Streptococcus iniae and primers, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of real-time monitoring of unsuitable pathogens, complicated operation, and long time consumption, and achieve accurate High reliability, low detection cost, and easy operation

Inactive Publication Date: 2015-10-28
天津市水产技术推广站
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of Streptococcus iniae is mainly carried out through bacterial isolation, biochemical identification, serological reaction and PCR technology. personnel, not suitable for real-time monitoring of pathogens in actual production
There is no report on the detection technology of Streptococcus iniae LAMP

Method used

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  • Primer and method for rapid detection of Streptococcus iniae
  • Primer and method for rapid detection of Streptococcus iniae
  • Primer and method for rapid detection of Streptococcus iniae

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0030] Example 1. Isolation and Identification of Tuli Fish Source Streptococcus iniae

[0031] (1) Pick the eyeballs, spleen and kidney tissues of the diseased Tucui, streak them on a BHI medium plate, place them in an incubator at 28°C for overnight culture, and then pick the dominant colonies for pure culture;

[0032] (2) Gram staining was used to observe the morphology of the isolated strains, and the biochemical characteristics of the bacteria were identified by referring to the "Common Bacteria Identification Manual"; the 16S rDNA of the bacteria was amplified, compared and analyzed after sequencing, and a system using 16S rDNA as a genetic marker was constructed development tree;

[0033] (3) Take the isolated bacterial strains and carry out mass culture. After collecting the bacterial liquid and centrifuging, adjust the concentration of the bacterial liquid to 3×10 7 cfu / ml, adopt the mode of intraperitoneal injection to carry out artificial infection experiment on h...

Embodiment 2

[0035] Example 2. Establishment of a rapid detection method for Streptococcus iniae

[0036] (1) Template preparation:

[0037] Genomic DNA of strain 0523 was extracted using a DNA extraction kit (Tiangen Rapid DNA Extraction and Detection Kit KG203) and used as a reaction template.

[0038] (2) Design and synthesis of primers

[0039] Blast software was used to analyze the gene sequence of Streptococcus iniae, and the nucleotide sequence of co-hemolysin gene (GenBank accession number: KC132870.1), a virulence gene shared by Streptococcus iniae, was screened out. According to the principle of LAMP technology primer design, LAMP primers were designed and synthesized for this fragment,

[0040] stin-F3: 5'-TGCTGTTCAAGCCATTGT-3' (SEQ ID NO.1)

[0041] stin-B3: 5'-GATGACTTCACATAAATTGTAGC-3' (SEQ ID NO.2)

[0042] stinFIP: 5'-TGGATCAGCAATTCGGATAACTAATTTTTAACTCAATTAAGCCACAAAGT-3' (SEQ ID NO. 3)

[0043] stinBIP: 5'-GATGCAATAAAAGCACAAGTCCAAGGATCCTCTATCACTAGCTTTTAAATCTGC-3' (SEQ ID...

Embodiment 3

[0053] Embodiment 3. a kind of streptococcus iniae rapid detection method comprises the steps:

[0054] (1) Extract the DNA of the sample to be tested:

[0055] 100 μl of blood from the fish to be tested was extracted under sterile conditions, and the DNA of the sample to be tested was extracted using a commercial DNA extraction kit.

[0056] (2) adopt the LAMP reaction system of 25 μ l in the step (3) of embodiment 2; With the whole genome DNA of Streptococcus iniae 0523 as the template of positive control, with sterile molecular biology grade ultrapure water as the template of negative control, Place it at 62°C for 50 minutes, then place it at 85°C for 15 minutes to obtain the test solution;

[0057] (3) Use agarose electrophoresis with a mass fraction of 1% to detect whether the test solution produces continuous ladder-like DNA bands, and if so, it is positive;

[0058] Then use steps (1) (2) to obtain another test solution, add 1 μl fluorescent dye SYBR Green I working s...

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Abstract

The invention discloses a primer for rapidly detecting Strepstococcus iniae and a detection method of Strepstococcus iniae. The primer comprises an outer primer and an inner primer, wherein the outer primer comprises an outer primer upstream primer as shown in SEQ ID NO. 1 and an outer primer downstream primer as shown in SEQ ID NO. 2; the inner primer comprises an inner primer upstream primer as shown in SEQ ID NO. 3 and an inner primer downstream primer as shown in SEQ ID NO. 4. According to the primer and the detection method disclosed by the invention, the problems, existing in the detection of Strepstococcus iniae by the prior art, of long period, high detection cost, impossibility in applying on-site detection and the like are solved; the detection method is simple and rapid, high in accuracy and sensitivity, the lowest concentration for detected Strepstococcus iniae is 3* 10<2>cfu / ml, due to no need of professional instrument and equipment, for example, a PCR (Polymerase Chain Reaction) instrument, the detection cost is low, the bacterial culture does not be needed, samples required for detection are less and minimally invasive sampling is achieved and thus the detection method is suitable for detecting ornamental fish with relatively high economic value.

Description

technical field [0001] The invention relates to a primer for rapid detection of Streptococcus iniae and a method thereof, belonging to the field of rapid detection of pathogenic bacteria in ornamental fish. Background technique [0002] Streptococcus iniae belongs to Bacilli, Lactobacillales, Streptococcus, and Streptococcus. It is a hemolytic Gram-positive coccus. It can cause infection of tilapia, barramundi, rainbow trout, channel catfish, hybrid striped bass, flounder and puffer fish, etc., resulting in high mortality. In recent years, Streptococcus iniae has been found many times in cultured ornamental fish, causing symptoms such as protruding eyes, opercular hemorrhage, and ascites in infected Tuli fish and Luohan fish. It is highly contagious and has a mortality rate of more than 30%. The value of ornamental fish lies in its ornamental quality. Once Streptococcus iniae infection occurs, even if it is treated in time to save the lives of some fish, its ornamental valu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q2531/119
Inventor 徐晓丽姚学良包海岩李灏丁子元
Owner 天津市水产技术推广站