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Special primer for assisted identification of Streptococcus suis type 2 and Streptococcus suis type 7 and application thereof

A technology for identifying Streptococcus suis and pigs, applied in the determination/inspection of microbes, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of ineffective rapid diagnosis of SS2

Inactive Publication Date: 2011-08-10
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In recent years, foreign scholars have mainly used restriction fragment length polymorphism analysis, immunochromatography, indirect fluorescent antibody test, ELISA, multi-site enzyme electrophoresis, pulsed field gel electrophoresis, random amplified nucleic acid Fragment polymorphism analysis and in situ molecular hybridization and other research methods, these methods are very effective and necessary as scientific research methods, but they are weak as a rapid diagnosis of SS2

Method used

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  • Special primer for assisted identification of Streptococcus suis type 2 and Streptococcus suis type 7 and application thereof
  • Special primer for assisted identification of Streptococcus suis type 2 and Streptococcus suis type 7 and application thereof
  • Special primer for assisted identification of Streptococcus suis type 2 and Streptococcus suis type 7 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The design of the specific type primer of embodiment 1, Streptococcus suis

[0039] Design SS2-specific primer pairs in the genera conserved sequence and CPS2J gene primer sequence region as follows:

[0040] SS-CPS2J-F: 5'-CGGTTACCAAATGGTGGTG-3' (SEQ ID NO: 1 of the Sequence Listing);

[0041] SS-CPS2J-R: 5'-ACTTTTGCAGCTCAGATTCTTG-3' (SEQ ID NO: 2 of the Sequence Listing).

[0042] The length of the target amplified fragment is 211bp (see sequence 5 in the sequence listing for the target sequence).

[0043] Design SS7-specific primer pairs in the genera conserved sequence and CPS7H gene primer sequence region as follows:

[0044] SS-CPS7H-F: 5'-GGCAGCTCTAACACGAAATAAG-3' (sequence 3 of the sequence listing);

[0045] SS-CPS7H-R: 5'-TCTTACACAATTGAAGTAATATCGC-3' (SEQ ID NO: 4 of the Sequence Listing).

[0046] The length of the target amplified fragment is 347bp (see sequence 6 in the sequence listing for the target sequence).

Embodiment 2

[0047] Embodiment 2, the preparation of sample

[0048] 1. Preparation of double positive samples

[0049] Each Du×Da×Chang three-way cross-breed pig was inoculated with 1ml of oral palatal tonsil at a concentration of 10 4 cfu / ml of Streptococcus suis type 2 bacteria solution and 1ml concentration of 10 10 cfu / ml Streptococcus suis type 7 bacterial fluid.

[0050] On the 5th day after inoculation and infection, tonsil test paper was collected, and the bacteria were isolated for morphological and physiological characteristics identification. Pigs with Streptococcus suis type 2 and Streptococcus suis type 7 were identified as double positive samples.

[0051] 2. Preparation of Streptococcus suis type 2 positive samples

[0052] Each Du×Da×Chang three-way cross-breed pig was inoculated with 1ml of oral palatal tonsil at a concentration of 10 4 cfu / ml Streptococcus suis type 2 bacterial fluid.

[0053] On the 5th day after inoculation and infection, tonsil test paper was col...

Embodiment 3

[0057] Embodiment 3, the application of the specific type primer of Streptococcus suis

[0058] The tonsil test papers of the double positive sample prepared in Example 2, the positive sample of Streptococcus suis type 2 and the positive sample of Streptococcus suis type 7 were taken respectively for detection.

[0059] 1. Genomic DNA extraction

[0060] (1) Add 3 times the volume of erythrocyte lysate to the blood, mix it upside down, and place it at room temperature for 5 minutes. The buffer is sufficient to suspend the precipitate;

[0061] (2) Take 100 μL of the solution obtained in step (1), add 50 μL of lysozyme (100 μg / mL), and treat at 37° C. for 1 hour.

[0062] (3) Add 200 μL lysis buffer to each tube and shake for 15 seconds.

[0063] (4) Add 100 μL of 5mol / L NaCl aqueous solution to each tube, mix thoroughly, and centrifuge at 12,000 rpm for 10 min to remove protein complexes and cell walls and other residues, and take the supernatant.

[0064] (5) Transfer the...

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Abstract

The invention discloses a special primer for assisted identification of Streptococcus suis type 2 and Streptococcus suis type 7 and application thereof. The special primer provided by the invention comprises DNA shown as a sequence 1 in a sequence table, DNA shown as a sequence 2 in the sequence table, DNA shown as a sequence 3 in the sequence table and DNA shown as a sequence 4 in the sequence table. The special primer provided by the invention can be used for assisted identification and discrimination of the Streptococcus suis type 2 and Streptococcus suis type 7. The special primer provided by the invention has the advantages of specificity, sensitivity, quick speed and the like in detection, can prevent troubles, and has far reaching importance for preventing and controlling human and animals from infecting Streptococcus suis.

Description

technical field [0001] The invention relates to a special primer for auxiliary identification of Streptococcus suis type 2 and Streptococcus suis type 7 and application thereof. Background technique [0002] Streptococcus suis (SS) is an important zoonotic pathogen that can infect pigs of all ages. The diseased pigs are usually characterized by sepsis, meningitis, arthritis, and lymphadenitis. According to the capsular polysaccharide antigen (CPS) of Streptococcus suis, it can be divided into 35 serotypes, among which the pathogenic Streptococcus suis serotypes that can cause human and animal diseases are mainly type 1 (SS1), type 2 (SS2) ), Type 7 (SS7) and Type 9 (SS9). The distribution of different serotypes is different. In Canada, types 2, 1 / 2, 3, 4, and 8 are the most prevalent; in Italy, type 2 is the most widespread, followed by types 1, 9, 1 / 2, 3, 7, 4, and 8 ; Netherlands and France are dominated by types 2 and 9; Brazil is the most popular type 2, followed by ty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12N15/11
Inventor 刘文军毕玉海李晶
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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