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Primer group for detecting Yersinia pestis, rapid diagnosis kit and detection method

A rapid diagnosis and kit technology, applied in the field of biological identification of Yersinia pestis, to achieve the effects of mild reaction conditions, improved specificity and high detection sensitivity

Active Publication Date: 2012-10-10
GUANGZHOU HUAFENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Due to its own advantages, LAMP has been used in the detection of pathogenic microorganisms and the diagnosis of infectious diseases, such as: detection of severe acute respiratory syndrome coronavirus (SARS-CoV), detection of mycobacteria, adenoviral conjunctivitis detection, fungal detection, etc., there are no relevant reports on the use of LAMP in the detection of hepatitis A virus

Method used

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  • Primer group for detecting Yersinia pestis, rapid diagnosis kit and detection method
  • Primer group for detecting Yersinia pestis, rapid diagnosis kit and detection method
  • Primer group for detecting Yersinia pestis, rapid diagnosis kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of rapid diagnosis kit for Yersinia pestis

[0056] (1) Use DNA synthesizer to synthesize the following sequence primers

[0057] The nucleotide sequence of the outer primer F3 is shown in SEQ ID NO:1;

[0058] The nucleotide sequence of outer primer B3 is shown in SEQ ID NO: 2;

[0059] The nucleotide sequence of the inner primer FIP is shown in SEQ ID NO: 3;

[0060] The nucleotide sequence of the inner primer BIP is shown in SEQ ID NO:4.

[0061] (2) Purchase DNA polymerase: Bst DNA polymerase (large fragment) and place it in a container.

[0062] (3) Preparation of reaction solution: The formula of the reaction solution contains 2mmol dNTP, 25mmol Tris-Cl, 12.5mmol potassium chloride, 12.5mmol ammonium sulfate, 10mmol magnesium sulfate, 0.04mol TritonX-100, 1mol betaine, internal The primers FIP / BIP each 2mol and the outer primer F3 / B3 each 0.25mol are prepared and placed in a container.

[0063] (4) Preparation of sample pretreatment solution: The formula...

Embodiment 2

[0075] Example 2 Preparation of a rapid diagnostic kit for Yersinia pestis

[0076] The formula of the reaction solution is: each 1L reaction solution contains 1.6mmol dNTP, 20mmol Tris-Cl, 10mmol potassium chloride, 10mmol ammonium sulfate, 8mmol / L magnesium sulfate, 0.01mol TritonX-100, 0.8mol betaine, and internal primer FIP / BIP each 1.6mol and outer primer F3 / B3 each 0.2mol.

[0077] The color developing fluid is EvaGreen.

[0078] Others are the same as in Example 2.

Embodiment 3

[0079] Example 3 Application of Yersinia pestis gene rapid diagnosis kit

[0080] In this example, the kit prepared in Example 1 was used to perform rapid diagnosis of Yersinia pestis genes on the following 27 strains.

[0081] 1. Test strain

[0082] 1. Pseudomonas aeruginosa (ATCC 27853); 2. Staphylococcus aureus (ATCC 25923); 3. Staphylococcus aureus (ATCC 29533); 4. Escherichia coli (ATCC 29522); 5. Vibrio vulnificus (ATCC) 27562); 6. Escherichia coli (8099); 7. Enteroinvasive Escherichia coli (EIEC); 8. Pathogenic Escherichia coli (EPEC); 9. Shigella flexneri (51142); 10. Sonnenzi Gagella (51592); 11. Enterohemorrhagic Escherichia coli (EHEC); 12. Enteroadhesive Escherichia coli (EAggEC); 13. Typhoid bacillus ("O" type); 14. Typhoid bacillus ("H" type) ; 15. Typhimurium; 16. Yersinia enterocolitis; 17. Paratyphoid A; 18. Bruceella; 19. Klebsiella pneumonia; 20. Serratia; 21. Vibrio cholerae Bacteria; 22. Pseudomonas aeruginosa; 23. Acinetobacter; 24. Vibrio parahaemolyticus; ...

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Abstract

The invention discloses a primer group for detecting Yersinia pestis, a rapid diagnosis kit and a detection method, wherein the primer group consists of the following four primers: an external primer F3, an external primer B3, an inner primer FIP and an inner primer BIP. The kit consists of the primer group, Bst DNA polymerase, sample pretreatment solution, stabilizing solution, reaction solution, colored solution and positive control solution, and the seven kinds of solutions are placed in a vessel. Both the primer group and the kit can detect the Yersinia pestis with high efficiency and high specificity, as well as are based on loop-mediated isothermal amplification technology, apply six segments, four primers and one constant temperature, complete an amplification reaction within 1 hour, and are low in detection cost, short in detection time, high in yield and specificity, obvious in negative and positive result color developing difference, high in verification rate, obvious and reliable in verification.

Description

Technical field [0001] The invention relates to the biological identification of Yersinia pestis, in particular to a primer set for detection of Yersinia pestis, a rapid diagnostic kit and a detection method. Background technique [0002] Yersinia (Yersinia) is now classified as Enterobacteriaceae. It is the pathogen of animal infectious diseases. Humans get sick through contact with infected animals or contaminated food. At present, the genus Yersinia is roughly divided into Yersinia pestis, Y. pseudotuberculosis (Y. pseudotuberculosis), Y. enterocolitica (Y. entererocolitica), and Yersinia intermedia (Yersinia pestis). Y.intermedia), the first three species are highly pathogenic to humans. Among them, Yersinia pestis causes a natural foci disease in rodents, which is highly infectious and has a high mortality rate, which is easy to cause a pandemic. [0003] At present, there are a variety of detection methods for Yersinia pestis, from the national standard (GB / T 18936-2003), w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/01
Inventor 曹以诚陈清杜正平柯雪梅冯雪梅陈洵陈胤瑜谢丽丽
Owner GUANGZHOU HUAFENG BIOTECH
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