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

A technology for rapid diagnosis of Yersinia pestis, applied in the field of biological identification of Yersinia pestis, to achieve the effects of reducing background influence, mild reaction conditions, and improving specificity

Active Publication Date: 2010-09-08
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 c

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 Yersinia pestis rapid diagnostic kit

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

[0057] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0058] Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0059] Internal primer FIP, the nucleotide sequence of which is shown in SEQ ID NO: 3;

[0060] The nucleotide sequence of the internal 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, 2 mol each of primers FIP / BIP and 0.25 mol each of outer primers F3 / B3 were prepared and placed in containers.

[0063] (4) Prepare the sample pretreatm...

Embodiment 2

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

[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, internal primer FIP 1.6 mol each of BIP and 0.2 mol each of outer primer F3 / B3.

[0077] The chromogenic solution is EvaGreen.

[0078] Others are the same as embodiment 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 for the rapid diagnosis of Yersinia pestis genes for the following 27 strains.

[0081] 1. Test strains

[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. Songnei Shigella (51592); 11. Enterohaemorrhagic Escherichia coli (EHEC); 12. Enteroadhesive Escherichia coli (EAggEC); 13. Salmonella typhi (“O” type); 14. Salmonella typhi (“H” type) ; 15. Typhimurium; 16. Yersinia enterocolitica; 17. Paratyphoid A; 18. Brucella; 19. Klebsiella pneumoniae; 20. Serratia; 21. Cholera arc 22. Pseudomonas aeruginosa; 23. Acinetobacter; 24. Vibrio parahaemolyticus; , prov...

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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 was originally the pathogenic bacteria 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 (Yersinia pestis), Yersinia pseudotuberculosis (Y. pseudotuberculosis), Y. enterocolitica (Y. enterocolitica) and Yersinia intermedia ( Y.intermedia), the first three are highly pathogenic to humans. Among them, Yersinia pestis is a natural foci disease in rodents, which is highly contagious and has a high fatality rate, and it is easy to cause a pandemic. [0003] At present, there are many detection methods for Yersinia pestis, ranging from the national standar...

Claims

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Application Information

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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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