LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus

A staphylococcus, aureus technology, applied in the direction of microorganism-based methods, microbial determination/testing, microorganisms, etc., can solve the problems of low sensitivity, poor specificity, time-consuming and labor-intensive, etc., and achieve high sensitivity, simple steps, and repeatability high effect

Inactive Publication Date: 2013-01-09
WUHAN ZHENFU PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The traditional culture method for detection of Staphylococcus aureus requires isolation, screening, biochemical identification, and serological identification if necessary, which generally takes 4 to 6 days, which is time-consuming and laborious, and has the disadvantages of low sensitivity, poor specificity, false positives, time-consuming and laborious, etc.
Various conventional PCR methods have the advantages of strong sensitivity, high specificity, simplicity, and rapidity, and there are many reports on their application in the detection of Staphylococcus aureus. The equipment and equipment limit its application in grass-roots units

Method used

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  • LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus
  • LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus
  • LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] 1. The composition and preparation of the new rapid visual detection of Staphylococcus aureus LAMP kit, reagent composition:

[0078] a) LAMP reaction mixture

[0079] The LAMP reaction mixture consists of 2.5 μl of LAMP 10×buffer, 2.0 μl of 10 μmol / L internal primers FIP and BIP, 1.0 μl of 10 μmol / L external primers F3 and B3, 3.0 μl of 10 mmol / L dNTPs, 50 mmol / LMgSO 4 3.0 μl, 5.0mol / L betaine, 4.5 μl, Bst DNA polymerase large fragment 1.0 μl, the total volume of the reaction solution is 20 μl.

[0080] Wherein the primer sequence is as follows:

[0081] Upstream outer primer F3: 5'-GCGATTGATGGTGATACGGTTA-3';

[0082] Downstream outer primer B3: 5′-GCCACGTCCATATTTATCAGTTCT-3′;

[0083] Upstream internal primer FIP: 5′-GGATGCTTTGTTTCAGGTGTATCATGTACAAAGGTCAACCAATGAC-3′;

[0084] Downstream internal primer BIP: 5′-TATGGTCCTGAAGCAAGTGCAGACCTTTGTCAAACTCGACTTC-3′;

[0085] b) Standard positive template

[0086] The standard positive template pMD18-T-nuc is a pMD18-T-n...

Embodiment 2

[0096] 1. The composition and preparation of the new rapid visual detection of Staphylococcus aureus LAMP kit, the difference from the kit described in Example 1 is that the specific primer sequences are different. The primer sequences of this kit are as follows:

[0097] Upstream outer primer F3: 5′-ACATAAAGAACCTGCGACA-3′;

[0098] Downstream outer primer B3: 5'-CTTTGTCAAACTCGACTTCA-3';

[0099] Upstream internal primer FIP: 5′-CTGAATGTCATTGGTTGACCTTTGTAATTAAAGCGATTGATGGTGAT-3′;

[0100] Downstream inner primer BIP:

[0101] 5′-CACCTGAAACAAAGCATCCTAAAAAGCATTTTCTACCATCTTTTTCGTA-3′

[0102] 2. The method for detecting Staphylococcus aureus using the PCR kit to detect Staphylococcus aureus in industrial food rapidly based on the loop-mediated isothermal amplification technology is exactly the same as the method described in Example 1, and the repeated test was repeated 3 times, and the obtained The detection results are the same, the experimental group has precipitation, and ...

Embodiment 3

[0104] 1. The composition and preparation of the new rapid visual detection of Staphylococcus aureus LAMP kit, the difference from the kit described in Example 1 is that the specific primer sequences are different. The primer sequences of this kit are as follows:

[0105] Upstream outer primer F3: 5'-GCGACATTAATTAAAGCGATTG-3';

[0106] Downstream outer primer B3: 5'-CTTTGTCAAACTCGACTTCAA-3';

[0107] Upstream inner primer FIP:

[0108] 5'-GCTTTGTTTCAGGTGTATCAACCAAATTAATGTACAAAGGTCAACCAATG-3';

[0109] Downstream internal primer BIP: 5′-AAGGTGTAGAGAAATATGGTCCTGATTCTTTGCATTTTCTACCATCT-3′;

[0110] 2. The method for detecting Staphylococcus aureus using the PCR kit to detect Staphylococcus aureus in industrial food rapidly based on the loop-mediated isothermal amplification technology is exactly the same as the method described in Example 1, and the repeated test was repeated 3 times, and the obtained The detection results are the same, the experimental group has precipitation...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus. The LAMP kit for rapid detection of staphylococcus aureus comprises LAMP reaction fluid, a standard positive template and a negative quality control standard. The LAMP reaction fluid contains large fragments in Bst DNA (deoxyribonucleic acid) polymerase, primers, LAMP 10Xbuffer, dNTPs (deoxyribonucleotide triphosphates) solution, MgSO4 solution and betaine. The primers include forward primers and reverse primers. The LAMP kit for rapid detection of staphylococcus aureus has the advantages that the LAMP kit is high in specificity, high in sensitivity, high in speed, simple and high in reliability, results are recognizable to naked eyes, and the like. The LAMP kit is applicable to rapid qualitative detection of staphylococcus aureus in industrial foods, and can be a substitute for commonly used traditional culture methods and serodiagnosis methods.

Description

technical field [0001] The invention relates to a LAMP kit for rapid detection of Staphylococcus aureus, which belongs to the field of food detection. Background technique [0002] Staphylococcus aureus is a Gram-positive spherical bacterium. Arranged in grape clusters under the microscope, Staphylococcus aureus has no spores, flagella, and most of them have no capsule. is a common pathogen that causes food poisoning. Commonly found on the skin surface and upper respiratory tract mucosa. Staphylococcus aureus is the most common pathogenic bacteria in human suppurative infection, which can cause local suppurative infection, pneumonia, pseudomembranous enteritis, pericarditis, etc., and even systemic infection such as sepsis and sepsis. [0003] The loop-mediated isothermal amplification (LAMP) technology developed in recent years has been widely used in the qualitative detection of genes due to its high sensitivity, fast speed, and strong specificity, and has become the cu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12R1/445
Inventor 王业富郑虎卢晅
Owner WUHAN ZHENFU PHARMA CO LTD
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