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Primer and probe composition and kit

A primer-probe and composition technology, applied in the field of molecular biology, can solve the problems of high nutritional requirements, failure, slow growth, etc., and achieve the effects of improving detection throughput, improving efficiency and high specificity

Inactive Publication Date: 2019-08-06
宁波基内生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional staphylococcus identification is mainly based on culture methods, including conventional Gram staining, isolation culture, catalase test, plasma coagulase test, etc. These methods usually take 3 to 5 days to detect, and are often delayed due to the use of antibiotics. Inhibition, high nutrient requirements, and slow growth lead to assay failure

Method used

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  • Primer and probe composition and kit
  • Primer and probe composition and kit
  • Primer and probe composition and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] The composition of the kit:

[0066] 1. Nucleic acid extraction solution: 2% (M / V) Chelex-100, 1% (V / V) Tris-HCl, 1M, pH9.0;

[0067] 2. PCR reaction enzyme system: 5U / μL Taq DNA polymerase and 2U / μL uracil-N-glycosylase (UNG enzyme) mixed in a volume ratio of 3:1;

[0068] 3. Internal standard: a plasmid containing the internal standard int gene fragment;

[0069] 4. Negative control substance: double distilled water purified by Millipore water purifier;

[0070] 5. Positive control substance: T vector plasmid carrying coagulase-negative Staphylococcus target gene (sodA), T vector plasmid carrying Staphylococcus aureus target gene (femA) and T vector carrying methicillin resistance gene (mecA) Plasmid or Escherichia coli engineering bacteria containing the above-mentioned plasmid, as a reference substance, the bacterial concentration of the bacterial liquid is 10 5 CFU / mL;

[0071] 6. The first primer-probe mixture: composed of deoxyribonucleoside triphosphate dN(U...

Embodiment 2

[0081] Kit detection method establishment

[0082] 1. Preparation of reference substance

[0083] Take 50 μL each of the positive control substance and the negative control substance and place them in 1.5mL (or 0.5mL) centrifuge tubes (shake and mix for 10 seconds after the frozen reagent has melted). Then 12,000rpm for 5min, take 2μL of the supernatant for PCR reaction.

[0084] 2. Reaction system preparation

[0085] Take 16 μL of the first primer-probe mixture and 0.2 μL of the PCR reaction enzyme system, shake and mix for a few seconds, and centrifuge at 3000 rpm for a few seconds; take 16 μL of the second primer-probe mixture and 0.2 μL of the PCR reaction enzyme system, shake and mix For a few seconds, centrifuge at 3000rpm and mix for a few seconds.

[0086] 3. PCR amplification

[0087] Add 2 μL each of the treated supernatants of the negative control and positive control to the PCR tubes of the reaction system mixture described in step 2, and add 2 μL of internal st...

Embodiment 3

[0095] Kit specificity experiments

[0096] 1. Experimental materials (see Table 1)

[0097] Table 1 Experimental material of the present invention

[0098]

[0099]

[0100] 2. Experimental method

[0101] Using the kit described in Example 1 and the experimental method established in Example 2, the above-mentioned 17 kinds of pathogenic bacteria were detected respectively, and the specificity analysis and evaluation of this kit were carried out.

[0102] 3. Experimental results

[0103] Detect 15 common pathogenic bacteria (Enterococcus faecalis, Enterococcus faecium, Diphtheria bacillus, Haemophilus influenzae, Proteus, Proteus mirabilis, Enterobacter cloacae, Acinetobacter baumannii, Neisseria meningitidis, Streptococcus sanguinis, Escherichia coli, Klebsiella oxytoca, Burkholderia cepacia, Pseudomonas aeruginosa, Candida albicans), the results were all negative, proving that the kit The detection specificity was good (see Table 2).

[0104] Table 2 The specific...

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Abstract

The invention discloses a primer and a probe composition and a kit. The primer and probe composition are used for detecting coagulase negative staphylococcus, Staphylococcus aureus and methicillin resistance genes; a coagulase negative staphylococcus detection target is a sodA gene, the staphylococcus aureus detection target is a femA gene, and the methicillin resistance gene is a mecA gene; The primer and probe composition include a forward primer, a reverse primer, and a probe for respectively detecting the sodA gene, the femA gene, and the mecA gene, which are shown as SEQ ID NO. 1 to SEQ ID NO. 9. The primer and a probe composition use real-time fluorescent quantitative PCR to detect the coagulase negative staphylococcus, staphylococcus aureus and methicillin resistance genes. The detection cycle is greatly shortened, the efficiency and the detection flux are improved, the detection requirement is catered for, and the specificity, sensitivity and accuracy are high, at the same time, no detection process is generated after amplification in the experiment, and the pollution problem in the experiment can be effectively solved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer and probe composition and kit. Background technique [0002] Since the late 1980s and early 1990s, infections caused by Gram-positive bacteria such as Staphylococcus have been on the rise. Taking bloodstream infection as an example, coagulase-negative Staphylococcus and Staphylococcus aureus have become important hospital-acquired infections. pathogen. [0003] Coagulase-negative Staphylococcus (CNS) gets its name because it does not produce coagulase. So far, more than 40 species of coagulase-negative Staphylococcus have been identified. With the development of modern medicine, it has been gradually confirmed. The multi-pathogenic role of coagulase-negative staphylococci, and further confirmed in clinical investigations that this type of bacterial infection has an increasing trend; Staphylococcus aureus is a coagulase-positive staphylococcus that can cause a wide range...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/14C12Q1/6851C12N15/11C12R1/44C12R1/445
CPCC12Q1/689C12Q1/6851C12Q2600/166C12Q2600/106C12Q2600/16C12Q2545/101C12Q2531/113C12Q2563/107C12Q2537/143
Inventor 倪剑锋史俊颖张博学高华山童慧珊刘春燕杨实虞承启
Owner 宁波基内生物技术有限公司
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