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Method, primer pair, target probe, internal standard probe and kit for detecting Cronobacter sakazakii

A technology of internal standard probes and detection methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of long detection time, time-consuming, heavy workload, etc., and achieve short detection time and high results The effect of simple judgment and reduced inspection cost

Active Publication Date: 2014-02-12
BRIGHT DAIRY & FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the defects of long detection time of the existing traditional technology, false positives and false negatives produced by ordinary PCR detection methods, and long time-consuming and heavy workload of analysis methods of genetic characteristics, and provide a method for detecting Detection methods, primer pairs, target probes, internal standard probes and kits for Rhinobacter

Method used

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  • Method, primer pair, target probe, internal standard probe and kit for detecting Cronobacter sakazakii
  • Method, primer pair, target probe, internal standard probe and kit for detecting Cronobacter sakazakii
  • Method, primer pair, target probe, internal standard probe and kit for detecting Cronobacter sakazakii

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Primer, Internal Standard Sequence, and Probe Synthesis

[0044] Synthesize primers, internal standard sequences (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.) and probes (Shanghai Biotechnology Co., Ltd.) capable of PCR amplifying Cronobacter sakazakii specific gene sequence (CS44) company), the sequence is as follows:

[0045] SEN2-L (primer): 5'-aatgcacaagcgcgatttc-3' (SEQ ID NO: 1)

[0046] SEN2-R (primer): 5'-tgcaaaaggcgctgtgataa-3' (SEQ ID NO: 2)

[0047] Probe-1 (target probe): 5'-FAM-tgctcgcccaatctcaacgcc-BHQ1-3' (SEQ ID NO:3)

[0048] Probe-2 (internal standard probe): 5’-HEX-ccttagtaaaaccggtacctc-BHQ1-3’SEQ ID NO:4)

[0049] Internal standard template:

[0050] 5'-aatgcacaagcgcgatttccacgccttagtaaaaccggtacctctacaattatcacagcgccttttgca-3' (SEQ ID NO: 5)

Embodiment 2

[0052] Detection of Cronobacter sakazakii

[0053] 1. Suggestions and reaction parameters for PCR detection reaction system

[0054] Using the primers and probes as described in Example 1, the genomic DNA of the standard strain of Cronobacter sakazakii ATCC29544 (purchased from ATCC) was used as a template, and the PCR reaction reagent was provided by Promega Probe qPCR Master Mix (Catalog No. A6102), by adjusting and optimizing the concentration of primers, target probes and internal standard probes in the reaction system, the PCR reaction system can achieve the best reaction system. The concentration of the primers is 400 nM, and the concentration of the genomic DNA of the sample to be tested is 20 ng / μL.

[0055] Add target probes and internal standard probes with an original concentration of 10 μM to different PCR reaction tubes, and serially dilute the concentrations of target probes and internal standard probes from 100 nM to 500 nM (100 nM, 150 nM, 200 nM, 250 nM, 30...

Embodiment 3

[0071] The 30 food samples (including powdered milk and vegetables) were purchased from farmers' markets and large supermarkets in Shanghai. Add 50g of each of the 30 samples to 450mL buffer peptone water (buffer peptone water, BPW, pH8.0) enrichment medium (ingredients include: peptone 10.0g, sodium chloride 5.0g, disodium hydrogen phosphate 9.0g , Potassium dihydrogen phosphate 1.5g, distilled water 1000mL, pH7.2±0.2; the preparation method is: add each ingredient into distilled water, stir well, let stand for about 10min, boil to dissolve, adjust pH, autoclave at 121℃, 15min) After enrichment at 37°C for 18 hours, take 1 mL of each sample and put it into a 1.5 mL centrifuge tube, and extract genomic DNA by boiling method, centrifuge at 12,000 rpm for 5 min, and take 2.5 μL of supernatant as a PCR template. Sterile water was used as a negative control for fluorescent quantitative PCR detection.

[0072] The reaction system for PCR detection is as shown in Example 1; the rea...

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Abstract

The invention discloses a method, a primer pair, a target probe, an internal standard probe and a kit for detecting Cronobacter sakazakii. The method comprises the following steps: (1) adding the deoxyribose nucleic acid (DNA) of a sample to be detected, the primer pair, the target probe, the internal standard probe and an internal standard template into a polymerase chain reaction (PCR) system, and performing fluorescent quantitative PCR amplification, wherein the sequences of the primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, the sequence of the target probe is shown as SEQ ID NO.3, the sequence of the internal standard probe is shown as SEQ ID NO.4, and the sequence of the internal standard template is shown as SEQ ID NO.5; (2) acquiring fluorescent signals in a PCR process. The method is short in detection time, low in cost and high in practicality; a detection result is specific and is easy to determine; a false negative result can be well avoided.

Description

technical field [0001] The invention relates to a detection method for detecting Cronobacter sakazakii, a primer pair, a target probe, an internal standard probe and a kit. Background technique [0002] Cronobacter sakazakii (Cronobacter sakazakii), also known as Enterobacter sakazakii, belongs to the genus Cronobacter, is a Gram-negative non-spore-forming bacterium parasitic in the intestinal tract of humans and animals. Clinical studies have found that Cronobacter sakazakii is the most common opportunistic pathogen in clinical infections of the genus Cronobacter. Strains of Cronobacter sakazakii can cause fatal infections in infants, with a mortality rate as high as 40%-80%. It often results in severe clinical symptoms in infants, such as brain abscess, meningitis, necrotizing enterocolitis, and systemic sepsis. There have been reports of Cronobacter sakazakii isolates in powdered infant formula. Cronobacter sakazakii strains are important opportunistic pathogens that e...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06C12N15/11C12R1/01
CPCC12Q1/04C12Q1/686C12Q2545/101C12Q2561/113C12Q2563/107
Inventor 陈万义任婧杭锋吴正钧
Owner BRIGHT DAIRY & FOOD
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