Primer probe combination, kit and method for detecting cronobacter based on RAA technology

A Cronobacter, technical detection technology, applied in the direction of microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of time-consuming and laborious, impossible on-site detection, easy pollution, etc., and achieve simple reaction Fast, good application prospects, high sensitivity effect

Pending Publication Date: 2021-05-14
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional detection method takes a long time to detect, and the detection steps are cumbersome, time-consuming and labor-intensive. Therefore, in recent years, many researchers have explored the rapid detection technology of Cronobacter
[0004] Compared with microbial culture methods, molecular biology methods can effectively shorten the detection time. At present, molecular detection technologies for Cronobacter mainly include ordinary PCR, real-time fluorescent PCR, LAMP, gene chips, etc., but these methods also have disadvantages: common The PCR operation is cumbersome and requires electrophoresis; the fluorescent PCR equipment is large and can only be used in the laboratory, and cannot be used for on-site detection; the LAMP isothermal amplification speed is fast but the specificity is not high, and it is easy to contaminate; the detection sensitivity of the gene chip is often not high enough. The cost is not cheap
[0005] Therefore, there is still a lack of a sensitive, simple, rapid and low-cost Cronobacter detection method that can perform both laboratory detection and on-site rapid detection.

Method used

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  • Primer probe combination, kit and method for detecting cronobacter based on RAA technology
  • Primer probe combination, kit and method for detecting cronobacter based on RAA technology
  • Primer probe combination, kit and method for detecting cronobacter based on RAA technology

Examples

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Embodiment 1

[0041] The present embodiment provides a combination of primers and probes based on RAA technology to detect Cronobacter, including a primer pair and a probe, the primer pair includes an upstream primer and a downstream primer, and the sequence of the upstream primer is as in Table 1 As shown in SEQ ID NO.1, the sequence of the downstream primer is shown in SEQ ID NO.2 in Table 1, and the sequence of the probe is shown in SEQ ID NO.5 in Table 1.

[0042] The primer probe design and screening process of this embodiment Cronobacter are as follows:

[0043](1) Design of primers and probes: The sequences of Cronobacter ompA and MMS were obtained from the NCBI GenBank database, and the sequences of fluorescent RAA primers and probes were designed according to the principles of RAA primers and probes design. Primers and probes were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the specific sequences are shown in Table 1 below.

[0044] Table 1 Cronobacter RAA primer ...

Embodiment 2

[0049] According to the primer screening results of Example 1, this example provides a test kit for detecting Cronobacter based on RAA technology, which includes: upstream primer OMP-F1, downstream primer OMP-R1, probe OMP-P1, Buffer, magnesium acetate and purified water, wherein the buffer was purchased from the fluorescent RAA amplification kit of Jiangsu Qitian Gene Biotechnology Co., Ltd.

Embodiment 3

[0051] This example provides a method for detecting Cronobacter based on RAA technology. The specific steps are as follows: use the kit in Example 2 to perform real-time fluorescent RAA amplification. The basic reaction system for RAA amplification is: buffer 25 μL, upstream primers And downstream primer (10 μM) each 2.1 μL, probe (10 μM) 0.6 μL, magnesium acetate 2.5 μL, DNA template plus purified water 17.7 μL, the final volume is 50 μL. According to the real-time fluorescent RAA amplification curve, analyze whether the sample to be tested contains Cronobacter. During operation, mix the buffer, upstream and downstream primers, probes, and purified water and centrifuge them, then pack them into the fluorescent basic reaction unit, flick gently to make the lyophilized powder fully redissolved evenly, centrifuge briefly, open the reaction unit, and pour Magnesium acetate was added to the tube cap of each reaction unit, and then DNA template was added to each reaction unit, thor...

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Abstract

The invention belongs to the technical field of microbiological detection, and particularly discloses a primer probe combination, a kit and a method for detecting cronobacter based on an RAA technology. The primer probe combination comprises a primer pair and a probe. The primer pair comprises an upstream primer and a downstream primer, the sequence of the upstream primer is as shown in SEQ ID NO.1, the sequence of the downstream primer is as shown in SEQ ID NO.2, and the sequence of the probe is as shown in SEQ ID NO.5. When the kit and the method are used for detecting cronobacter, the specificity is good, the sensitivity is high, the accuracy is high, the operation is simple, the cost is low, laboratory detection and on-site rapid detection can be carried out, and the kit and the method have a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a primer-probe combination, a kit and a method for detecting Cronobacter based on RAA technology. Background technique [0002] Cronobacter spp. is an important food-borne pathogen, which has been isolated from infant formula, meat products, fruits and vegetables, grains and other foods. Cronobacter spp. has been associated with cases of neonatal meningitis, necrotizing enterocolitis, sepsis, bloody dysentery, and brain abscess. For infants, especially premature infants weighing less than 2500g, there is a high risk of infection. Studies have shown that Cronobacter can survive in infant milk powder for a long time, so the timely detection of Cronobacter is an important means to prevent its harm. [0003] The traditional Cronobacter detection method is the microbial culture method, and my country has also formulated a national standard for Cronobacter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2563/107C12Q2522/101C12Q2531/119
Inventor 黄新新何宇平古淑青郭德华
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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