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Salmonella detection primer group, method and kit based on RPA-LbCas12a-TTECDS system

A technology for Salmonella and detection primers, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., and can solve problems such as lack of homology

Inactive Publication Date: 2021-07-30
常州市疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, the amino acid sequences of invA and fimY show little homology to other prokaryotic proteins known in the GenBank database

Method used

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  • Salmonella detection primer group, method and kit based on RPA-LbCas12a-TTECDS system
  • Salmonella detection primer group, method and kit based on RPA-LbCas12a-TTECDS system
  • Salmonella detection primer group, method and kit based on RPA-LbCas12a-TTECDS system

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Experimental program
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Embodiment 1

[0039] 1. Materials and methods

[0040] 1.1 Strain screening and DNA extraction

[0041] Standard strains Salmonella typhimurium ATCC700720, Staphylococcus aureus ATCC25923, Listeria monocytogenes ATCC15313, Shigella flexneri ATCC12022, and Escherichia coli ATCC25922 were purchased from the ATCC strain bank. Cucumbers were purchased from a local supermarket. 50 strains of Salmonella clinical specimens in Changzhou were collected through the pathogen identification network. Genomic DNA of Salmonella was extracted by boiling method.

[0042] 1.2 Primer design and RPA reaction

[0043] The full-length nucleotide sequence of the invA gene:

[0044]gtgctgctttctctacttaacagtgctcgtttacgacctgaattactgattctggtactaatggtgatgatcatttctatgttcgtcattcc attacctacctatctggttgatttcctgatcgcactgaatatcgtactggcgatattggtgtttatggggtcgttctacattgacagaatcctca gtttttcaacgtttcctgcggtactgttaattaccacgctctttcgtctggcattatcgatcagtaccagtcgtcttatcttgattgaagccgatg ccggtgaaattatcgccacgttcgggcaattcgttattggcgatagcc...

Embodiment 2

[0076] The application also provides a fast, ultra-sensitive, high-precision Salmonella detection kit based on RPA-LbCas12a-TTECDS, its working principle is as follows Figure 7 shown. In order to improve the detection sensitivity, crRNA at 6 different positions was selected as the best crRNA fluorescence, and the efficiency of crRNA at different positions of the target gene was different. For rapid detection technology, getting enough fluorescence value as soon as possible is crucial for detection. Therefore, it is particularly necessary to screen crRNA, and at the same time, 7 DNA bases are added to the 3'-end of crRNA to form a chimeric DNA-RNA. This has several benefits: First, it improves target specificity by changing the binding energy between crRNA and target. Secondly, the chimeric DNA-RNA form is more convenient for product storage. Finally, the chimeric DNA-RNA form can improve the cleavage activity of high-efficiency crRNA, increase the fluorescence intensity by ...

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Abstract

The invention provides a salmonella detection primer group, a salmonella detection method and a salmonella detection kit based on an RPA-LbCas12a-TTECDS system, and relates to the field of salmonella detection. Based on the combination of a recombinase polymerase amplification technology and a CRISPR gene detection technology, salmonella is detected through an optimized and modified crRNA double-target gene combination, crRNA is screened and optimized aiming at an invA gene and a fimY gene of the salmonella respectively, and upstream and downstream primers for RPA reaction are designed according to nucleotide sequences at two ends of a matched sequence of the crRNA, so that the primer group is obtained. According to the primer group and the crRNA, the RPA-LbCas12a-TTECDS system is designed, and t hesalmonella detection method and kit are established according to the system. The detection method and the kit provided by the invention are used for detecting actual samples, and have the advantages of strong specificity, rapid detection, high sensitivity and high precision.

Description

technical field [0001] The invention relates to the field of salmonella detection, in particular to a primer set, method and kit for salmonella detection based on the RPA-LbCas12a-TTECDS system. Background technique [0002] Salmonella is one of the most common pathogens in foodborne infections and poses a global public health challenge. The main clinical manifestations of mild patients are vomiting and diarrhea, and severe patients may die of infection. Accurate and rapid detection of Salmonella is a necessary means to prevent large-scale public health events. Conventional culture methods require pre-enrichment, selective isolation and biochemical identification of samples. This traditional detection method is cumbersome and time-consuming, requiring 2 to 3 working days to screen suspected Salmonella colonies, and then 1 to 2 working days for biochemical testing. However, it cannot meet the requirement of rapid detection of a large number of samples, so alternative techn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12N15/113
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107C12Q2521/327Y02A50/30
Inventor 毛旭建赵莹杜强屠博文黎俊宏王凤鸣
Owner 常州市疾病预防控制中心
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