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RPA detection primer set of reverse tolerance gene mcr-8, reagent kit and method

An mcr-8, detection kit technology, applied in the field of molecular biology, can solve the problems of time-consuming, labor-intensive, low sensitivity, etc., and achieve the effects of convenient detection, high sensitivity and high sensitivity

Inactive Publication Date: 2020-11-27
山东省兽药质量检验所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some of these techniques are time-consuming and labor-intensive, some have low sensitivity, and some require the help of large precision instruments

Method used

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  • RPA detection primer set of reverse tolerance gene mcr-8, reagent kit and method
  • RPA detection primer set of reverse tolerance gene mcr-8, reagent kit and method
  • RPA detection primer set of reverse tolerance gene mcr-8, reagent kit and method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Synthesis and Copy Number Determination of Recombinant Plasmids of mcr-8 and Other Colistin Resistance Genes

[0043] According to the mcr-8 reference sequence in the GenBank database, the sequence information of the mcr-8 gene (MH598530.1) was determined, synthesized by Huada Gene and cloned into the pUC vector, named pUC-mcr-8, and transformed into the pUC-mcr-8 plasmid To the competent cell DH5α, the plasmid was extracted after overnight culture and the concentration was determined.

[0044] According to the taxonomic status of the colistin resistance gene plasmid, the mode of lateral transmission and the kinship of the adjacent resistance gene, mcr-1 (MF084991.1), mcr-2 (NG_051171.1) and mcr-3 ( NG_055505.1) three drug-resistant gene sequences were used as controls to construct control plasmids pUC-mcr-1, pUC-mcr-2 and pUC-mcr-3.

[0045] Calculate the copy number of recombinant plasmids such as pUC-mcr-8 according to the following formula:

[0046] Copy number (c...

Embodiment 2

[0048] RPA primer (probe) set design

[0049] According to the mcr-8 gene sequence, compared with the mcr-1, mcr-2 and mcr-3 nucleic acid sequences, specific conserved regions were selected, and multiple sets of primer pairs and probes were designed. After preliminary screening, one probe was screened out. and 3 upstream primers for the upstream region of the probe and 3 downstream primers for the downstream region of the probe. Primers and probes were synthesized by Shanghai Sangong (Table 1 and Table 2).

[0050] Table 1

[0051] Primer name Primer sequence (5'-3') mcr-8-F1 CTATTTCTACTTGGCATTGTACCAGCAATTATC, SEQ ID NO 4; mcr-8-F2 TCCGTTCCTTTTTCTTTCCACTCTATTTCTACTT, SEQ ID NO 1; mcr-8-F3 TGAATTAACATCATACTTATCCGTTCCTTTTC, SEQ ID NO 5; mcr-8-R1 GGTAACCATGGCTAATAAGACTACAGCTATGCA, SEQ ID NO 6; mcr-8-R2 CCTTATAAAACACCATGGTAACCATGGCTAATA, SEQ ID NO 2; mcr-8-R3 TTAATCTGCATATTGTTTCGTATGAGAGATGCG, SEQ ID NO 7.

[0052] Table ...

Embodiment 3

[0057] RPA Primer Set (Probe) Specificity

[0058] Using pUC-mcr-8, pUC-mcr-1, pUC-mcr-2 and pUC-mcr-3 as templates, RPA detection was carried out, and the specificity of primer pairs and probes was determined by fluorescence signals. As a result, only pUC-mcr-8 had a specific fluorescent signal, indicating that the method had good specificity (see attached figure 2 ).

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Abstract

The invention discloses a RPA detection primer set of a polymyxin reverse tolerance gene mcr-8, and relates to the technical field of molecular biology. The RPA detection primer set comprises a pair of primers and a probe. The invention provides an mcr-8 detection reagent kit, a detection method of non-diagnosis and treatment purpose, and an application of the primer set. The RPA detection primerset only needs to be provided with a fluorescence detection instrument having the temperature control function, and quick, real-time, acute, accurate and convenient detection can be performed on the mcr-8 under the condition of 39 DEG C.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer set, a kit and a method for detecting RPA of drug resistance gene mcr-8. Background technique [0002] Polymyxin is a polycationic polypeptide drug, which has good curative effect on infections caused by drug-resistant Gram-negative bacteria, especially in super-bacteria such as Carbapenem-resistantenteriaceae (CRE). In the treatment of drug-resistant bacteria, drugs are often used as the last line of defense. The use of polymyxin in clinical medicine, animal husbandry and agriculture is increasing year by year and the situation of irrational drug use poses a serious threat to public health security and the development of animal husbandry. Therefore, it is of great significance to establish a rapid detection method for polymyxin-resistant strains for targeted drug use. In 2016, the plasmid-mediated polymyxin resistance gene mcr-1 was discovered and proved to ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2600/106
Inventor 刘少宁李有志陈智徐恩民马艳芳牛华星
Owner 山东省兽药质量检验所
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