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CN-121975958-A - Pseudomonas aeruginosa drug resistance gene detection primer group based on multiplex PCR-time-of-flight mass spectrometry, kit and kit using method

CN121975958ACN 121975958 ACN121975958 ACN 121975958ACN-121975958-A

Abstract

The invention discloses a pseudomonas aeruginosa drug resistance gene detection primer group based on multiple PCR-time-of-flight mass spectrometry, a kit and a kit using method. The pseudomonas aeruginosa drug-resistant gene comprises armA, rmtB, rmtC, mexA, blaVIM, blaNDM, blaIMP, blaKPC, blaOXA and blaGES, the detection primer group comprises an upstream amplification primer and a downstream amplification primer of each gene and a single-base extension primer, the nucleotide sequences of the upstream amplification primer and the downstream amplification primer of each gene are respectively shown as SEQ ID NO. 1-NO. 20, and the nucleotide sequences of the single-base extension primers of each gene are respectively shown as SEQ ID NO. 21-NO. 30. By adopting the kit comprising the primer group, whether the pseudomonas aeruginosa is infected or not can be obtained within 40 minutes, and if the pseudomonas aeruginosa is infected, the pseudomonas aeruginosa drug resistance condition can be obtained. The detection process has higher sensitivity and specificity, the accuracy rate of detection on samples can reach 92-97%, and the method can be used for rapid, accurate, high-sensitivity and high-throughput detection of pseudomonas aeruginosa antibiotic resistant strains, and is beneficial to application and popularization in hospitals and basic departments.

Inventors

  • CHE TUANJIE
  • MA LI
  • WU KANGLI
  • ZHENG XIAOLING
  • FANG CHUNYAN
  • XIN QIAN
  • LI YUAN

Assignees

  • 兰州百源基因技术有限公司

Dates

Publication Date
20260505
Application Date
20251229

Claims (10)

  1. 1. The pseudomonas aeruginosa drug resistance gene detection primer group based on multiplex PCR-time-of-flight mass spectrometry is characterized in that the pseudomonas aeruginosa drug resistance genes comprise armA, rmtB, rmtC, mexA, blaVIM, blaNDM, blaIMP, blaKPC, blaOXA and blaGES; the detection primer group comprises an upstream amplification primer and a downstream amplification primer of each gene and a single-base extension primer; The nucleotide sequences of the amplification primers at the upper and the lower stream of each gene are respectively shown as SEQ ID NO. 1-SEQ ID NO.20, and the nucleotide sequences of the single-base extension primers of each gene are respectively shown as SEQ ID NO. 21-SEQ ID NO. 30.
  2. 2. The primer set for detecting the drug-resistant gene of pseudomonas aeruginosa based on multiplex PCR-time-of-flight mass spectrometry according to claim 1, wherein the primer set is designed by designing a single base extension primer with a length of 14-28 bases in a conserved sequence region of an amplification region between an upstream amplification primer and a downstream amplification primer designed for each gene, allowing extension of a designed base at the 3' -end of the extension primer as a specific sequence marker of the gene, and the total length after single base extension is not more than 29 bases, wherein the molecular weight difference between each extension primer and an extension product is at least 16Da.
  3. 3. A pseudomonas aeruginosa drug-resistant gene detection kit based on multiplex PCR-time-of-flight mass spectrometry is characterized by comprising a mixed solution of upstream and downstream amplification primers of each gene, a mixed solution of single-base extension primers of each gene, a PCR amplification reagent and an extension reagent.
  4. 4. The pseudomonas aeruginosa drug resistance gene detection kit based on multiplex PCR-time-of-flight mass spectrometry according to claim 3, wherein the PCR amplification reagent comprises PCR buffer, PCR enzyme and deionized water, and the extension reagent comprises extension buffer, extension stop solution and extension enzyme.
  5. 5. The kit for detecting the drug-resistant gene of pseudomonas aeruginosa based on multiplex PCR-time-of-flight mass spectrometry according to claim 4, wherein the mixed solution of the amplification primers is prepared by adding 80 μl deionized water into 1 μl of each of the upstream and downstream amplification primers of 100 μΜ, and the final concentration of the mixed solution is 1 μΜ.
  6. 6. The kit for detecting the drug-resistant gene of pseudomonas aeruginosa based on multiplex PCR-time-of-flight mass spectrometry according to claim 4, wherein the single base extension primer mixture is prepared by mixing 1 μl of single base extension primers of each gene with a concentration of 100 μΜ, and the final concentration of the mixture is 10 μΜ.
  7. 7. The method of using a kit for non-diagnostic purposes based on multiplex PCR-time-of-flight mass spectrometry for pseudomonas aeruginosa drug resistance gene according to any one of claims 3-6, comprising the steps of: (1) The PCR amplification reaction, namely mixing an amplification primer mixed solution with a PCR amplification reagent to form a PCR reaction system by taking DNA of a sample to be detected as a template, and carrying out multiplex PCR amplification reaction to obtain an amplification product; (2) Performing shrimp alkaline phosphatase reaction, namely performing dephosphorylation reaction on the amplification product in the step (1) by using shrimp alkaline phosphatase SAP to obtain a dephosphorylation product; (3) Single base extension reaction, namely preparing an extension reaction system by using an extension reagent and single base extension primer mixed solution, and carrying out single base extension reaction on the dephosphorylated product in the step (2) to obtain an extension product; (4) Desalting resin, namely desalting and purifying the extension product in the step (3) by using desalting resin to obtain a purified extension product; (5) And mass spectrum detection, namely carrying out molecular weight detection on the purified extension product by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrum system, and rapidly judging whether the drug-resistant gene sequence of the pseudomonas aeruginosa exists or not according to the difference of the mass-charge ratios of the nucleic acid fragments as a result detection signal.
  8. 8. The method of using a kit for non-diagnostic purposes based on multiplex PCR-time-of-flight mass spectrometry according to claim 7, wherein the PCR amplification reaction is performed in step (1) under conditions of 95℃for 5min, 95℃for 30s,60℃for 30s,72℃for 1min,45 cycles, 72℃for 5min, and constant temperature of 4 ℃.
  9. 9. The method for using the pseudomonas aeruginosa drug resistance gene detection kit based on multiple PCR-time-of-flight mass spectrometry according to claim 8, wherein in the step (2), the condition of the enzymolysis reaction is 37 ℃ for 40min, 85 ℃ for 5min and constant temperature is 4 ℃.
  10. 10. The method of using a kit for non-diagnostic purposes based on multiplex PCR-time-of-flight mass spectrometry according to claim 9, wherein the conditions for the extension reaction in step (3) are 95℃30s, 95℃5s,52℃5s (5 internal cycles), 80℃5s (5 internal cycles), 40 external cycles, 72℃3min, and constant temperature of 4 ℃.

Description

Pseudomonas aeruginosa drug resistance gene detection primer group based on multiplex PCR-time-of-flight mass spectrometry, kit and kit using method Technical Field The invention relates to the technical field of molecular biology, in particular to a pseudomonas aeruginosa drug resistance gene detection primer group based on multiplex PCR-time-of-flight mass spectrometry, a kit and a kit using method. Background Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA), pseudomonas aeruginosa, is a non-fermenting gram-negative bacillus that is widely distributed in nature, can survive in a variety of environments, and can inhabit soil, water, plants and human bodies. The result of 2022CHINET bacterial drug resistance monitoring shows that PA in clinical isolates is the fourth position in gram negative bacilli and the first position in non-zymophytes. Results from a clinical retrospective cohort study involving 5 countries, 12 emergency hospitals showed that the mortality rates in the hospital for HAP and VAP patients from PA infection were as high as 40.1% and 41.9%, respectively. 10% of infections in intensive care areas are associated with pseudomonas aeruginosa, which causes pneumonia, sepsis, etc., which show high morbidity and mortality. In 2017, the world health organization listed pseudomonas aeruginosa as one of the bacteria that urgent need to develop new antibacterial drug therapies. Drug resistance monitoring of fine bacteria in CHINET in 2021 showed that PA accounted for 7.96% of the total detected strain, where PA accounted for 34.9% of non-fermented sugar gram-negative bacilli. Various resistance genes are available for PA, such as the beta-lactamase genes (tem, shv, oxa, veb, imp, vim), aminoglycoside modifying enzyme genes (aac, aad, aph). The former may lead to bacterial resistance to β -lactam antibiotics, such as penicillins and cephalosporins, leading to failure of antibiotic treatment, and the latter may lead to bacterial resistance to aminoglycoside antibiotics, both of which are currently the primary drugs for clinical treatment of PA infections. Aminoglycoside antibiotics have been shown to have a good therapeutic effect on PA, play an important role in the treatment of PA infection, and have been widely used as first-line drugs. However, with the clinical application of the medicines, the drug resistance of PA is increased. In recent years, the phenomenon of PA becoming resistant to aminoglycoside antibiotics has been reported. The Aminoglycoside Modifying Enzyme (AMEs) gene and the 16S rRNA methylase gene in the drug resistance mechanism are the main reasons for mediating the high drug resistance of the aminoglycoside antibiotics. The bacteria produce 16SrRNA methylase and the aminoglycoside resistant drugs are main drugs for preventing or treating gram-negative bacteria infection in recent years, and the aminoglycoside drug action target of the bacteria can resist all the aminoglycoside drugs after being methylated. Because the 16S r RNA methylase gene and the aminoglycoside modification enzyme gene are mostly positioned on the same plasmid, the bacterial strain is easy to horizontally spread among different bacteria, and the drug-resistant strain is caused to spread, so that the monitoring of bacterial drug resistance and the research of drug-resistant mechanism are enhanced. Some pseudomonas aeruginosa have been found to produce an ultra-broad spectrum of beta-lactamases (ESBLs) that makes them highly resistant to most beta-lactam antibiotics, including penicillins, cephalosporins, carbapenems and aztreonam. With the wide application of carbapenem antibacterial drugs, the detection rate of carbapenem-resistant pseudomonas aeruginosa (CRPA) is continuously increased, and the method brings serious challenges to clinical anti-infection treatment. Infection of multi-drug resistant bacteria is closely related to the death rate of inpatients, and has become an important point of the prevention and control work of nosocomial infection. Treatment and prevention of PA bacterial infection has been a concern for researchers, and corresponding therapeutic drugs and methods have been reported in recent years. Therefore, it is necessary to develop a kit for rapidly detecting PA drug resistance gene in clinical diagnosis, epidemiological investigation and laboratory mechanism investigation. Disclosure of Invention Aiming at the problems of small detection flux, low speed or high cost of the existing drug-resistant gene detection method, the invention provides a pseudomonas aeruginosa drug-resistant gene detection primer group based on multiplex PCR-time-of-flight mass spectrometry, a kit and a kit using method, and realizes the antibiotic drug resistance condition corresponding to 10 genes in total by one-time analysis armA, rmtB, rmtC, mexA, blaVIM, blaNDM, blaIMP, blaKPC, blaOXA and blaGES. In order to achieve the purpose, the invention adopts the following technical scheme: The invention provides