Nucleic acid reagent, reagent kit, system and method for detecting escherichia coli and klebsiella pneumoniae and toxicity and reverse tolerance of escherichia coli and klebsiella pneumoniae

A technology for Klebsiella pneumoniae and Escherichia coli, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of time-consuming, laborious detection, poor detection accuracy, and low detection sensitivity , achieve high conservatism and specificity, and save time

Active Publication Date: 2019-11-12
北京卓诚惠生生物科技股份有限公司
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Problems solved by technology

[0004] However, among the currently commonly used clinical bacterial resistance detection methods, factors such as the rate of specimen submission, drug use, specimen collection quantity, culture medium, and culture methods will affect the detection results of bacterial resistance

Method used

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  • Nucleic acid reagent, reagent kit, system and method for detecting escherichia coli and klebsiella pneumoniae and toxicity and reverse tolerance of escherichia coli and klebsiella pneumoniae
  • Nucleic acid reagent, reagent kit, system and method for detecting escherichia coli and klebsiella pneumoniae and toxicity and reverse tolerance of escherichia coli and klebsiella pneumoniae
  • Nucleic acid reagent, reagent kit, system and method for detecting escherichia coli and klebsiella pneumoniae and toxicity and reverse tolerance of escherichia coli and klebsiella pneumoniae

Examples

Experimental program
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Example Embodiment

[0075] Example 1 Detection method and detection result judgment

[0076] 1. Synthesis of primers and probes

[0077] According to the primer sequence shown in Table 1 and the probe sequence shown in Table 2, sequence synthesis was performed. In the probe, FAM is 6-carboxyfluorescein, HEX is hexachloro-6-methylfluorescein, CY5 is 5H-indocyanine, ROX is 6-carboxy-X-rhodamine, and VIC is a dye purchased from ABI , BHQ1, BHQ2 and BHQ3 are quenching groups.

[0078] Table 1

[0079]

[0080]

[0081] Table 2

[0082] Detection target Probe sequence Probe sequence SEQ ID NO Klebsiella pneumoniae KP-P ROX-cctacaccagctccgaccgtaccaa-BHQ229 Escherichia Coli uidA-P CY5-tcggcatccggtcagtggcagt-BHQ330 iroB iroB-P FAM-agtctcggcaccgtcaaacc-BHQ131 magA magA-P VIC-tcgccgcaaatacgagaagtgtagt-BHQ132 peg344 peg344-P CY5-cctccgtgatgaggatgaacgaaagtgaag-BHQ333 iucA iucA-P CY5-ctatactgcgccaccagccgcgacatgat-BHQ334 rmpA rmpA-P VIC-cagggaaatggggagggtacaaaat-BHQ135 rmpA2 rmpA2-P FAM-cagggaaatgggatggtta...

Example Embodiment

[0104] Example 2 Verification of the lowest detection limit

[0105] The Escherichia coli species gene, Klebsiella pneumoniae species gene, iroB virulence factor, magA virulence factor, peg344 virulence factor, iucA virulence factor, rmpA virulence factor, rmpA2 virulence factor, KPC The gene fragments of drug resistance gene, NDM drug resistance gene, IMP drug resistance gene, VIM drug resistance gene, and OXA-48 drug resistance gene were connected to the PUC57 vector and sequenced and checked to obtain each recombinant plasmid. The construction of the above recombinant plasmids and the sequencing and proofreading work were completed by Shanghai Shenggong Biotechnology Company.

[0106] The concentration of the recombinant plasmid containing the above target gene fragments was quantified to 10 10 Copy / μL, and perform serial dilutions to obtain a concentration of 10 5 Copy / μL, 10 4 Copy / μ, 10 3 Copy / μL, 10 2 Copy / μL, 10 1 Copy / μL, 10 0 Copy / μL of test sample.

[0107] According to t...

Example Embodiment

[0109] Example 3 Specificity verification

[0110] Select Enterobacter cloacae (purchased from China Medical Culture Collection, number 45301), Serratia marcescens (purchased from China Medical Culture Collection, number 41002), Salmonella paratyphi A (purchased from China Medical Culture Collection Center, No. 50001), Salmonella Paratyphi B (purchased from China Medical Culture Collection, No. 50004), Bacillus mycoides (purchased from China Medical Culture Collection, No. 32012), Wei Bacillus spp (purchased from China Medical Culture Collection, number 32011), Bacillus subtilis (purchased from China Medical Culture Collection, number 32041), Staphylococcus aureus (purchased from China Medical Culture Collection, No. 26003), Vibrio parahaemolyticus (purchased from the Chinese Medical Culture Collection, No. 21617), Listeria monocytogenes (purchased from the Chinese Medical Culture Collection, No. 54002), Cholera Vibrio (purchased from China Medical Culture Collection, number 110...

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Abstract

The invention relates to a nucleic acid reagent, reagent kit, system and method for detecting escherichia coli and klebsiella pneumoniae and toxicity and reverse tolerance of the escherichia coli andthe klebsiella pneumoniae. The nucleic acid reagent comprises a primer as shown in SEQIDNO.1-26 and a probe as shown in SEQIDNO.29-41, which are in mutual independent storage or mutual discretionary mixed storage. Through the primer and the probe disclosed by the invention, the nucleic acid reagent, reagent kit, system and method for detecting the escherichia coli and klebsiella pneumoniae and 6 virulence factors and 5 reverse tolerance genes, carried by the escherichia coli and the klebsiella pneumoniae, are constructed, quick, comprehensive, sensitive, excellent and automatic testing resultjudgment can be realized, and the sensibility, the specificity and the simplicity for detection for target detection genes can be notably improved.

Description

technical field [0001] The disclosure relates to the field of biotechnology, in particular, to a nucleic acid reagent, kit and method for detecting Escherichia coli, Klebsiella pneumoniae and their virulence factors and drug resistance genes. Background technique [0002] Klebsiella pneumoniae is a common opportunistic pathogenic bacteria in clinical practice. It has the characteristics of high mucus phenotype, high invasiveness and high lethality. Its pathogenicity is related to multiple virulence factors. Klebsiella pneumoniae and Escherichia coli producing extended-spectrum β-lactamases (ESBLs) and carbapenemases are pathogenic bacteria with drug resistance. my country's CHINET bacterial resistance surveillance data show that pneumonia The antibiotic resistance rate of Klebsiella and Escherichia coli has been increasing year by year, from 3% in 2005 to 19.2% in 2016. A great challenge has come. [0003] At present, the clinically commonly used bacterial resistance detecti...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107C12Q2545/113
Inventor 杨启文杨海英李雪王雷姜怀德徐英春张志强
Owner 北京卓诚惠生生物科技股份有限公司
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