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Nucleic acid reagents, kits, systems and methods for detecting Escherichia coli and Klebsiella pneumoniae and their toxicity and drug resistance

A technology for Klebsiella pneumoniae and Escherichia coli, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of poor detection accuracy, time-consuming and laborious, Klebsiella pneumoniae Fewer virulence factor detection methods, etc., to achieve the effect of saving time, high conservation and specificity

Active Publication Date: 2021-02-05
北京卓诚惠生生物科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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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, so the detection accuracy At the same time, these detection methods also have problems such as cumbersome operation, time-consuming and laborious, and low detection sensitivity; moreover, there are still few clinical detection methods for Klebsiella pneumoniae virulence factors

Method used

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  • Nucleic acid reagents, kits, systems and methods for detecting Escherichia coli and Klebsiella pneumoniae and their toxicity and drug resistance
  • Nucleic acid reagents, kits, systems and methods for detecting Escherichia coli and Klebsiella pneumoniae and their toxicity and drug resistance
  • Nucleic acid reagents, kits, systems and methods for detecting Escherichia coli and Klebsiella pneumoniae and their toxicity and drug resistance

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Experimental program
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Effect test

Embodiment 1

[0075] Embodiment 1 detection method and detection result judgment

[0076] 1. Primer and probe synthesis

[0077] Sequence synthesis was performed according to the primer sequences shown in Table 1 and the probe sequences shown in Table 2. 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-BHQ2 29 Escherichia coli uidA-P CY5-tcggcatccggtcagtggcagt-BHQ3 30 iROB iroB-P FAM-agtctcggcaccgtcaaacc-BHQ1 31 magA magA-P VIC-tcgccgcaaatacgagaagtgtagt-BHQ1 32 peg344 peg344-P CY5-cctccgtgatgaggatgaacgaaagtgaag-BHQ3 33 iucA iucA-P CY5-ctatactgcgccaccagccgcgacatgat-BHQ3 34 ...

Embodiment 2

[0104] Embodiment 2 minimum detection limit verification

[0105] Escherichia coli gene, Klebsiella pneumoniae gene, iroB virulence factor, magA virulence factor, peg344 virulence factor, iucA virulence factor, rmpA virulence factor, rmpA2 virulence factor, KPC The gene fragments of the 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 the recombinant plasmids. The construction and sequencing proofreading of the above recombinant plasmids were completed by Shanghai Sangon Biotechnology Company.

[0106] The concentrations of the recombinant plasmids containing the above target gene fragments were quantified as 10 10 copies / μL, and were serially diluted to obtain a concentration of 10 5 copies / μL, 10 4 copies / μ, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 copies / μL of test sample.

[0107] According to the dete...

Embodiment 3

[0109] Example 3 specificity verification

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

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Abstract

The present disclosure relates to a nucleic acid reagent, kit, system and method for detecting Escherichia coli and Klebsiella pneumoniae and their virulence factors and drug resistance genes. The primer shown in SEQ ID NO.1-26 and the probe shown in SEQ ID NO.29-41 are mixed. The present disclosure establishes nucleic acid reagents, kits, systems and methods for detecting Escherichia coli, Klebsiella pneumoniae and the six virulence factors and five drug resistance genes carried by them through the above-mentioned primers and probes, The method can realize rapid, comprehensive, sensitive, specific and automatic determination of detection results, and significantly improves the sensitivity, specificity and simplicity of simultaneous detection of the above-mentioned target detection genes.

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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Patent Type & Authority Patents(China)
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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