CN109536626B - Nucleic acid reagent, kit, system and method for detecting gram-negative bacteria and/or gram-negative bacteria drug resistance - Google Patents

Nucleic acid reagent, kit, system and method for detecting gram-negative bacteria and/or gram-negative bacteria drug resistance Download PDF

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CN109536626B
CN109536626B CN201811641979.5A CN201811641979A CN109536626B CN 109536626 B CN109536626 B CN 109536626B CN 201811641979 A CN201811641979 A CN 201811641979A CN 109536626 B CN109536626 B CN 109536626B
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王雷
杨海英
林笑冬
王晓艳
张志强
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Beijing Applied Biological Technologies Co ltd
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Abstract

The present disclosure relates to a nucleic acid reagent, a kit, a system and a method for detecting gram-negative bacteria and/or gram-negative bacteria drug resistance, wherein the nucleic acid reagent comprises primers shown in SEQ ID No.1-60 and probes shown in SEQ ID No.63-92, which are respectively stored independently or randomly mixed with each other. The nucleic acid reagent, the kit, the system and the method for detecting 6 pathogens such as Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Bacillus cloacae, Serratia marcescens, Acinetobacter baumannii and the like and the common 24 drug resistance genes carried by the pathogens are established through the primers and the probes, so that the rapid, comprehensive, sensitive, specific and automatic detection result judgment can be realized, and the sensitivity, the specificity and the simplicity of simultaneously detecting various gram-negative bacteria and the drug resistance of the gram-negative bacteria are obviously improved.

Description

Nucleic acid reagent, kit, system and method for detecting gram-negative bacteria and/or gram-negative bacteria drug resistance
Technical Field
The present disclosure relates to the field of biotechnology, and in particular, to a nucleic acid reagent, a kit, a system and a method for detecting gram-negative bacteria and/or gram-negative bacteria drug resistance.
Background
The gram-negative bacteria account for 60% of the pathogenic bacteria spectrum causing infection, and mainly comprise Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Bacillus cloacae, Serratia marcescens and Acinetobacter baumannii. With the high use of antibiotics, gram-negative bacteria develop resistance. Drug resistance is also called drug resistance, and generally refers to that after bacteria contact with drugs for many times, the sensitivity of the drugs is reduced or even disappears, so that the curative effect of the drugs on drug-resistant bacteria is reduced or ineffective. After the drug-resistant bacteria appear, the drug-resistant bacteria are quickly transferred among different regions, same species and different species of bacteria, so that drug-resistant strains appear in individuals or regions which do not use the drug, and the drug-resistant rate tends to rise year by year. And with the increase of the variety of drugs, the drug resistance spectrum of the drug-resistant bacteria is wider and wider, which has become a great trouble in clinical diagnosis and treatment.
At present, the identification method of gram-negative bacteria mainly comprises bacteria isolation culture, biochemical reaction and serological method. The final result obtained by the whole detection needs about 2-5 days, which is time-consuming and labor-consuming. The clinically common bacterial drug resistance detection methods comprise bacterial drug resistance phenotype detection, drug sensitivity test, plasmid elimination test and plasmid fingerprint technology. These detection methods can systematically and comprehensively identify drug-resistant genes, but they have drawbacks of the respective techniques.
Disclosure of Invention
The purpose of the present disclosure is to provide a nucleic acid reagent, a kit, a system and a method for rapidly and accurately detecting gram-negative bacteria and/or gram-negative bacteria drug resistance.
To achieve the above object, a first aspect of the present disclosure: a nucleic acid reagent for detecting gram-negative bacteria and/or gram-negative bacteria resistance is provided, wherein the nucleic acid reagent comprises primers shown in SEQ ID NO.1-60 and probes shown in SEQ ID NO.63-92 which are respectively stored independently or randomly mixed with each other.
Optionally, the amount of the primer represented by SEQ ID NO.2-60 is 0.9-1.1. mu.M, 0.3-0.6. mu.M, 0.9-1.1. mu.M, 0.3-0.5. mu.M, 0.9-1.1. mu.M, 0.5-1. mu.M, 0.9-1.1. mu.M, 0.4-0.8. mu.M, 0.9-1.1. mu.M, 0.3-0.6. mu.M, 0.9-1.1. mu.M, 0.9-1. mu.0.9-1. mu.0.1. mu.M, 0.9-1.0.9-0.1. mu.0.9-0.9-1. mu.9-0.0.1. mu.0.9-0.1. mu.0.0.9-0.9-0.1, 0.0.9-0.9-0.1, 0.9-0.0.0.0.9-0.1. mu.9-0.1, 0.9-0.1. mu.9-0.9-0.0.9-0.9-0.1. mu.1. mu.M, 0.1. mu.0.9-0.M, 0.9-1, 0.9-0.1. mu.9-1. mu.0.9-0.0.9-0.M, 0.1. mu.1, 0.9-1. mu.9-0.M, 0.9-0.1, 0.9-1. mu.9-1, 0.0.1, 0.0.0.1. mu.1, 0.9-0.1. mu.0.9-0.0.0.0.9-0.M, 0.9-1. mu.0.9-0.0.0.1. mu.9-0.9-0.5-0.9-1. mu.9-1, 0.0.0.9-1, 0.M, 0.9-1. mu.1. mu.0.0.0.M, 0.9-0.1-1-0.9-1-0.0.0.9-1, 0.0.0.0.0.M, 0.1-1-0.0.0.0.0.0.0.0.0.1-1-0.1, 0.0.0.0.9-0.9-1-0.0.9-0.M, 0.5-0.0.0.0.0.1-0.9-0.0.0.0.0.0.0.0.0.0.0.0.0.0.9-1-0.5-0.0.0.9-1-0.0.0.0.0.0.0.0.0.0.5-0.0.0.0.0.0.0.0.0.0.0.0.5-0., 0.9 to 1.1. mu.M, 0.4 to 0.8. mu.M, 0.9 to 1.1. mu.M, 0.3 to 0.6. mu.M, 0.9 to 1.1. mu.M, 0.3 to 0.5. mu.M, 0.9 to 1.1. mu.M, 0.5 to 1. mu.M, 0.9 to 1.1. mu.M, 0.3 to 0.6. mu.M, 0.9 to 1.1. mu.M, 0.3 to 0.5. mu.M, 0.9 to 1.1. mu.M, 0.5 to 1.1. mu.M, 0.4 to 0.8. mu.M, 0.9 to 1.1. mu.M, 0.3 to 0.6. mu.M, 0.9 to 1.1. mu.1. mu.M, 0.3 to 0.5. mu.5. mu.M, 0.9 to 1.1. mu.1. mu.M, 0.5 to 1. mu.1. mu.M, 0.9 to 1. mu.M, and 0.9 to 1. mu.92. mu.63 to 1. mu.1. mu.M, respectively, and 0.63 to 1. mu.63 to 1. mu.M of a probe, respectively, wherein the content of the probe ID is 0.63 to 1 [ mu.1 [ mu.M, respectively, and [ mu.M ] M, respectively, as expressed by [ mu.63 to [ mu.1 [ mu ] M ] ID.
Optionally, the nucleic acid reagent further comprises a positive internal quality control;
the positive endoplasmic control contains a primer shown by SEQ ID NO.61-62 and a probe shown by SEQ ID NO. 93.
Optionally, the nucleic acid reagent comprises tube a and tube B; the tube A contains primers shown in SEQ ID NO.1-32 and 61-62 and probes shown in SEQ ID NO.63-78 and 93; the B tube contains primers shown in SEQ ID NO.33-62 and probes shown in SEQ ID NO. 79-93.
Alternatively, the probes shown in SEQ ID NO.63-66,79-82 have a first fluorescent label; the probes shown in SEQ ID NO.67-70,83-86 have a second fluorescent label; the probes shown in SEQ ID NO.71-74,87-89 have a third fluorescent label; the probes shown in SEQ ID NO.75-78,90-93 have a fourth fluorescent label; the first fluorescent label, the second fluorescent label, the third fluorescent label and the fourth fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a VIC fluorescent label, a CY5 fluorescent label and a ROX fluorescent label.
Optionally, the gram-negative bacteria comprise at least one of klebsiella pneumoniae, escherichia coli, pseudomonas aeruginosa, enterobacter cloacae, serratia marcescens, acinetobacter baumannii, and the gram-negative bacteria resistance comprises at least one of gram-negative bacteria aminoglycoside resistance, gram-negative bacteria penicillin cephalosporin resistance, gram-negative bacteria sulfonamide resistance, and gram-negative bacteria trimethoprim resistance.
In a second aspect of the present disclosure: there is provided a kit for detecting gram-negative bacteria and/or gram-negative bacteria resistance, the kit comprising a nucleic acid reagent according to the first aspect of the disclosure, and optionally, the kit further comprises at least one of a reaction system buffer, DNA polymerase, DMSO, magnesium ions, BSA, dntps, and water
A third aspect is disclosed: there is provided the use of a nucleic acid reagent according to the first aspect of the disclosure in the preparation of a kit for the detection of gram-negative bacteria and/or gram-negative bacteria resistance.
A fourth aspect of the present disclosure: providing a system for detecting gram-negative bacteria and/or gram-negative bacteria drug resistance, which comprises a PCR instrument, a computing device and an output device, wherein the PCR instrument is provided with an A tube detector and a B tube detector, the A tube detector and the B tube detector are respectively a nucleic acid reagent storage container loaded with the nucleic acid reagents, the PCR instrument comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel, the first fluorescence channel, the second fluorescence channel, the third fluorescence channel and the fourth fluorescence channel are different and are respectively and independently a FAM fluorescence channel, a VIC fluorescence channel, a CY5 fluorescence channel or a ROX fluorescence channel; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
if the blank control, the positive control and the positive internal control are established, the detection result is valid;
if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains the Escherichia coli; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains Klebsiella pneumoniae; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains acinetobacter baumannii; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains pseudomonas aeruginosa; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains serratia marcescens; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 62 ℃, determining that the sample contains enterobacter cloacae; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 66 ℃, determining that the sample contains blaOXA-1; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 70 ℃, determining that the sample contains blaOXA-10; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaIMP-4; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains blaIMP-6; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaIMP-8; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaTEM; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaVEB; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains the blaSVV; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaVIM; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaCTX; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 1; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 2; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains aadA 3; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains aadA 4; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 65, determining that the sample contains the Sull; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains sul 3; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 5; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 6; if the third fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains sut 1; if the third fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains sat 1; if the third fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 1; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains dfrA 14; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 17; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains dfrA 15; if the fourth fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
determining that the sample has gram-negative bacterial aminoglycoside resistance if the sample contains at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA 6; determining that the sample has gram negative bacterial resistance to penicillin cephalosporins if the sample contains at least one of blaOXA-1, blaOXA-10, blaIMP-4, blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSVV, blaVIM, and blaCTX; if the sample contains at least one of sull, sul3, sut1 and sat1, judging that the sample has sulfonamide resistance of gram-negative bacteria; when the sample contains at least one of dfrA1, dfrA14, dfrA17 and dfrA15, the sample is judged to have the drug resistance of gram-negative bacteria trimethoprim.
The fifth aspect of the present disclosure: a method for detecting gram negative bacteria and/or gram negative bacteria resistance is provided, wherein the method comprises: carrying out PCR amplification on DNA of a sample to be detected by adopting the nucleic acid reagent; the PCR instrument for carrying out the PCR amplification comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel; the first, second, third, and fourth fluorescent channels are different from each other and are each independently a FAM, VIC, CY5, or ROX fluorescent channel; and the following discrimination is made:
if the blank control, the positive control and the positive internal control are established, the detection result is valid;
if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains the Escherichia coli; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains Klebsiella pneumoniae; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains acinetobacter baumannii; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains pseudomonas aeruginosa; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains serratia marcescens; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 62 ℃, determining that the sample contains enterobacter cloacae; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 66 ℃, determining that the sample contains blaOXA-1; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 70 ℃, determining that the sample contains blaOXA-10; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaIMP-4; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains blaIMP-6; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaIMP-8; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaTEM; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaVEB; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains the blaSVV; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaVIM; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaCTX; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 1; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 2; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains aadA 3; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains aadA 4; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 65, determining that the sample contains the Sull; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains sul 3; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 5; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 6; if the third fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains sut 1; if the third fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains sat 1; if the third fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 1; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains dfrA 14; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 17; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains dfrA 15; if the fourth fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
determining that the sample has gram-negative bacterial aminoglycoside resistance if the sample contains at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA 6; determining that the sample has gram negative bacterial resistance to penicillin cephalosporins if the sample contains at least one of blaOXA-1, blaOXA-10, blaIMP-4, blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSVV, blaVIM, and blaCTX; if the sample contains at least one of sull, sul3, sut1 and sat1, judging that the sample has sulfonamide resistance of gram-negative bacteria; when the sample contains at least one of dfrA1, dfrA14, dfrA17 and dfrA15, the sample is judged to have the drug resistance of gram-negative bacteria trimethoprim.
The beneficial effect of this disclosure lies in:
the method can quickly realize screening and identification of drug resistance genes of 6 gram-negative bacteria and 24 gram-negative bacteria in a sample to be detected, avoids complex operations of methods such as serology, pathogen culture and the like, and achieves the following detection effects:
(I) 2-tube simultaneous detection of target pathogens and drug resistance
The detection method established by the disclosure can screen 30 genes of 6 gram-negative bacteria and 24 gram-negative bacteria drug resistance genes and the like through 2 tubes. The related pathogen species and the drug resistance information can be quickly and simply obtained, and time, labor and reagent cost are saved.
(II) high sensitivity
The detection method established by the disclosure can realize simultaneous detection of 30 genes, and the detection sensitivity of each target gene in a reaction system can reach 102CFU/ml, comparable to the sensitivity of single real-time fluorescent PCR detection.
(III) high specificity
In the detection method established by the disclosure, all primers are subjected to BLAST comparison analysis, have high conservation and specificity, can distinguish detection targets from each other, and can also distinguish from other bacteria with similar species and same living environment, including Shigella, Salmonella, Proteus, pertussis, Citrobacter, Yersinia enterocolitica, stenotrophomonas maltophilia, Citrobacter, and the like.
(IV) accuracy of sample results
Due to the special design of the sampler, the sample entering each reactor can be uniform, and the reaction of each tube is repeated for 2 times, so that the accuracy of the sample detection result is improved.
(V) the operation is quick and convenient
The clinical samples can be directly placed in a ParaDNA reactor through a sampler for detection to obtain reliable results, so that expensive and time-consuming sample extraction steps are avoided, and emergency detection except a professional laboratory is realized.
(VI) Emergency site treatment
The nucleic acid reagent disclosed by the invention is based on a Hybeacon technology ParaDNA system, is small in volume and convenient to carry, and can be used for clinical emergency sites.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
The first aspect of the disclosure: a nucleic acid reagent for detecting gram-negative bacteria and/or gram-negative bacteria resistance is provided, wherein the nucleic acid reagent comprises primers shown in SEQ ID NO.1-60 and probes shown in SEQ ID NO.63-92 which are respectively stored independently or randomly mixed with each other.
The method can detect the drug resistance of gram-negative bacteria and/or gram-negative bacteria by using ParaDNA and Hybeacon probe technology, can simultaneously and accurately identify 6 pathogens such as Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acidum cloacae, Serratia marcescens, Acinetobacter baumannii and the like, and can also simultaneously identify 24 common drug-resistant genes carried by 6 pathogens, mainly comprises 6 aminoglycoside drug-resistant genes such as aadA1, aadA2, aadA3, aadA4, aadA5 and aadA6 and the like, 10 penicillin cephalosporin drug-resistant target genes such as blaOXA-1, blaOXA-10, blaIMP-4, blaIMP-865-6, blaIMP-8, blaBLaTEM, blaVEB, blaSVV, blaVIM, blaCTX and the like, and can also comprises 4 Sadfra drug-resistant target genes such as sulfr 3, sut1, t1 and the like, and can also comprises a5 and a 3. Has important significance for monitoring the drug resistance dynamic of bacteria, guiding the clinical reasonable use of antibacterial drugs and effectively controlling infection.
The Hybeacon probe technology has high requirements on the probe, and the Tm value of the probe is particularly important; in addition, the effect of the combination of the probe and the primer also has an important influence on the amplification effect. In the design process of the primers and the probes, the problem of co-amplification of the primers and the probes of different target genes in a reaction system is considered, namely, the Tm value, the difference value of the Tm values of the probes corresponding to the targets, GC content and the like are evaluated, the conditions of hairpin structures, dimers and the like are avoided, the alternative primers and the probe segments can be ensured to respectively and comprehensively cover the enteroviruses, and the primers and the probes have good specificity and high coverage.
Further, the amount of the primer represented by SEQ ID NO.2-60 may be 0.9-1.1. mu.M, 0.3-0.6. mu.M, 0.9-1.1. mu.M, 0.3-0.5. mu.M, 0.9-1.1. mu.M, 0.5-1. mu.M, 0.9-1.1. mu.M, 0.4-0.8. mu.M, 0.9-1.1. mu.M, 0.3-0.6. mu.M, 0.9-1.0.9-1. mu.M, 0.9-1. mu.0.9-1. mu.M, 0.9-1.9-1. mu.0.9-1, 0.9-1. mu.9-0.0.9-1. mu.0.9-0.M, 0.9-1. mu.9-0.1, 0.9-0.1, 0.9-0.1. mu.0.0.0.9-0.9-1, 0.9-1, 0.9-0.1. mu.0.9-0.9-0.1, 0.9-1. mu.0.1, 0.0.9-1. mu.0.9-1, 0.9-0.0.9-0.M, 0.9-1. mu.9-0.1, 0.9-1, 0.9-0.1, 0.9-1-0.M, 0.9-1-0.0.0.9-0.M, 0.9-0.0.0.0.9-0.1-1-0.M, 0.9-0.0.0.9-1-0.9-1-0.0.0.0.9-0.9-1-0.0.0.M, 0.9-0.0.9-0.0.M, 0.0.0.9-0.M, 0.0.0.0.9-1-0.M, 0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.9-0.9-1-0.9-0.0.9-0.0.0.0.0.0.0.0.0.0.9-0.5-0.0.0.0.0.0.0.0.0.0.0.0.0.0.9-1-0.0.0.0.0.0.M, 0.0.0.0.9-0.0.0.0.0.0.0.0.0.0.0.0, 0.5 to 1 μ M, 0.9 to 1.1 μ M, 0.4 to 0.8 μ M, 0.9 to 1.1 μ M, 0.3 to 0.6 μ M, 0.9 to 1.1 μ M, 0.3 to 0.5 μ M, 0.9 to 1.1 μ M, 0.5 to 1 μ M, 0.9 to 1.1 μ M, 0.4 to 0.8 μ M, 0.9 to 1.1 μ M, 0.3 to 0.6 μ M, 0.9 to 1.1 μ M, 0.3 to 0.5 μ M, 0.9 to 1.1 μ M, 0.5 μ M and 0.63 to 1.92 μ M, respectively, and the respective contents of the probes are expressed by ID of SEQ ID, 0.9 to 1 μ M.
According to the present disclosure, the nucleic acid reagent may further include a positive internal quality control for the sake of quality control. Further, the positive internal quality control can contain primers shown in SEQ ID NO.61-62 and a probe shown in SEQ ID NO. 93. In this case, the content of the primers shown by SEQ ID NO.61 to 62 may be 0.9 to 1.1. mu.M and 0.4 to 0.6. mu.M, respectively, and the content of the probe shown by SEQ ID NO.93 may be 0.1 to 0.3. mu.M, respectively, relative to 0.5. mu.M of the primer shown by SEQ ID NO. 1. By adding the positive internal quality control, false negative detection results caused by misoperation, PCR inhibitors and the like can be effectively prompted.
According to the present disclosure, in order to enhance the accuracy of the detection result, the nucleic acid reagent may be divided into two tubes, i.e., the nucleic acid reagent may include a tube a and a tube B; the tube A can contain primers shown in SEQ ID NO.1-32,61-62 and probes shown in SEQ ID NO.63-78, 93; the B tube may contain primers shown by SEQ ID NO.33-62 and probes shown by SEQ ID NO. 79-93.
Further, the arrangement and combination of the fluorescent labels may be performed according to the respective Tm values of the probes, so that the amplification of different probes in the same system can be recognized separately. For example, as one embodiment, the probes shown in SEQ ID Nos. 63-66,79-82 have a first fluorescent label; the probes shown in SEQ ID NO.67-70,83-86 have a second fluorescent label; the probes shown in SEQ ID NO.71-74,87-89 have a third fluorescent label; the probes shown in SEQ ID NO.75-78,90-93 have a fourth fluorescent label; the first fluorescent label, the second fluorescent label, the third fluorescent label and the fourth fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a VIC fluorescent label, a CY5 fluorescent label and a ROX fluorescent label. As a particularly preferred embodiment, the probes shown in SEQ ID NO.63-66,79-82 have FAM fluorescent labels; the probes shown in SEQ ID NO.67-70 and 83-86 have VIC fluorescent labels; the probes shown in SEQ ID NO.71-74,87-89 have CY5 fluorescent label; the probes shown in SEQ ID NO.75-78,90-93 have ROX fluorescent labels. In order to enhance the peak effect, the target probe may be a double-labeled probe. In the probe, FAM is 6-carboxyfluorescein, JOE is 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein, CY5 is 5H-indocyanine, ROX is 6-carboxy-X-rhodamine, and VIC is a dye purchased from ABI company.
According to the present disclosure, the gram-negative bacteria may include at least one of klebsiella pneumoniae, escherichia coli, pseudomonas aeruginosa, enterobacter cloacae, serratia marcescens, acinetobacter baumannii. The gram-negative bacteria drug resistance can comprise at least one of gram-negative bacteria aminoglycoside drug resistance, gram-negative bacteria penicillin cephalosporin drug resistance, gram-negative bacteria sulfanilamide drug resistance and gram-negative bacteria trimethoprim drug resistance.
In a second aspect of the present disclosure: there is provided a kit for detecting gram-negative bacteria and/or gram-negative bacteria resistance, the kit comprising the nucleic acid reagent of the first aspect of the disclosure, and optionally, the kit further comprises at least one of a reaction system buffer, DNA polymerase, DMSO, magnesium ions, BSA, dntps, and water.
The kit disclosed by the invention can realize rapid, accurate, sensitive, specific and automatic detection result judgment, and obviously improves the sensitivity, specificity and simplicity of simultaneously detecting the drug resistance of gram-negative bacteria and/or gram-negative bacteria.
A third aspect of the disclosure: there is provided the use of a nucleic acid reagent according to the first aspect of the disclosure in the preparation of a kit for the detection of gram-negative bacteria and/or gram-negative bacteria resistance.
A fourth aspect of the present disclosure: providing a system for detecting gram-negative bacteria and/or gram-negative bacteria resistance, comprising a PCR instrument having an A-tube detector and a B-tube detector, the A-tube detector and the B-tube detector being nucleic acid reagent storage containers loaded with the above-mentioned nucleic acid reagents including the A-tube and the B-tube, respectively, the PCR instrument comprising a first fluorescence channel, a second fluorescence channel, a third fluorescence channel, and a fourth fluorescence channel, the first fluorescence channel, the second fluorescence channel, the third fluorescence channel, and the fourth fluorescence channel being different from each other and each independently being a FAM fluorescence channel, a VIC fluorescence channel, a CY5 fluorescence channel, or a ROX fluorescence channel; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
if the blank control, the positive control and the positive internal control are established, the detection result is valid;
if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains the Escherichia coli; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains Klebsiella pneumoniae; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains acinetobacter baumannii; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains pseudomonas aeruginosa; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains serratia marcescens; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 62 ℃, determining that the sample contains enterobacter cloacae; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 66 ℃, determining that the sample contains blaOXA-1; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 70 ℃, determining that the sample contains blaOXA-10; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaIMP-4; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains blaIMP-6; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaIMP-8; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaTEM; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaVEB; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains the blaSVV; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaVIM; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaCTX; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 1; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 2; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains aadA 3; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains aadA 4; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 65, determining that the sample contains the Sull; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains sul 3; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 5; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 6; if the third fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains sut 1; if the third fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains sat 1; if the third fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 1; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains dfrA 14; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 17; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains dfrA 15; if the fourth fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
determining that the sample has gram-negative bacterial aminoglycoside resistance if the sample contains at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA 6; determining that the sample has gram negative bacterial resistance to penicillin cephalosporins if the sample contains at least one of blaOXA-1, blaOXA-10, blaIMP-4, blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSVV, blaVIM, and blaCTX; if the sample contains at least one of sull, sul3, sut1 and sat1, judging that the sample has sulfonamide resistance of gram-negative bacteria; when the sample contains at least one of dfrA1, dfrA14, dfrA17 and dfrA15, the sample is judged to have the drug resistance of gram-negative bacteria trimethoprim.
The fifth aspect of the present disclosure: a method for detecting gram negative bacteria and/or gram negative bacteria resistance is provided, wherein the method comprises: carrying out PCR amplification on DNA of a sample to be detected by adopting the nucleic acid reagent comprising the tube A and the tube B; the PCR instrument for carrying out the PCR amplification comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel; the first, second, third, and fourth fluorescent channels are each different and each independently selected from a FAM fluorescent channel, a VIC fluorescent channel, a CY5 fluorescent channel, or a ROX fluorescent channel; and the following discrimination is made:
if the blank control, the positive control and the positive internal control are established, the detection result is valid;
if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains the Escherichia coli; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains Klebsiella pneumoniae; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains acinetobacter baumannii; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains pseudomonas aeruginosa; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains serratia marcescens; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 62 ℃, determining that the sample contains enterobacter cloacae; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 66 ℃, determining that the sample contains blaOXA-1; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 70 ℃, determining that the sample contains blaOXA-10; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaIMP-4; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains blaIMP-6; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaIMP-8; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaTEM; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaVEB; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains the blaSVV; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaVIM; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaCTX; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 1; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 2; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains aadA 3; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains aadA 4; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 65, determining that the sample contains the Sull; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains sul 3; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 5; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 6; if the third fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains sut 1; if the third fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains sat 1; if the third fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 1; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains dfrA 14; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 17; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains dfrA 15; if the fourth fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
determining that the sample has gram-negative bacterial aminoglycoside resistance if the sample contains at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA 6; determining that the sample has gram negative bacterial resistance to penicillin cephalosporins if the sample contains at least one of blaOXA-1, blaOXA-10, blaIMP-4, blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSVV, blaVIM, and blaCTX; if the sample contains at least one of sull, sul3, sut1 and sat1, judging that the sample has sulfonamide resistance of gram-negative bacteria; when the sample contains at least one of dfrA1, dfrA14, dfrA17 and dfrA15, the sample is judged to have the drug resistance of gram-negative bacteria trimethoprim.
Wherein, the sample to be detected can be a patient anus swab sample, and the PCR amplification condition can be as follows: 98.0 deg.C, 1min (98.0 deg.C, 5 s; 58.0 deg.C, 5 s; 72.0 deg.C, 5 s; 49 cycles) 98.0 deg.C, 1 min; 35.0 deg.C for 1 min; the melting curve is 35.0-80.0 ℃, and the rise rate is 0.5 ℃/s. .
The method disclosed by the invention can quickly, sensitively and specifically realize the systematic screening of 6 pathogens such as Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Bacillus cloacae, Serratia marcescens, Acinetobacter baumannii and the like and common drug-resistant genes carried by the 6 pathogens, the detection flow is simple, the result is automatically interpreted and reliable, and the time, the labor and the reagent cost are saved.
The present disclosure is further illustrated in detail below by way of examples, but the present disclosure is not limited thereto.
In the following examples, the reagents were all commercially available, and the primers and probes were synthesized by Biosearch (USA).
Examples
1. Primer and probe synthesis
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, JOE is 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein, CY5 is 5H-indocyanine, ROX is 6-carboxy-X-rhodamine, and VIC is a dye purchased from ABI company. The parentheses in the probe sequences in Table 2 indicate that the bases on the left side of the parentheses have fluorescent labels, and the contents of the parentheses indicate the selection of the fluorescent labels.
TABLE 1
Figure BDA0001931316170000071
Figure BDA0001931316170000081
TABLE 2
Figure BDA0001931316170000082
Figure BDA0001931316170000091
2. Sample processing
After the patient's anal swab sample is collected by conventional methods, it is processed with a commercial extraction kit.
And collecting the treated anus swab sample by using a sampler matched with the ParaDNA, and directly placing the sample in a reactor of the ParaDNA for amplification.
3. Detection system for constructing Hybeacon probe technology
Polymerase Phire Hot Start II DNA Polymerase (cat # F122L), Mg2+The dNTPS is purchased from ThermoFisher company, and other biochemical reagents are imported separately packaged or domestic analytical purifiers; the fluorescence detector is ParaDNA.
The reaction system was prepared as follows: the total volume is 15 μ L, the buffer is 13 μ L, and comprises 0.5-0.75U/μ L polymerase, 1mM dNTP, 10-10 mM dNTP5For each μ L of DNA template, the final concentration of the forward primer was 500nM, the final concentration of the reverse primer was 1 μ M, and the final concentration of each probe was 200 nM.
The kit is divided into two reaction tubes, namely a tube A and a tube B, wherein the tube A contains primers shown by SEQ ID NO.1-32 and 61-62 in the table 1 and probes shown by SEQ ID NO.63-78 and 93 in the table 2, and the tube B contains primers shown by SEQ ID NO.33-62 in the table 1 and probes shown by SEQ ID NO.79-93 in the table 2.
Placing the PCR tube into a fluorescent quantitative PCR instrument, selecting FAM, VIC, CY5 and ROX as reporter groups, and carrying out the following reaction procedures: 98 ℃, 60s, (98 ℃, 5s, 58 ℃, 5s, 72 ℃, 5s, 49 cycles); dissolution curve analysis: the temperature is 98 ℃, 60s, 35 ℃, 60s, and the reduction rate is 1.0 ℃/s; fluorescence was collected at 80 ℃ for 5s with a liter of 0.5 ℃/s.
If the blank control, the positive control and the positive internal control are established, the detection result is valid;
if the A tube FAM fluorescence channel has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains Escherichia coli; if the A tube FAM fluorescence channel has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains Klebsiella pneumoniae; if the A tube FAM fluorescence channel has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains acinetobacter baumannii; if the A tube FAM fluorescence channel has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains pseudomonas aeruginosa; if the VIC fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains serratia marcescens; if the A tube VIC fluorescence channel has a dissolution peak curve corresponding to the Tm value of 62 ℃, determining that the sample contains enterobacter cloacae; if the VIC fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 66 ℃, determining that the sample contains blaOXA-1; if the VIC fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 70 ℃, determining that the sample contains blaOXA-10; if the CY5 fluorescence channel A has a dissolution peak curve corresponding to the Tm value of 58 ℃, the sample is judged to contain blaIMP-4; if the CY5 fluorescence channel of tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains blaIMP-6; if the CY5 fluorescence channel A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaIMP-8; if the CY5 fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaTEM; if the tube A ROX fluorescence channel has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaVEB; if the tube A ROX fluorescence channel has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains blaSVV; if the tube A ROX fluorescence channel has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaVIM; if the tube A ROX fluorescence channel has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaCTX; if the tube A ROX fluorescence channel has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
if the B tube FAM fluorescence channel has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 1; if the B tube FAM fluorescence channel has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 2; if the B tube FAM fluorescence channel has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains aadA 3; if the B tube FAM fluorescence channel has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains aadA 4; if the B-tube VIC fluorescence channel has a dissolution peak curve corresponding to a Tm value of 65, determining that the sample contains the Sull; if the VIC fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains sul 3; if the VIC fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 5; if the VIC fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 6; if the CY5 fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, the sample is judged to contain sut 1; if the CY5 fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains sat 1; if the CY5 fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 1; if the tube B ROX fluorescence channel has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains dfrA 14; if the tube B ROX fluorescence channel has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 17; if the tube B ROX fluorescence channel has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains dfrA 15; if the tube B ROX fluorescence channel has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
determining that the sample has gram-negative bacterial aminoglycoside resistance if the sample contains at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA 6; determining that the sample has gram negative bacterial resistance to penicillin cephalosporins if the sample contains at least one of blaOXA-1, blaOXA-10, blaIMP-4, blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSVV, blaVIM, and blaCTX; if the sample contains at least one of sull, sul3, sut1 and sat1, judging that the sample has sulfonamide resistance of gram-negative bacteria; when the sample contains at least one of dfrA1, dfrA14, dfrA17 and dfrA15, the sample is judged to have the drug resistance of gram-negative bacteria trimethoprim.
4. Specificity verification
Shigella, salmonella, proteus, pertussis, Citrobacter, Yersinia enterocolitica, stenotrophomonas maltophilia and Citrobacter (provided by bacterial disease prevention and control in the Chinese disease prevention and control center) are selected as specificity evaluation samples, and after a sampler in system detection is adopted to collect an anus swab sample, the anus swab sample is detected on ParaDNA by utilizing reaction conditions established and optimized in the early stage.
The result shows that under the condition that positive control is established, the target to be detected has no specific dissolution peak, and the nucleic acid reagent disclosed by the invention can effectively distinguish the detected target from the non-detected target and has better specificity.
5. Minimum detection limit verification
Test samples for evaluation: selecting Escherichia coli containing drug resistance gene as representative strain, adjusting bacterial suspension of 5 strains to 108CFU/mL, bacterial genomic DNA was extracted separately. The templates were each diluted in gradient to 107CFU/mL，106CFU/mL，105CFU/mL，104CFU/mL，103CFU/mL，102CFU/mL, 10CFU/mL test samples.
The results show that the lowest detection limits of the kit for detecting the target bacteria and the drug-resistant genes reach 102CFU/mL, the lowest detection limit of the total kit is 1 copy of the target molecule detected per reaction.
6. Coverage verification
Anal swab samples were selected as templates for coverage assessment. The test was carried out according to the above-mentioned reaction system and reaction procedure.
The results show that the detection can be covered for all samples.
7. Shelf life test of kit
Respectively taking strong positive 105CFU/mL and Weak Positive 103CFU/mL template of Klebsiella pneumoniae containing drug resistance gene as evaluationWith the test sample, on day 0, 9 portions were divided and frozen in a refrigerator at-70 ℃. And (3) storing the assembled kit at the temperature of-20 ℃, and performing storage period tests on the kit with the time periods of 0, 10, 15, 30, 60, 90, 120, 150, 180 and 360 days respectively.
The results show that the kit disclosed by the invention is stored in a refrigerator at the temperature of-20 ℃, and the detection is positive in different storage periods, which indicates that the storage period of the kit is at least one year.
Comparative example
1. Primer and probe synthesis
Sequence synthesis was performed according to the primer and probe sequences shown in tables 3 and 4.
TABLE 3
Figure BDA0001931316170000101
Figure BDA0001931316170000111
TABLE 4
Figure BDA0001931316170000112
Figure BDA0001931316170000121
2. Specificity verification was performed according to the method of example. The results showed that the reaction results of the primers and the probes of the comparative examples were negative.
3. Minimum detection limit verification the minimum detection limit verification is performed according to the method of the embodiment. The lowest detection limit of the examples versus the comparative examples is shown in table 5 below.
TABLE 5
Figure BDA0001931316170000122
Figure BDA0001931316170000131
As can be seen from Table 5, the kit disclosed by the invention has stronger detection capability for trace amounts of Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella cloaca, Serratia marcescens, Acinetobacter baumannii and drug-resistant gene nucleic acid carried by the Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and the drug-resistant gene nucleic acid in a sample compared with a comparative example.
4. Coverage verification
Coverage verification was performed as per the method of the examples. The coverage ratio of examples to comparative examples is shown in table 6 below.
TABLE 6
Detecting an object Examples Comparative example
Escherichia coli All 120 strains were positive 118 positive
Klebsiella pneumoniae All 80 plants were positive 77 all positive
Acinetobacter baumannii All 100 plants are positive All 90 plants were positive
Pseudomonas aeruginosa All 90 plants were positive All 86 plants were positive
Serratia marcescens All 80 plants were positive All 74 strains were positive
Enterobacter cloacae All 120 strains were positive 109 positive strains
aadA1 89 all positive All 72 plants were positive
aadA2 All 120 strains were positive 118 positive
aadA3 All 82 plants were positive 77 all positive
aadA4 87 all positive 78 all positive
aadA5 All 90 plants were positive All 86 plants were positive
aadA6 All 80 plants were positive All 74 strains were positive
blaOXA-1 113 all positive 109 positive strains
blaOXA-10 76 all positive All 72 plants were positive
blaIMP-4 All 120 strains were positive 118 positive
blaIMP-6 All 80 plants were positive All 80 plants were positive
blaIMP-8 All 111 strains were positive All 108 strains were positive
blaTEM All 90 plants were positive All 86 plants were positive
blaVEB All 82 plants were positive All 80 plants were positive
blaSHV All 120 strains were positive 109 positive strains
blaVIM 89 all positive 76 all positive
blaCTX All 120 strains were positive 119 positive
sull All 80 plants were positive 77 all positive
sul3 All 100 plants are positive All 90 plants were positive
sut1 All 90 plants were positive All 86 plants were positive
Sat1 All 80 plants were positive All 75 plants were positive
dfrA1 All 120 strains were positive 118 positive
dfrA14 All 82 plants were positive 73 all positive
dfrA15 All 119 plants were positive 118 positive
dfrA17 All 40 plants were positive All 38 strains were positive
As can be seen from Table 6, the detection coverage of the kit of the present disclosure is much greater than that of the comparative example.
As can be seen from the comparison of the examples and the comparative examples, the method can detect 6 pathogens such as Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Bacillus cloacae, Serratia marcescens, Acinetobacter baumannii and the like at one time, can also identify multiple drug-resistant genes carried by the 6 pathogens at the same time, and has the advantages of high specificity, lower minimum detection limit and wider coverage.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, for example, the a-tube detection target and the B-tube detection target can be interchanged. In order to avoid unnecessary repetition, various possible combinations will not be separately described in this disclosure.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Sequence listing
<110> Beijing Zhuozcheng Biotech GmbH
<120> nucleic acid reagents, kits, systems and methods for detecting gram-negative bacteria and/or gram-negative bacteria resistance
<130> 12326ABT
<160> 186
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tttcaccgaa gttcatgcc 19
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggaatttcgc cgattttgc 19
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aggccaacaa gaagtacaac c 21
<210> 4
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcaacccaac gatcctg 17
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ccctagaccc tcaatatgca c 21
<210> 6
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
accgagtcca caacgat 17
<210> 7
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
caacctgaag gacgatt 17
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ccttcttggc cttgtcgag 19
<210> 9
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atgcaggccg cgtttc 16
<210> 10
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
acagattcac gtccggctc 19
<210> 11
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tgcgtcagat cgtgtcca 18
<210> 12
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cttcagttgc cgcgttgt 18
<210> 13
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gcagcaaaga tgaaatc 17
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aggtggttgt aaataatgtt 20
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aataaaagat gaaaaatgat gaa 23
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cttctttact cgcctttatc gg 22
<210> 17
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atgttatcag tttcaata 18
<210> 18
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggaaaccatc ttcgtatttt agatggg 27
<210> 19
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tcgggcaatg tagacagt 18
<210> 20
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
caccattggc ttcggtca 18
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tgacactcca tttacggcta 20
<210> 22
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tagttaattc agacgcatac gtg 23
<210> 23
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gacactccat ttacggcta 19
<210> 24
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gatagatcga gaattaagcc act 23
<210> 25
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
cgcctatctg attgacactc c 21
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ttgttaattc agatgcatac gtg 23
<210> 27
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ggacagttgg gtgcac 16
<210> 28
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gcagcaaaga tgaaatc 17
<210> 29
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
actatcgcca gcaggatct 19
<210> 30
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
agttcgccga ccgtca 16
<210> 31
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ccgactttac cagattgcc 19
<210> 32
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tcacggacaa tgagacca 18
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
agtatcgact caactatcag a 21
<210> 34
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
tgtggcttca ggccgccatc c 21
<210> 35
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
agcaacgatg ttacgcagca g 21
<210> 36
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
cattgccaat aacccgattg g 21
<210> 37
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gcaagtagcg tatgcgctca c 21
<210> 38
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gcccgaggca tagactgtac a 21
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gtgggtcgat gtttgatgtt a 21
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
atattttcaa tttaactccc 20
<210> 41
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gatgttatgg agcagcaacg at 22
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
caaatcgcaa atatgcagta 20
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gcaacgatgt tacgcagcag g 21
<210> 44
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
agctagaatt ttgtgtatca a 21
<210> 45
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
attttatctc tncggtgttn aatat 25
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gaatggatgt agacactcga g 21
<210> 47
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
ttcaaggcat ctgataaaga c 21
<210> 48
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
agcaccaact cttgcagcag a 21
<210> 49
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gatcgacaac ttaaacgcga t 21
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tatctttagg gttcagtttt g 21
<210> 51
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
gtgacccgac gcctgcgcag c 21
<210> 52
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
cgtacgccgc ccaggccgac a 21
<210> 53
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
ggagcagcaa cgatgttacg c 21
<210> 54
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
atatagctac cattagtgat a 21
<210> 55
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
atgagaacct tgaaagtatc a 21
<210> 56
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
tgctcccctt tcgcggacca g 21
<210> 57
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
gttacgcagc agggcagtcg c 21
<210> 58
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
atatctgggc catttccgat a 21
<210> 59
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
ggagcagcaa cgatgttacg c 21
<210> 60
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gacactgcag aaatcaatga t 21
<210> 61
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
gattcatggc tcagaacgaa c 21
<210> 62
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
cgctttactc atcccgttg 19
<210> 63
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
ccctttctgt tacgccaa 18
<210> 64
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
agaagacaac cgcattatta cccgctc 27
<210> 65
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
cttgccgttc aggcgaattg at 22
<210> 66
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ctgcgctgct gcgtcgctt 19
<210> 67
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
ctgctgtcta cgcccggtg 19
<210> 68
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
atgtacctcg cccgcttcac gtt 23
<210> 69
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
aatatccttc aaagacgtat tcaaa 25
<210> 70
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
ctggcgcgct ccgaacgtgt aactta 26
<210> 71
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
ccacctatcc gcccgtgct 19
<210> 72
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
ccgcccggct gcgctat 17
<210> 73
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
ccgcgcccac aaacaattga ct 22
<210> 74
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
gggtacacga actggat 17
<210> 75
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
tctggggaca ctcgccggtc a 21
<210> 76
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
ttgtgaacag caaacgccgg gttatt 26
<210> 77
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
tagccgcgcc atcaacgac 19
<210> 78
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gagagcagtc aaattctt 18
<210> 79
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
cgccactcga acgacgttg 19
<210> 80
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
gccaatgaac cggaatcag 19
<210> 81
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
cctgaccgaa gcagcggtg 19
<210> 82
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
acgagcaggg agtcgccct 19
<210> 83
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
gtcagaaaag gcgaatcggt agtgg 25
<210> 84
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
aaagtaggcc gcaggacac 19
<210> 85
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
ggtgacattt taatttacct gggt 24
<210> 86
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gcggagaaat cgcaccttt 19
<210> 87
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gcatatttaa caattgaaa 19
<210> 88
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
cgtacgatcg cctctcgtg 19
<210> 89
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
caaaacaaag taacctctg 19
<210> 90
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
tgcgaagcga aaaaggcgt 19
<210> 91
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
accccaagga atatcgtga 19
<210> 92
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
gtcgcctaaa acaaagttag 20
<210> 93
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
ctttccgcrc gcttgcat 18
<210> 94
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
cgcaactggc tgacgat 17
<210> 95
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
tcgggacgga ttatagttat g 21
<210> 96
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
gtcccggcgc cggcggagcc 20
<210> 97
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
tcacctgctg cacgcggtgg gc 22
<210> 98
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
aatgacattg caagcaattg ct 22
<210> 99
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
tgcttgtccg ttcaggcgca att 23
<210> 100
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
gaggaaggca tccgcgaagt gatgagc 27
<210> 101
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
acgacgaagg cgacggcggt ccgg 24
<210> 102
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
ctttaaaagc ctgctttcgc agg 23
<210> 103
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
ctggcgtttc agca 14
<210> 104
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
gttgctggtg gcggcgttgc 20
<210> 105
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
tcggtattct ggacccaact aaagt 25
<210> 106
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
gagcgccatc tcgaaccgac g 21
<210> 107
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
ggtcaccgta accagcaaat c 21
<210> 108
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
ttagccatat gaactcggaa t 21
<210> 109
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
aatcttctgc tcacccggaa t 21
<210> 110
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
ccagaacctt gaccgaacgc a 21
<210> 111
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
gcgtaacgcg cttgctgctt g 21
<210> 112
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
tgatgttatg gagcagcaac g 21
<210> 113
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
ccactaccga ttacgccatt t 21
<210> 114
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
taagggagtt aaattgaaaa t 21
<210> 115
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
gagtagttgc tcacctttta c 21
<210> 116
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
aacgatgtta cgcagcaggg c 21
<210> 117
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
ggcagatttc gctcatctgc c 21
<210> 118
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
ttgccacgtg tcaaacgaga a 21
<210> 119
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
gcaatgagct catccacaga c 21
<210> 120
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
gaaaacaaaa gttggaatgc t 21
<210> 121
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
gctattggga attttaaagg t 21
<210> 122
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
agcattgcta ccgcagcaga g 21
<210> 123
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
caaccaaacc atgtttagga a 21
<210> 124
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
tttccatagc gacagcacag g 21
<210> 125
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
atgcatacgt gggaatagat t 21
<210> 126
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
ggagcggctt tgcctgattt a 21
<210> 127
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
gagtgtcaat cagataggcg t 21
<210> 128
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
tacatcgaac tggatctcaa c 21
<210> 129
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
accgagttgc tcttgcccgg c 21
<210> 130
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
tgctcagagg agcatgacgt a 21
<210> 131
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
attttccata caccagattt c 21
<210> 132
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
gaattgtgaa tcagcaaaac g 21
<210> 133
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
cagcggcagg gtggctaaca g 21
<210> 134
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
cgcatatcgc aacgcagtcg 20
<210> 135
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
ccgcgagaag tgccgctgtg t 21
<210> 136
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
aatcagcgcg ttgaaattaa g 21
<210> 137
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
ccgcactaag ctcagccagc g 21
<210> 138
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
tgtttggtct gtgtttatta c 21
<210> 139
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
gggtcccctc ccccacttgg g 21
<210> 140
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
acttaacatc atgggtgcgg a 21
<210> 141
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
agtgtcacgg aaatcattct t 21
<210> 142
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
tttcaattga ttaaataatg c 21
<210> 143
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
aacagaaggt aggtggcagg g 21
<210> 144
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
gtgcctgctc gtgccgttcg g 21
<210> 145
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
cggtgtccgc gatggcgtcg c 21
<210> 146
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
tggtagctat atcgaagaat g 21
<210> 147
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
cttgcgtcca accaacagcc a 21
<210> 148
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
cataccctgg tccgcgaaag g 21
<210> 149
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
tggggagtgc gcccatagat t 21
<210> 150
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
aagtatcgtg aaactatcac taat 24
<210> 151
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
ccaaagggga acaattactc ttca 24
<210> 152
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
attgaaaata tcattgattt c 21
<210> 153
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
cttttactga ccacgggata t 21
<210> 154
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
acgaacgctg gcggcgtgcc taacac 26
<210> 155
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
ggaaccaaag ggggcgagcg ta 22
<210> 156
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
atgggtacag ggaggat 17
<210> 157
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
ggcccgcgcg gcggcgcgtc ag 22
<210> 158
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
acgcaaactt ggtgtagata ttgataa 27
<210> 159
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
cggcagcctg cccgccgaag aggtg 25
<210> 160
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
cactgtaagg cgacaacgac gacaacgccc c 31
<210> 161
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
gcgcgcaatg gaagcgcctc t 21
<210> 162
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
tccgcagtgg atggcggcct g 21
<210> 163
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
tctcgttgct gcgatgggag c 21
<210> 164
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
acggcgcagt ggcggttttc a 21
<210> 165
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
cgcagcaggg cagtcgccct a 21
<210> 166
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
cagaaaatgg cgtaatcggt a 21
<210> 167
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
gacacaacgc aggtcacatt g 21
<210> 168
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
ctcttcaaat gacgtattca a 21
<210> 169
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
gcgtagttgt gctctggaat g 21
<210> 170
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
tgatgaaggc gtttatgttc a 21
<210> 171
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
tggtttgtgg agcgcggcta t 21
<210> 172
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
gtgtttatgt tcatacatcg t 21
<210> 173
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
tcgccccgaa gaacgttttc c 21
<210> 174
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
gatagccgct aacattaata a 21
<210> 175
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
cgggttattc ttatttgtcg c 21
<210> 176
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
ccgtccaatg gtctcattgt c 21
<210> 177
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
acagagcgag agcgataagc a 21
<210> 178
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
tactgtcctt gcctgttcta g 21
<210> 179
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
atacatttct gctgcaagag t 21
<210> 180
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
tcccgatctc aaaactgaac c 21
<210> 181
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
ggcctgggcg gcgtacgggc t 21
<210> 182
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
ccctgatatt ccatggagtg c 21
<210> 183
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
attgacctac aatcagtggc t 21
<210> 184
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
gagttatcgg aaatggccca gatattccat 30
<210> 185
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
agaaaatggc gtaatcggta g 21
<210> 186
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
cgtgggtaat ctaccctacg gatcggg 27

Claims (7)

1. A nucleic acid reagent for detecting gram-negative bacteria and gram-negative bacteria drug resistance, wherein the nucleic acid reagent comprises primers shown in SEQ ID NO.1-60 and probes shown in SEQ ID NO. 63-92;
wherein, the nucleic acid reagent also comprises a positive internal quality control; the positive endoplasmic control contains a primer shown by SEQ ID NO.61-62 and a probe shown by SEQ ID NO. 93;
the nucleic acid reagent is stored in the tube A and the tube B; the tube A contains primers shown in SEQ ID NO.1-32 and 61-62 and probes shown in SEQ ID NO.63-78 and 93; the B tube contains primers shown in SEQ ID NO.33-62 and probes shown in SEQ ID NO. 79-93.
2. The nucleic acid reagent according to claim 1, wherein the content of the primer represented by SEQ ID NO.2-60 is 0.9-1.1. mu.M, 0.3-0.6. mu.M, 0.9-1.1. mu.M, 0.3-0.5. mu.M, 0.9-1.1. mu.M, 0.5-1. mu.M, 0.9-1.1. mu.M, 0.4-0.8. mu.M, 0.9-1.1. mu.M, 0.3-1.6. mu.M, 0.9-1.0.9-1. mu.1. mu.0.1. mu.M, 0.5-1. mu.M, 0.9-1. mu.1. mu.9-1, 0.0.9-1. mu.9-1. mu.M, 0.0.9-1. mu.1, 0.9-1. mu.0.0.0.0.9-1. mu.1, 0.9-1, 0.0.9-1. mu.1. mu.9-1. mu.0.9-1, 0.9-1, 0.0.9-1, 0.1. mu.M, 0.9-1. mu.M, 0.1. mu.1.9-1. mu.0.9-1, 0.9-1. mu.1. mu.M, 0.9-1, 0.1. mu.M, 0.1. mu.9-1. mu.0.9-1, 0.1. mu.1. mu.M, 0.0.M, 0.1. mu.1. mu.M, 0.M, 0.1. mu.1. mu.0.9-1. mu.0.9-0.9-1. mu.1. mu.M, 0.0.1.1.M, 0.M, 0.1. mu.1. mu.M, 0.M, 0.1. mu.1, 0.1. mu.M, 0.M, 0.9-1. mu.M, 0.1, 0.1.M, 0.1. mu.M, 0.M, 0.1. mu.M, 0.M, 0.1.1. mu.1.1. mu.M, 0.1. mu.1. mu.M, 0.1. mu.1.1. mu.M, 0.1.0.0.1.1. mu.1.1. mu.0.9-0.1.1. mu.1.0.0.1.M, 0.0.1.0.0.1. mu.M, 0.9-0.0.9-0.M, 0.1.M, 0.9-0.M, 0.1. mu.0.9-0.1. mu.1. mu, 0.3 to 0.5. mu.M, 0.9 to 1.1. mu.M, 0.5 to 1. mu.M, 0.9 to 1.1. mu.M, 0.4 to 0.8. mu.M, 0.9 to 1.1. mu.M, 0.3 to 0.6. mu.M, 0.9 to 1.1. mu.M, 0.3 to 0.5. mu.M, 0.9 to 1.1. mu.M, 0.5 to 1. mu.M, 0.9 to 1.1. mu.M, 0.3 to 0.6. mu.M, 0.9 to 1.1. mu.M, 0.3 to 0.5. mu.M, 0.9 to 1.1. mu.M, 0.4 to 0.8. mu.M, 0.9 to 1.1. mu.M, 0.3 to 0.6. mu.M, 0.9 to 1.1. mu.M, 0.9 to 1. mu.1. mu.M, 0.9 to 0.1. mu.1. mu.M, 0.4 to 0.8. mu.8. mu.1. mu.M, 0.1. mu.1 to 0.1. mu.M, 0.1 to 0.9 to 1. mu.1, 0.1 to 0.1. mu.M, 0.1 to 0.63 to 1. mu.M, 0.1 to 1 to 0.1. mu.M, 0.63 to 1. mu.M, 0.1 to 1. mu.1 to 0.1. mu.M, respectively, and 0.1 to 0.63 to 1. mu.1. mu.M of an ID of a probe, each independently of each independently expressed by the content of each of the probe, and the content of each independently expressed by ID of each, respectively, 0.1 to 0.3 to 0.1 to 1 to 0.1 to 1 to 1.9 to 1 [ mu.9 to 1 [ mu.1 [ mu.M, respectively.
3. The nucleic acid reagent according to claim 1, wherein the probe represented by SEQ ID No.63-66,79-82 has a first fluorescent label; the probes shown in SEQ ID NO.67-70,83-86 have a second fluorescent label; the probes shown in SEQ ID NO.71-74,87-89 have a third fluorescent label; the probes shown in SEQ ID NO.75-78,90-93 have a fourth fluorescent label; the first fluorescent label, the second fluorescent label, the third fluorescent label and the fourth fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a VIC fluorescent label, a CY5 fluorescent label and a ROX fluorescent label.
4. The nucleic acid agent as claimed in any one of claims 1 to 3, wherein the gram-negative bacteria include Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Serratia marcescens and Acinetobacter baumannii, and the gram-negative bacteria resistance includes gram-negative bacteria aminoglycoside resistance, gram-negative bacteria penicillin cephalosporin resistance, gram-negative bacteria sulfonamide resistance and gram-negative bacteria trimethoprim resistance.
5. A kit for detecting gram-negative bacteria and gram-negative bacteria resistance, comprising the nucleic acid reagent of any one of claims 1 to 4, and optionally further comprising at least one of reaction system buffer, DNA polymerase, DMSO, magnesium ions, BSA, dntps, and water.
6. Use of the nucleic acid reagent of any one of claims 1 to 4 in the preparation of a kit for detecting gram-negative bacteria and gram-negative bacteria resistance.
7. A system for detecting gram-negative bacteria and gram-negative bacteria resistance, comprising a PCR instrument having an a-tube detector and a B-tube detector, the a-tube detector and the B-tube detector being nucleic acid reagent storage containers loaded with the nucleic acid reagent of any one of claims 1 to 4, respectively, the PCR instrument comprising a first fluorescence channel, a second fluorescence channel, a third fluorescence channel, and a fourth fluorescence channel, the first fluorescence channel, the second fluorescence channel, the third fluorescence channel, and the fourth fluorescence channel being different from each other and each independently being a FAM fluorescence channel, a VIC fluorescence channel, a CY5 fluorescence channel, or a ROX fluorescence channel; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
if the blank control, the positive control and the positive internal control are established, the detection result is valid;
if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains the Escherichia coli; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains Klebsiella pneumoniae; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains acinetobacter baumannii; if the first fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains pseudomonas aeruginosa; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains serratia marcescens; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 62 ℃, determining that the sample contains enterobacter cloacae; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 66 ℃, determining that the sample contains blaOXA-1; if the second fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 70 ℃, determining that the sample contains blaOXA-10; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaIMP-4; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains blaIMP-6; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaIMP-8; if the third fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaTEM; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains blaVEB; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains the blaSVV; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains blaVIM; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains blaCTX; if the fourth fluorescence channel of the tube A has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 1; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 2; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains aadA 3; if the first fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains aadA 4; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 65, determining that the sample contains the Sull; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, determining that the sample contains sul 3; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains aadA 5; if the second fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains aadA 6; if the third fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains sut 1; if the third fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains sat 1; if the third fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 1; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 58 ℃, determining that the sample contains dfrA 14; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 65 ℃, determining that the sample contains dfrA 17; if the fourth fluorescence channel of the B tube has a dissolution peak curve corresponding to the Tm value of 61 ℃, determining that the sample contains dfrA 15; if the fourth fluorescence channel of the tube B has a dissolution peak curve corresponding to the Tm value of 69 ℃, the quality control of the positive endoplasm is judged to be qualified;
determining that the sample has gram-negative bacterial aminoglycoside resistance if the sample contains at least one of aadA1, aadA2, aadA3, aadA4, aadA5 and aadA 6; determining that the sample has gram negative bacterial resistance to penicillin cephalosporins if the sample contains at least one of blaOXA-1, blaOXA-10, blaIMP-4, blaIMP-6, blaIMP-8, blaTEM, blaVEB, blaSVV, blaVIM, and blaCTX; if the sample contains at least one of sull, sul3, sut1 and sat1, judging that the sample has sulfonamide resistance of gram-negative bacteria; when the sample contains at least one of dfrA1, dfrA14, dfrA17 and dfrA15, the sample is judged to have the drug resistance of gram-negative bacteria trimethoprim.
CN201811641979.5A 2018-12-29 2018-12-29 Nucleic acid reagent, kit, system and method for detecting gram-negative bacteria and/or gram-negative bacteria drug resistance Active CN109536626B (en)

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CN111349721B (en) * 2020-03-19 2022-10-04 北京卓诚惠生生物科技股份有限公司 Nucleic acid reagent, kit, system and method for detecting respiratory tract infection pathogen
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CN1687459A (en) * 2005-04-15 2005-10-26 北京博奥生物芯片有限责任公司 Authenticating gram positive bacteria species and method for testing drug resistant gene and dedicating kit
CN104561340A (en) * 2015-01-23 2015-04-29 杭州迪安医学检验中心有限公司 Primers and kit for detecting aminoglycoside drug-resistance genes of bacteria
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KR20180033112A (en) * 2017-10-23 2018-04-02 가톨릭대학교 산학협력단 Probe for detecting drug resistant gram-positive pathogens, probe set and a method for detecting drug resitant gram-positive pathogens using them

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CN1687459A (en) * 2005-04-15 2005-10-26 北京博奥生物芯片有限责任公司 Authenticating gram positive bacteria species and method for testing drug resistant gene and dedicating kit
CN104561340A (en) * 2015-01-23 2015-04-29 杭州迪安医学检验中心有限公司 Primers and kit for detecting aminoglycoside drug-resistance genes of bacteria
CN105420371A (en) * 2015-12-21 2016-03-23 张明 Multi-pathogen and drug resistance gene detection method
KR20180033112A (en) * 2017-10-23 2018-04-02 가톨릭대학교 산학협력단 Probe for detecting drug resistant gram-positive pathogens, probe set and a method for detecting drug resitant gram-positive pathogens using them

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