CN116004351A - Antibiotic resistance gene detection kit and application method thereof - Google Patents
Antibiotic resistance gene detection kit and application method thereof Download PDFInfo
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to an antibiotic resistance gene detection kit, which comprises a kit cover, a kit body, an eight-pipe-connecting groove, a positive internal control pipe, a reagent reaction pipe and a negative control reaction pipe, wherein two eight-pipe-connecting grooves which are distributed front and back are formed in the kit body; the invention adopts a probe method fluorescence quantitative detection method, takes the gene of bacterial 16SrRNA as an internal reference gene, designs gene detection primers and fluorescence labeling probes aiming at 21 antibiotic resistance genes, distributes the gene detection primers and the fluorescence labeling probes into seven detection tubes, each tube is provided with three different labeled antibiotic resistance gene detection probes and corresponding primers, and simultaneously, each tube is provided with the fluorescence detection probes and the corresponding primers of the internal reference gene. The kit is suitable for a fluorescent quantitative PCR instrument and a digital PCR instrument which can simultaneously distinguish four fluorescent signals of FAM, VIC, ROX and CY 5. The invention can improve the detection flux, reduce the detection cost and has higher detection sensitivity and specificity.
Description
Technical Field
The invention relates to the field of germ detection kits, in particular to an antibiotic resistance gene detection kit and a use method thereof.
Background
Since the 20 th century, a large number of natural antibiotics including penicillin, streptomycin, etc. have been discovered successively, whereby humans have opened the antibiotic era, and have an absolute advantage in combating bacteria. However, with an excessive dependence on the use of antibiotics, antibiotic resistance genes (Antibiotics resistance genes, ARGs) are constantly present and accumulate in large amounts in environmental microorganisms, resulting in an increasing problem of microbial resistance. The horizontal and vertical transfer of ARGs between microorganisms makes the power of the primary antibiotic drugs diminished, even ineffective. The antibiotic resistance gene is used as a new environmental pollutant, affects biosafety and seriously threatens human health.
Therefore, rapid identification and quantification of ARGs is an effective means of monitoring environmental microbial resistance or evaluating microbial infection resistance in humans and animals, and has important roles in monitoring ARGs contamination and guiding the use of antibiotics in humans and animals.
Researchers have found that 21 antibiotic resistance genes have higher detection rates and have clear relevance with microbial resistance through extensive literature studies and environmental microbial antibiotic resistance gene detection, and the 21 antibiotic resistance genes are tetG1, tetW, tnpA-2 (tetracycline resistance), sul1, sul2 (sulfanilamide resistance), blaTEM-1, ampc (beta-lactam resistance), ermX (MLSB resistance), acrB (tigecycline resistance), aac (6') -lb-cr, aadA-01, strA (aminoglycoside resistance), vanTC-02, vanXO (vancomycin resistance), mexE, qac (multidrug resistance), intI-1, ISCR1 (mobile transfer element), cmlA1, cmlB (chloramphenicol resistance) and fluorine chloramphenicol resistance, respectively.
At present, a dye method fluorescence quantitative PCR is generally adopted for detection, a detection sample is often transferred to a laboratory during detection, the laboratory conventional detection instrument is large in size and inconvenient to move, the sample needs to be kept at low temperature in the transportation process, biological safety leakage is easy to occur, and the sample is inappropriately stored and is subjected to the risk of change. The dye method fluorescence quantitative PCR is low in specificity, so that false positive is easy to detect, the result accuracy is low, one reaction hole can only detect one target gene of one sample, three target genes can be detected in one hole, the flux is improved by three times, and the detection comprehensive cost is lower.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a low-cost, high-flux, high-sensitivity, high-specificity, safe and convenient germ antibiotic resistance gene detection kit.
Specifically, the invention provides an antibiotic resistance gene detection kit.
Including reagent lid, kit body, eight allies oneself with the tube bank, positive internal control article groove, positive internal control article pipe, reagent reaction tube and negative control reaction tube, the top rear swing joint of kit body has the reagent lid, just the inside of kit body has been seted up two and has been distributed eight allies oneself with the tube bank, every eight the inside of allies oneself with the tube bank all is equipped with seven from left to right the reagent reaction tube that arranges in proper order with be located the single of one end negative control reaction tube, the inside one side of kit body has been seted up positive internal control article groove, the inside in positive internal control article groove is equipped with positive internal control article pipe.
Preferably, the positive internal control is added in advance for comparison, and the positive internal control is a DNA fragment of a plurality of synthesized known gene sequences.
Preferably, the positive internal control comprises an antibiotic resistance gene positive internal control and an internal control positive internal control of an internal reference gene.
Preferably, the final concentration of each positive internal control DNA fragment pre-added in the positive internal control tube is 10 6 Copy/μL。
Preferably, the nucleotide sequence corresponding to the positive internal control of the antibiotic resistance gene is SEQ ID NO.1-SEQ ID NO.21.
Preferably, the antibiotic resistance genes include tetG1, tetW, tnpA-2, sul1, sul2, blaTEM-1, ampc, ermX, acrB, aac (6') -lb-cr, aadA-01, strA, vanTC-02, vanXO, mexE, qac, intI-1, ISCR1, cmlA1, cmlB, floR.
Preferably, the gene detection method of the kit is probe-method fluorescence quantitative PCR or probe-method digital PCR.
Preferably, three target resistance gene detection probes which do not interfere with each other and three corresponding target resistance gene detection primers are added in each reagent reaction tube in advance, the 5 'end of each target resistance gene detection probe is respectively connected with a fluorescent reporter group marked by different fluorescent dyes (FAM/VIC/ROX), the 3' end of each probe is connected with a BHQ1 fluorescence quenching group, and all target resistance gene detection probes and corresponding target resistance gene detection primers are added in advance in a negative control reaction tube.
Preferably, the sequence of the pre-added target resistance gene detection probe, the sequence of the corresponding target resistance gene detection primer, and the fluorophore to which the corresponding target resistance gene detection probe is attached in each reagent reaction tube are selected from the combinations shown in the following table:
preferably, the combination of the three added target resistance gene detection probes and the corresponding resistance genes of the target resistance gene detection primers in each of the seven reagent reaction tubes is: tetG1, tetW, tnpA-2, sul1, sul2, blaTEM-1, ampc, ermX, acrB, aac (6') -lb-cr, aadA-01, strA, vanTC-02, vanXO, mexE, qac, intI-1, ISCR1, cmlA1, cmlB, floR.
Preferably, the final concentration of each target resistance gene detection primer added in advance in each of the reagent reaction tube and the negative control reaction tube is 0.8. Mu. Mol/L, the final concentration of each target resistance gene detection probe is 0.3. Mu. Mol/L,
preferably, the kit uses a reference gene for control detection.
Preferably, the reference gene is a gene of 16S rRNA of the microorganism.
Preferably, an internal reference gene detection primer and an internal reference gene detection probe are added in each reagent reaction tube and each negative control reaction tube in advance, an internal reference gene positive internal control product mixed by equal mass is added in the positive internal control product tube in advance, the 5 'end of the internal reference gene detection probe is connected with a fluorescence report group (CY 5), and the 3' end of the internal reference gene detection probe is connected with a fluorescence quenching group (BHQ 1) marked by quenching fluorescent dye.
Preferably, the sequence of the reference gene detection primer, the sequence of the reference gene detection probe, the sequence of the reference gene positive internal control and the fluorescent group connected with the reference gene detection probe are shown in the following table:
preferably, the reagent reaction tube, the negative control reaction tube and the positive internal control tube are added with a reaction premix containing PCR enzyme in advance.
Preferably, the method comprises the following steps: 2 mu L of each detected qualified microorganism DNA is added into the first seven reagent reaction tubes of the eight-joint tube, and no sample or 2 mu L of reagent-grade non-nucleic acid water is added into the negative control reaction tube; after the sample is added, amplification reaction is carried out on a 16 Kong Bianxie type fluorescent quantitative PCR instrument comprising FAM, VIC, ROX, CY5 four-color fluorescent channels, FAM, VIC, ROX, CY5 fluorescent channel signals are collected, and when the Ct of the sample detection is less than 35, the corresponding target gene is detected, otherwise, the corresponding target gene is not detected.
Preferably, the amplification reaction is performed by the following steps: 95 ℃ for 15min; 3min at 95 ℃; cycling for 5 times at 95 ℃ for 5S to 60 ℃ for 40S; and (3) carrying out circulation for 50 times at 95 ℃ for 5S-60 ℃ for 40S, and collecting FAM, VIC, ROX, CY5 fluorescent channel signals at 60 ℃.
Preferably, when the quantitative determination is needed, the quantitative determination can be performed relative to the result of the reference gene, the quantitative determination can be performed by selecting the detection result of the positive internal control in the same batch, and the absolute quantitative determination can be performed by selecting the detection result of the digital PCR platform.
The invention has the following beneficial effects:
1. the probe method fluorescence quantitative method adopted by the invention has the advantage of higher detection specificity, simultaneously takes the 16S rRNA gene of bacteria as an internal reference gene, has the function of relative quantitative reference, can also carry out quality control on experimental operation, is simultaneously adaptive to a commonly used four-color fluorescence quantitative PCR instrument or four-color digital PCR instrument, has different fluorescence labels for 21 ARGs detection probes designed in the invention, and sets every three target genes as a group, and simultaneously detects 21 genes through one-time 7-tube reaction, thereby saving experimental reagents, improving detection flux and reducing detection cost.
2. In view of the fact that the microbial drug resistance is a relatively urgent situation, and the risk of biosafety leakage is easily introduced in the transfer process of the microorganism rich in antibiotic resistance genes, and the situation that the detection result and the truest state are distorted due to the fact that the transfer condition of the microorganism is unstable in the transfer process is likely to occur, the kit is designed for a 16-hole (2X 8) portable fluorescent quantitative PCR instrument, can easily realize detection experiments of sampling sites, and has the advantages of convenience and reality for emergency events and field investigation. Meanwhile, in the design of the kit, the pre-mixed solution is adopted, and the extracted DNA sample is only required to be added into a reaction tube of the kit, so that the operation is simple and convenient.
Drawings
FIG. 1 is a schematic diagram of the structure of a kit of the present invention;
FIG. 2 is a diagram showing amplification by a single gene qPCR reaction according to one embodiment of the present invention;
FIG. 3 is a graph showing qPCR amplification of mixed genes according to an embodiment of the present invention;
FIG. 4 is a graph showing qPCR amplification of a chicken manure sample mixed gene according to an embodiment of the present invention;
FIG. 5 is a graph showing the results of digital PCR of a seed sul1 plasmid standard according to one embodiment of the present invention.
In the figure: 1. a reagent box cover; 2. a kit body; 3. an eight-connecting pipe groove; 4. a positive internal control groove; 5. a positive internal control tube; 6. a reagent reaction tube; 7. negative control reaction tube.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments (e.g., detection on a digital PCR instrument using the subject kit) that would be available to one of ordinary skill in the art without the benefit of the inventive step are within the scope of the present invention based on the embodiments herein. All the raw materials in the present invention are not particularly limited in their sources, and may be commercially available or prepared according to conventional methods well known to those skilled in the art. The purity of all the raw materials in the present invention is not particularly limited, and the present invention preferably employs a conventional purity used in the field of analytical purity or composite materials.
As shown in fig. 1, the kit for detecting antibiotic resistance gene by using the fluorescent quantitative PCR method according to one embodiment of the present invention includes a kit cover 1, a kit body 2, an eight-tube slot 3, a positive internal control slot 4, a positive internal control slot 5, a reagent reaction tube 6 and a negative control reaction tube 7, wherein the kit cover 1 is movably connected to the rear of the top of the kit body 2, two eight-tube slots 3 distributed front and back are provided in the kit body 2, seven reagent reaction tubes 6 sequentially arranged from left to right and a single negative control reaction tube 7 positioned on the rightmost side are provided in the two eight-tube slots 3, the positive internal control slot 4 is provided on the right side of the interior of the kit body 2, and the positive internal control slot 4 is provided with the positive internal control tube 5.
Preferably, the positive internal control product tube 5 of the kit is pre-added with positive internal control products for comparison, wherein the positive internal control products are synthesized DNA fragments with known gene sequences, and comprise 21 positive internal control products of antibiotic resistance genes and 1 positive internal control product of reference genes (mixed into one tube, the total volume is 50 mu L), and the concentration of the positive internal control products of each gene is preferably 10 6 Copy/. Mu.L, the nucleotide sequences corresponding to the positive internal control of the antibiotic resistance gene are SEQ ID NO.1-SEQ ID NO.21, and the sequences are derived from the published CARD database.
Preferably, the 21 antibiotic resistance genes include tetG1, tetW, tnpA-2, sul1, sul2, blaTEM-1, ampc, ermX, acrB, aac (6') -lb-cr, aadA-01, strA, vanTC-02, vanXO, mexE, qac, intI-1, ISCR1, cmlA1, cmlB, floR.
At least one antibiotic resistance target gene detection primer and a target resistance gene detection probe are added in advance in each reagent reaction tube 6 and the recessive control reaction tube 7, the probes cannot affect and react with each other, the 5' end and the 3' end of each probe are respectively connected with a fluorescent group, wherein the 5' end of each target resistance gene detection probe in each reagent reaction tube 6 is connected with a different fluorescent group, the preferred primer, the nucleotide sequence of each probe and the fluorescent group connected with each probe are shown in the table 1, the final concentration of the primer is 0.8 mu mol/L, and the final concentration of each probe is 0.3 mu mol/L.
TABLE 121 antibiotic resistance Gene detection primers and probe sequences
Preferably, in the two rows of eight-linked tubes in the shown kit, three primers and probes of target resistance genes are added to each tube of the first seven reagent reaction tubes 6, and the corresponding relationship between the reaction tubes 6 and the target genes is as follows: the reaction tube No.1 corresponds to three genes of tetG1, tetW and tnpA-2, the reaction tube No.2 corresponds to three genes of sul1, sul2 and blaTEM-1, the reaction tube No. 3 corresponds to three genes of ampc, ermX, acrB, the reaction tube No. 4 corresponds to three genes of aac (6') -lb-cr, aadA-01 and strA, the reaction tube No. 5 corresponds to three genes of VanTC-02, vanXO and MexE, the reaction tube No. 6 corresponds to three genes of qac, intI-1 and ISCR1, the reaction tube No. 7 corresponds to three genes of cmlA1, cmlB and floR, and the recessive control reaction tube No. 7 is pre-added with primers and probes of all target resistance genes.
In a preferred embodiment, the kit is based on reference genes, an reference gene detection primer and an reference gene detection probe are also added in each of the reagent reaction tube 6 and the negative control reaction tube 7 in advance, an equal-mass mixed reference gene positive internal control is added in the positive internal control tube 5 in advance, the 5 'ends of the three target gene detection probes are respectively connected with one of FAM/VIC/ROX fluorescent reporter groups marked by fluorescent dyes, and the 3' end of each probe is connected with a BHQ1 fluorescent quenching group marked by quenching fluorescent dyes. Preferably, the kit uses the gene of the microbial 16S rRNA as an internal reference gene, and the corresponding internal reference gene positive internal control sequence, the detection upstream primer sequence, the detection downstream primer sequence and the detection probe sequence information are shown in Table 2.
Table 216S rRNA positive reference substance, detection primer and probe sequence
Preferably, the total volume of the reaction premix of the reagent tube 6 of the shown kit is 18. Mu.L, and the volume after adding the nucleic acid sample is 20. Mu.L, and the components of the kit are as follows: 1x Taqman fluorescent quantitative PCR reagent, primers and probes corresponding to the antibiotic resistance gene and the reference gene (16 SrRNA) (the final concentration of the primers is 0.8. Mu. Mol/L, and the final concentration of the probes is 0.3. Mu. Mol/L). The total volume of the reaction premix for the negative control tube 7 is 20 mu L, and the composition of the premix is as follows: 1X Taqman fluorescent quantitative PCR reagent, primers and probes corresponding to the antibiotic resistance gene and the reference gene (16 SrRNA) (the final concentration of the primers is 0.8 mu mol/L, and the final concentration of the probes is 0.3 mu mol/L).
One embodiment of the invention also relates to a method for using the antibiotic resistance gene detection kit, which comprises the following steps: 2 mu L of each qualified microorganism DNA (with the concentration of more than 10 ng/. Mu.L and A260/280:1.8-2.0) is added into the first seven reagent reaction tubes 6 of the eight-joint tube, and no sample or 2 mu L of reagent-grade non-nucleic acid water is added into the negative control reaction tube 7. The two-row eight-connection tube can be used for performing two technical repetitions on one sample and can be used for performing one technical repetition on 2 samples; after the sample was added, an amplification reaction was performed on a 16 Kong Bianxie fluorescent quantitative PCR instrument comprising a FAM, VIC, ROX, CY5 four-color fluorescent channel, the reaction procedure being: 95 ℃ for 15min; 3min at 95 ℃; cycling for 5 times at 95 ℃ for 5S to 60 ℃ for 40S; 95 ℃ 5S-60 ℃ 40S, and 50 times of circulation are carried out, and FAM, VIC, ROX, CY5 fluorescent channel signals are collected at 60 ℃; and if Ct is smaller than 35, the detection is carried out, otherwise, the detection is not carried out. When quantification is needed, the quantitative calculation can be carried out relative to the result of the reference gene, the quantitative calculation can be carried out on the detection result of the positive internal control product in the same batch, and the absolute quantification can be carried out on the detection result of the digital PCR platform (the calculation mode refers to the conventional fluorescent quantitative PCR quantitative calculation mode or the quantitative mode of the digital PCR platform).
The invention also preferably adopts the following four embodiments, and four schemes are selected: example 1 was performed without fusing probes, each qPCR reaction well detected a single antibiotic resistance gene, and fluorescent signals of the corresponding probes were collected for amplification curve fitting. Example 2 is to mix three different fluorescent-labeled probes for the corresponding antibiotic resistance genes and add the 16S rRNA reference gene. Example 3 is a reaction performed on a nucleic acid sample extracted from chicken manure. Example 4 to assist in verifying the copy number of a standard using digital PCR, the error in calculating the copy number using the conventional formula was eliminated.
Example 1:
the full-length gene of the antibiotic resistance gene is constructed into a cloning vector pUC57, transfected into competent cells DH5 alpha, and the constructed plasmid is extracted as a corresponding antibiotic resistance gene standard after screening and expanding culture. The construction of vector and plasmid standard is a routine means of biology, and this example is not described in detail.
Taking the constructed standard product as a qPCR reaction template to prepare a qPCR reaction system:
TABLE 3 Single gene qPCR reaction System
| Reagent name | Initial concentration | Sampling volume/. Mu. | Final concentration | |
| 2*Taqman permix | 2* | 10 | 1* | |
| Primer (F+R) | 10μM/each | 1.6 | 0.8 | |
| Probe with a probe tip | 10μM | 0.6 | 0.3 | |
| Plasmid standard | 0.1ng/ |
2 | 0.01ng/μL | |
| ddH 2 O | —— | 5.8 | —— |
The qPCR reaction procedure was:
the reaction procedure is: 95 ℃ for 15min; 3min at 95 ℃; cycling for 5 times at 95 ℃ for 5S to 60 ℃ for 40S; and (3) carrying out circulation for 50 times at 95 ℃ 5S-60 ℃ 40S, and collecting corresponding fluorescent signals at 60 ℃ according to the type of the fluorescent group modified at the 5' end of the probe of the reaction hole site.
Reaction results:
TABLE 4 Single gene qPCR detection results
| Sample Name | Detector | Ct |
| tetG1 | FAM | 13.2246 |
| tetW | VIC | 13.4693 |
| tnpA-2 | ROX | 13.3688 |
| sul1 | FAM | 13.3933 |
| sul2 | VIC | 13.293 |
| blaTEM-1 | ROX | 13.4153 |
| ampc | FAM | 13.4542 |
| ermX | VIC | 13.5815 |
| acrB | ROX | 13.3674 |
| aac(6')-lb-cr | FAM | 13.2778 |
| aadA-01 | VIC | 13.1954 |
| strA | ROX | 13.2075 |
| VanTC-02 | FAM | 13.2471 |
| vanXO | VIC | 13.0878 |
| MexE | ROX | 13.291 |
| qac | FAM | 13.3051 |
| intI-1 | VIC | 13.1729 |
| ISCR1 | ROX | 13.1644 |
| cmlA1 | FAM | 13.2463 |
| cmlB | VIC | 13.2706 |
| floR | ROX | 13.2681 |
| 16S rRNA | CY5 | 13.1777 |
Specific amplification diagrams are shown in FIG. 2.
Example 2:
the constructed plasmid standard was prepared according to the following rule, according to concentration 1:1:1: mixing well, wherein the No.1 reaction tube corresponds to four genes of tetG1, tetW, tnpA-2 and 16S rRNA plasmid standard, the No.2 reaction tube corresponds to four genes of sul1, sul2, blaTEM-1 and 16S rRNA plasmid standard, the No. 3 reaction tube corresponds to four genes of ampc, ermX, acrB and 16S rRNA plasmid standard, the No. 4 reaction tube corresponds to four genes of aac (6') -lb-cr, aadA-01, strA and 16S rRNA plasmid standard, the No. 5 reaction tube corresponds to four genes of VanTC-02, vanXO, mexE and 16S rRNA plasmid standard, the No. 6 reaction tube corresponds to four genes of qac, intI-1, ISCR1 and 16S rRNA plasmid standard, and the No. 7 reaction tube corresponds to four genes of qac, intI-1, flo R and 16S cmNA plasmid standard.
Mixing the primers and the probes according to the arrangement rule, wherein the ratio of the primers of different antibiotic resistance genes of the same tube is 1:1, the ratio of primer to probe is 8:3.
qPCR reaction is carried out on the mixed standard substance, primer and probe, and the qPCR system is as follows:
table 5 Mixed Gene qPCR reaction System
qPCR reaction procedure
The reaction procedure is: 95 ℃ for 15min; 3min at 95 ℃; cycling for 5 times at 95 ℃ for 5S to 60 ℃ for 40S; 95℃5S-60℃40S, and 50 cycles, FAM, VIC, ROX, CY-5 fluorescence signals were simultaneously collected at 60℃per well.
Reaction results:
TABLE 6 Mixed Gene qPCR reaction results
* NTC is a negative control, i.e. water without nucleic acid is used instead of the mixed plasmid standard, as follows.
Specific amplification diagrams are shown in FIG. 3.
Example 3:
0.3g of chicken manure sample with high abundance of antibiotic resistance genes is taken for nucleic acid sample extraction (the nucleic acid extraction is a routine experimental operation of molecular biology, and no tiredness is performed in the embodiment). The concentration of the extracted nucleic acid detected by Qkit is 89 ng/. Mu.L. The concentration of DNA was diluted to 10 ng/. Mu.L with non-nucleic acid water. Taking an eight-tube reaction system of the kit, sequentially adding 2 mu L of the diluted DNA sample into a 1-7-hole, and adding 2 mu L of non-nucleic acid water into a 8-hole. Amplification reactions were performed on a 16 Kong Bianxie fluorescent quantitative PCR instrument comprising a FAM, VIC, ROX, CY four-color fluorescent channel, the reaction procedure being: 95 ℃ for 15min; 3min at 95 ℃; cycling for 5 times at 95 ℃ for 5S to 60 ℃ for 40S; and (3) carrying out circulation for 50 times at 95 ℃ for 5S-60 ℃ for 40S, and collecting FAM, VIC, ROX, CY5 fluorescent channel signals at 60 ℃.
Reaction results:
TABLE 7 results of qPCR detection of chicken manure sample Mixed Gene
Specific amplification diagrams are shown in FIG. 4.
Example 4:
the current qPCR absolute copy number calculation method is to firstly carry out concentration gradient dilution on plasmid standard substances, respectively carry out qPCR reaction on standard plasmids subjected to the gradient dilution to obtain Ct values, and linearly fit the Ct values with the concentration of the plasmid standard substances. And calculating the relation between the concentration and the copy number through a relation. And further obtaining a linear relation between the ct value and the copy number.
The relationship between plasmid standard and copy number is as follows:
copies/ul＝(6.02*10 23 )*(ng/ul*10 -9 )/(DNA Length*660)＝(9.12*10 11 )*(ng/ul)/(DNA Length)
however, there is often a difference between the copy number obtained by this relation and the actual copy number of the plasmid, and if the difference is too large, the quantitative analysis of the subsequent detection is affected for the detection of the antibiotic resistance gene. The correction of the copy number by digital PCR is increased. The digital PCR annealing temperature was consistent with examples 1, 2 and 3. The comparative results were obtained as follows:
table 8 plasmid Standard monogenic digital PCR detection results and comparison of calculated copy number by equation method
In summary, the digital PCR was 2 orders of magnitude smaller than the copy number calculated by the formula method. The method can be well used for correcting the subsequent standard curve, so that the detection condition of the obtained antibiotic resistance gene is more practical, and a specific amplification diagram is shown in fig. 5.
Claims (19)
1. The utility model provides an antibiotic resistance gene detection kit, its characterized in that, including reagent lid (1), kit body (2), eight allies oneself with tub (3), positive interior accuse article groove (4), positive interior accuse article pipe (5), reagent reaction tube (6) and negative control reaction tube (7), the top rear swing joint of kit body (2) has reagent lid (1), just eight allies oneself with tub (3) that two front and back distributed have been seted up to the inside of kit body (2), every eight allies oneself with tub (3) the inside all is equipped with seven reagent reaction tube (6) that arrange in proper order from left to right and the single that is located one end negative control reaction tube (7), the inside one side of kit body (2) has been seted up positive interior accuse article groove (4), the inside in positive interior accuse article groove (4) is equipped with positive interior accuse article pipe (5).
2. An antibiotic resistance gene test kit according to claim 1, wherein: the positive internal control product tube (5) is pre-added with a positive internal control product for comparison, wherein the positive internal control product is a DNA fragment of a plurality of synthesized known gene sequences.
3. An antibiotic resistance gene test kit according to claim 2, wherein: the positive internal control products comprise an antibiotic resistance gene positive internal control product and an internal reference gene positive internal control product.
4. An antibiotic resistance according to claim 2A sex gene detection kit is characterized in that the final concentration of each positive internal control DNA fragment added in advance in a positive internal control tube (5) is 10 6 Copy/μL。
5. The kit for detecting antibiotic resistance gene according to claim 3, wherein the nucleotide sequence corresponding to the internal control positive for antibiotic resistance gene is SEQ ID NO.1-SEQ ID NO.21.
6. The kit of claim 1, wherein the antibiotic resistance gene comprises tetG1, tetW, tnpA-2, sul1, sul2, blaTEM-1, ampc, ermX, acrB, aac (6') -lb-cr, aadA-01, strA, vanTC-02, vanXO, mexE, qac, intI-1, ISCR1, cmlA1, cmlB, floR.
7. The kit for detecting antibiotic resistance genes according to claim 1, wherein the gene detection method of the kit is probe-method fluorescent quantitative PCR or probe-method digital PCR.
8. The antibiotic resistance gene detection kit according to claim 1, wherein three target resistance gene detection probes which do not interfere with each other and three corresponding target resistance gene detection primers are added in advance in each reagent reaction tube (6), the 5 'end of each target resistance gene detection probe is respectively connected with a fluorescent reporter group marked by a different fluorescent dye (FAM/VIC/ROX), the 3' end of each probe is connected with a BHQ1 fluorescence quenching group, and all target resistance gene detection probes and corresponding target resistance gene detection primers are added in advance in a negative control reaction tube (7).
9. The kit for detecting an antibiotic resistance gene according to claim 7, wherein the sequence of the target resistance gene detection probe, the sequence of the corresponding target resistance gene detection primer, and the fluorophore to which the corresponding target resistance gene detection probe is attached, which are previously added to each reagent reaction tube (6), are selected from the group consisting of:
10. the antibiotic resistance gene test kit according to claim 8, wherein the combination of the three added target resistance gene test probes and the corresponding resistance genes of the target resistance gene test primers in each of the seven reagent reaction tubes (6) is: tetG1, tetW, tnpA-2, sul1, sul2, blaTEM-1, ampc, ermX, acrB, aac (6') -lb-cr, aadA-01, strA, vanTC-02, vanXO, mexE, qac, intI-1, ISCR1, cmlA1, cmlB, floR.
11. The antibiotic resistance gene test kit according to claim 8, wherein each target resistance gene test primer is added in advance to each of the reagent reaction tube (6) and the negative control reaction tube (7) at a final concentration of 0.8. Mu. Mol/L and each target resistance gene test probe is added at a final concentration of 0.3. Mu. Mol/L.
12. The kit for detecting an antibiotic resistance gene according to claim 1, wherein the kit uses a reference gene for control detection.
13. The kit for detecting antibiotic resistance gene according to claim 12, wherein the reference gene is a gene of 16S rRNA of the microorganism.
14. An antibiotic resistance gene test kit according to any one of claims 1 to 13, wherein: an internal reference gene detection primer and an internal reference gene detection probe are added in each reagent reaction tube (6) and each negative control reaction tube (7) in advance, an internal reference gene positive internal control product mixed by equal mass is added in advance in a positive internal control product tube (5), the 5 'end of the internal reference gene detection probe is connected with a fluorescence report group (CY 5), and the 3' end of the internal reference gene detection probe is connected with a fluorescence quenching group (BHQ 1) marked by quenching fluorescent dye.
15. An antibiotic resistance gene test kit according to claim 14, wherein: the sequence of the reference gene detection primer, the sequence of the reference gene detection probe, the sequence of the reference gene positive internal control and the fluorescent group connected with the reference gene detection probe are shown in the following table:
16. an antibiotic resistance gene test kit according to claim 1, wherein: the reagent reaction tube (6), the negative control reaction tube (7) and the positive internal control tube (5) are added with a reaction premix containing PCR enzyme in advance.
17. A method of using the antibiotic resistance gene test kit of any one of claims 1-16, comprising the steps of: 2 mu L of each detected qualified microorganism DNA is added into the first seven reagent reaction tubes (6) of the eight-joint tube, and no sample or 2 mu L of reagent-grade non-nucleic acid water is added into the negative control reaction tube (7); after the sample is added, amplification reaction is carried out on a 16 Kong Bianxie type fluorescent quantitative PCR instrument comprising FAM, VIC, ROX, CY5 four-color fluorescent channels, FAM, VIC, ROX, CY5 fluorescent channel signals are collected, and when the Ct of the sample detection is less than 35, the corresponding target gene is detected, otherwise, the corresponding target gene is not detected.
18. The method of claim 17, wherein the amplification reaction is performed by the steps of: 95 ℃ for 15min; 3min at 95 ℃; cycling for 5 times at 95 ℃ for 5S to 60 ℃ for 40S; and (3) carrying out circulation for 50 times at 95 ℃ for 5S-60 ℃ for 40S, and collecting FAM, VIC, ROX, CY5 fluorescent channel signals at 60 ℃.
19. The method of claim 17, wherein the quantitative determination is performed by comparing the result of the reference gene with the result of the reference gene, or by selecting the same batch of positive internal control detection results, or by selecting the digital PCR platform detection results.
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