CN111733269A - Nucleic acid composition, kit and method for detecting staphylococcus aureus and klebsiella pneumoniae - Google Patents
Nucleic acid composition, kit and method for detecting staphylococcus aureus and klebsiella pneumoniae Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a nucleic acid composition, a kit and a method for detecting staphylococcus aureus and klebsiella pneumoniae, and relates to the field of determination or detection methods of microorganisms. The nucleic acid composition and the kit can detect staphylococcus aureus and klebsiella pneumoniae in experimental animal facilities, so as to obtain the health condition of experimental animals. The inspection method is beneficial to reducing the using amount of sentinel animals and saving the animal cost. In addition, the detection method provided by the invention has the advantages of high detection sensitivity, high detectable rate and good specificity, can screen the health condition of the whole experimental animal facility in one detection, and has short detection period.
Description
Technical Field
The invention relates to the field of determination or detection methods of microorganisms, and particularly relates to a nucleic acid composition, a kit and a method for detecting staphylococcus aureus and klebsiella pneumoniae.
Background
Staphylococcus aureus (1)Staphylococcus aureus) And Klebsiella pneumoniae (C.), (B.) (Klebsiella Pneumoniae) The method has great influence on the health of experimental mice and experimental results, the national standard GB14922.2-2011, the microbiological grade and detection of experimental animals, lists the pathogenic bacteria which must be eliminated by SPF-grade experimental mice, and the rapid, accurate and convenient method for detecting staphylococcus aureus and klebsiella pneumoniae of the experimental mice is very important for experimental animal facilities.
At present, the detection technologies for staphylococcus aureus and klebsiella pneumoniae of experimental mice mainly comprise the following steps: (1) the detection method of the culture is long in time consumption, needs subjective judgment of experienced detection personnel, has a large missing detection risk and is difficult to meet the detection of a large sample amount as a traditional detection method; (2) biochemical identification, according to different biochemical reactions of microorganisms, the types of the microorganisms can be accurately identified by using a biochemical reaction tube or a biochemical identification system, but the biochemical reaction has generally higher requirements on the purity of a sample, the complex sample composition can generate larger influence on the biochemical reaction result, and certain biochemical reactions still need longer time to observe the result; (3) immunology, mainly based on antigen-antibody specific recognition, commonly used detection methods include immunomagnetic bead separation (Yazdankhah et al, 1999; Lefantis et al, 2004) and immunosensor (Rowe et al, 1999), although immunology detection methods have high throughput, these immunology methods are based on antigen-antibody specific recognition, and a specific antibody can only be directed against a certain antigen of a pathogen, not the pathogen itself; (4) molecular detection of PhoenixThe PCR detection method for staphylococcus aureus in food established by Xiaobo et al (2014) has the sensitivity of 61.76 pg/mu L, can be used for the rapid detection of food-borne staphylococcus aureus, and the LAMP detection method for staphylococcus aureus in experimental animals is established by Wan 281567 and the like (2017), and the detection limit of pure culture of staphylococcus aureus can reach 9 × 103CFU/ml, the detection limit of the animal manure sample can reach 4.49 × 104CFU/ml is 10 times higher than the sensitivity of common PCR, and can effectively avoid the occurrence of false positive results, the multiple PCR detection method for staphylococcus aureus and klebsiella pneumoniae is established by the congratulatory wave and the like (2017), the minimum detection limit of the two bacteria is 10pg, the artificially infected excrement sample is detected, and the result is consistent with the detection result of the traditional detection method.
According to the test capability verification results of staphylococcus aureus and klebsiella pneumoniae of experimental animals implemented in the hospitals 2016 and 2017, 97 percent and 85 percent of test laboratories respectively adopt national standard test methods, which indicates that the methods commonly and preferentially adopted by the experimental animals for detecting staphylococcus aureus and klebsiella pneumoniae are the national standard test methods, and the sampling parts of the methods are blind contents or lesion tissues and the like.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a nucleic acid composition, a kit and a method for detecting staphylococcus aureus and klebsiella pneumoniae so as to solve the technical problems.
The invention is realized by the following steps:
in a first aspect, the invention provides a nucleic acid composition for detecting staphylococcus aureus and klebsiella pneumoniae. It includes: a first nucleic acid combination for detecting staphylococcus aureus and a second nucleic acid combination for detecting klebsiella pneumoniae; the first nucleic acid combination comprises a first primer pair and a first probe, the second nucleic acid combination comprises a second primer pair and a second probe, the sequences of the first primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, the sequence of the first probe is shown as SEQ ID NO.5, the sequences of the second primer pair are shown as SEQ ID NO.3 and SEQ ID NO.4, and the sequence of the second probe is shown as SEQ ID NO. 6.
The invention provides a nucleic acid composition for detecting staphylococcus aureus and klebsiella pneumoniae, wherein a first nucleic acid composition is designed according to the specificity of the staphylococcus aureus, the sensitivity of a detection sample reaches 50 copies/mu L, and the nucleic acid composition can be used for detecting the staphylococcus aureus in experimental animal facilities with high specificity. Compared with the existing bacterial culture method, the detection rate of the first nucleic acid combination is higher.
The second nucleic acid combination is designed according to the specificity of Klebsiella pneumoniae, can detect the Klebsiella pneumoniae and can reduce the content of the second nucleic acid combination to 2.03 × 10-3And detecting the standard substance at ng/. mu.L. Compared with the existing PCR detection method, the detection rate of the second nucleic acid combination is higher, the lowest detection limit is lower, and the specificity is better.
In a preferred embodiment of the present invention, the first probe and the second probe are labeled with different fluorescent reporter groups at their 5 'ends and with a quencher group at their 3' ends.
In some embodiments of the invention, the quencher labeled at the 3' end is a non-fluorescent quencher that does not itself produce fluorescence.
In a preferred embodiment of the invention, the fluorescent reporter group is HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, Texas Red or VIC, and the quencher group is MGB or BHQ.
The maximum fluorescence absorption wavelength of the fluorescence reporter group HEX is 535nm, and the name of the fluorescence reporter group HEX is hexachloro-6-methyl fluorescein.
The maximum fluorescence absorption wavelength of FAM is 495nm, and the name of Chinese is 6-carboxyfluorescein.
TET has a maximum fluorescence absorption wavelength of 521nm, and is named as tetrachloro-6-carboxyfluorescein in Chinese.
The JOE has a maximum fluorescence absorption wavelength of 520nm, and is named 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein in Chinese.
TAMRA has a maximum fluorescence absorption wavelength of 555nm, and is named 6-carboxytetramethylrhodamine by the Chinese name.
The maximum fluorescence absorption wavelength of ROX is 575nm, and the Chinese name is carboxyl-X-rhodamine.
The maximum fluorescence absorption wavelength of Texas Red is 589nm, the Chinese name is Texas Red.
The quenching group marked at the 3 'end can emit fluorescence with different wavelengths after absorbing the fluorescence energy emitted by the fluorescence reporter group marked at the 5' end, so that the background is high. Therefore, the inventor selects MGB as a quenching group, the MGB does not generate fluorescence, and the intensity of background signals can be greatly reduced, so that the detection sensitivity is improved.
It should be noted that, those skilled in the art can select other types of fluorescence reporter and quencher according to actual needs, and whatever fluorescence reporter and quencher are selected, they are within the scope of the present invention.
In a second aspect, the present invention provides a kit for detecting staphylococcus aureus and klebsiella pneumoniae, comprising the nucleic acid composition described above.
In a preferred embodiment of the present invention, the kit further comprises a reaction mixture, wherein the reaction mixture is a fluorescent quantitative pre-mix solution or a PCR pre-mix solution.
The kit provided by the invention can be used for fluorescence quantitative detection of staphylococcus aureus and klebsiella pneumoniae and can also be used for qualitative PCR detection.
In some embodiments of the invention, the reaction mixture is a qPCR probe method fluorescent quantitation premix; in some embodiments of the invention, the qPCR probe method fluorometrically quantifies the premix. For example, Vazyme AceQ U + Probe Mix.
The Vazyme AceQ U + Probe Mix contains a dUTP/Heat-label UDG anti-pollution system, can effectively remove pollutants in the system at room temperature, and simultaneously, when the temperature of the reaction system is raised to 50-55 ℃, the Heat-label UDG is quickly and completely inactivated, the integrity of cDNA can be maintained, and the detection sensitivity is not influenced.
In some embodiments of the invention, the PCR premix comprises a PCR buffer and an enzyme cocktail. The pH of the PCR premix was 8.3.
In some embodiments of the invention, the PCR buffer contains at least one of PCR buffer, cation, anion, and deoxyribonucleotide.
In some embodiments of the invention, the PCR buffer is Tris-HCl. The concentration of Tris-HCl is 40-400mM, and the effective buffering range is between pH7.0-9.2.
In some embodiments of the invention, the cation comprises a divalent cation and a monovalent cation; the monovalent cation comprises K+And NH4 +The divalent cation comprises Mg2+The anion comprises SO4 2-And Cl-。
The concentration of divalent cations is 15-40mM, the concentration of monovalent cations is 200-500mM, SO4 2-Is 15-40 mM.
In some embodiments of the invention, the deoxyribonucleotides include dATP, dCTP, dGTP, and dUTP. The concentrations of dATP, dCTP and dGTP are 0.4-4mM, and the concentration of dUTP is 1.5-8 mM.
The enzyme mixture comprises DNA polymerase and UNG enzyme.
The sample detected by the kit is mouse excrement or dust taken from facilities for feeding mice animals. In other embodiments, the detection sample of the kit may be adaptively selected according to actual conditions.
In some embodiments of the invention, the DNA polymerase is selected from at least one of Taq, Tfl, Pfu, or Tth DNA polymerase. The concentration of DNA polymerase is 1-100U/. mu.L.
In some embodiments of the invention, the UNG enzyme is a temperature sensitive UNG enzyme. The concentration of UNG enzyme is 0.1-5U/. mu.L.
The UNG enzyme can degrade uracil bases in existing U-DNA pollutants in a reaction system, and DNA strands are broken under the subsequent denaturation condition, so that amplification caused by the polluted DNA is eliminated, and the specificity and the accuracy of an amplification result are guaranteed. Simultaneously, UNG enzyme is inactivated, and the newly amplified product U-DNA can not be degraded.
The PCR buffer substance, cation, anion, deoxyribonucleotide and enzyme in the kit may be present in the kit in a partially mixed form or in a form of separate portions of each, or may be present in the kit in a mixed form. Whatever the form of its presence in the kit, it is within the scope of the invention.
In addition, in other embodiments, water is also included in the kit.
The concentrations of the PCR buffer substance, the cation, the anion, the deoxyribonucleotide and the enzyme in the kit can be adaptively adjusted according to actual requirements, and are not limited in the concentration range provided by the invention.
In other embodiments, the pre-Mix used for ordinary PCR may also be Vazyme2 × Taq Master Mix.
The concentration of the first primer pair of the first nucleic acid combination in the kit is 0.1-20 mu M, the concentration of the first probe is 0.1-20 mu M, the concentration of the second primer pair of the second nucleic acid combination in the kit is 0.1-20 mu M, and the concentration of the second probe is 0.1-20 mu M.
A method of testing for staphylococcus aureus and klebsiella pneumoniae, comprising: and (3) taking a nucleic acid sample to be detected as a template sequence, and carrying out PCR amplification detection by adopting the nucleic acid composition or the kit.
In a preferred embodiment of the present invention, the inspection method includes: the procedure for PCR amplification detection includes:
35-37 deg.C for 3-10 min; 95-96 deg.C for 6-8 min; 94-96 deg.C, 5-10s, 55-60 deg.C, 25-32s, and circulating 40-50 times. The amplification detection procedure is qPCR amplification detection procedure.
In a preferred embodiment of the present invention, the PCR amplification detection system comprises: a reaction system for detecting staphylococcus aureus and a reaction system for detecting klebsiella pneumoniae; wherein the concentration of the first primer pair in the reaction system for detecting staphylococcus aureus is 0.1-20 mu M, and the concentration of the first probe is 0.1-20 mu M; the concentration of the second primer pair in the reaction system for detecting the Klebsiella pneumoniae is 0.1-20 mu M, and the concentration of the second probe is 0.1-20 mu M.
In some embodiments of the invention, the nucleic acid sample to be tested is extracted from mouse feces or dust from the animal facility in which the mouse is kept.
In some embodiments of the invention, the animal facility in which the mice are raised is a cage.
In a preferred embodiment of the present invention, the method for extracting a nucleic acid sample to be tested from cage dust comprises: and (3) culturing the dust in a enrichment culture medium, collecting thalli and extracting DNA.
In some embodiments of the invention, the enrichment medium for staphylococcus aureus is 10% sodium chloride trypticase soy broth and the enrichment medium for klebsiella pneumoniae is selenite enrichment fluid.
In some embodiments of the invention, subjecting the dust to enrichment culture comprises: and (3) placing the dust in a bacterium enrichment culture medium, performing shake culture, and collecting bacterial liquid subjected to shake culture to obtain bacterial colony precipitates.
In some embodiments of the invention, the shake culture time is 10-24 h.
In some embodiments of the invention, the colony precipitate is collected by centrifugation at 8000-12000r/min for 3-10 min.
In some embodiments of the invention, the colony precipitate is digested with proteinase K and Tail Buffer at 50-60 ℃.
The detection method provided by the invention can be used for quickly detecting staphylococcus aureus and klebsiella pneumoniae of nucleic acid samples in experimental animal facilities, and has high detection sensitivity.
Alternatively, in some embodiments of the invention, the above methods are for the purpose of non-disease diagnosis.
The invention has the following beneficial effects:
the invention provides a nucleic acid composition, a kit and a method for detecting staphylococcus aureus and klebsiella pneumoniae, and the nucleic acid composition and the kit can detect the staphylococcus aureus and the klebsiella pneumoniae in experimental animal facilities, so as to obtain the health condition of experimental animals. The inspection method is beneficial to reducing the using amount of sentinel animals and saving the animal cost. In addition, the detection method provided by the invention has the advantages of high detection sensitivity, high detectable rate and good specificity, can screen the health condition of the whole experimental animal facility in one detection, and has short detection period.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph showing the amplification curves of Staphylococcus aureus in example 4 at different concentrations;
FIG. 2 is a fluorescence quantitative standard curve diagram of a standard substance of Staphylococcus aureus at different concentrations;
FIG. 3 is an electrophoretogram of common PCR products of standard Staphylococcus aureus at different concentrations;
FIG. 4 is a graph showing the amplification curves of the Klebsiella pneumoniae standards at different concentrations;
FIG. 5 is a fluorescence quantitative standard curve diagram of a Klebsiella pneumoniae standard at different concentrations;
FIG. 6 is an electrophoresis diagram of a common PCR product of a standard sample of Klebsiella pneumoniae at different concentrations;
FIG. 7 is a PCR detection electrophoretogram of Staphylococcus aureus in stool sample of Experimental example 1;
FIG. 8 is a qPCR detection amplification curve of Staphylococcus aureus in stool sample of Experimental example 1;
FIG. 9 is a PCR detection electrophoretogram of Staphylococcus aureus of dust sample of Experimental example 1;
FIG. 10 shows qPCR detection amplification curves for Staphylococcus aureus in dust sample of Experimental example 1 (A-sample Nos. 5, 17 and 30; B-sample Nos. 117, 120, 155 and 178; C-sample No. 195);
FIG. 11 is a PCR detection electrophoretogram of Klebsiella pneumoniae in a stool sample obtained in Experimental example 2;
FIG. 12 is a qPCR detection amplification curve of Klebsiella pneumoniae in the stool sample of Experimental example 2;
FIG. 13 is a PCR detection electrophoretogram of Klebsiella pneumoniae from a dust sample obtained in Experimental example 2;
FIG. 14 is a Klebsiella pneumoniae qPCR detection amplification curve (A-sample Nos. 14, 56 and 78; B-sample Nos. 124, 140, 145 and 175; C-sample No. 188) of the dust sample of Experimental example 2;
FIG. 15 is a graph of amplification curves of Staphylococcus aureus standards in Experimental example 3 at different concentrations using control primers;
FIG. 16 is a graph of fluorescence quantification standard of Staphylococcus aureus in Experimental example 3 at different concentrations using control primers;
FIG. 17 is a graph showing the amplification of a sample of Klebsiella pneumoniae in Experimental example 4 at different concentrations using control primers;
FIG. 18 is a graph showing the fluorescence quantitative standard of a sample of Klebsiella pneumoniae in Experimental example 4 at different concentrations using control primers;
FIG. 19 is a primer specific qPCR amplification curve in Experimental example 5;
FIG. 20 is a primer specific qPCR amplification curve in Experimental example 6;
FIG. 21 is a graph of qPCR detection of dust samples using control primers in Experimental example 7 (A-sample Nos. 5, 17 and 30; B-sample Nos. 117, 120 and 195);
FIG. 22 is a graph of qPCR detection of a stool sample using control primers in Experimental example 8;
FIG. 23 is a graph of qPCR assays performed on dust samples using control primers in Experimental example 9 (A-sample Nos. 14, 56 and 78; B-sample Nos. 124, 140, 145 and 175);
FIG. 24 is a graph of qPCR detection of fecal samples using control primers in Experimental example 10.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example provides a method for extracting target pathogen DNA from laboratory mouse cage dust.
200 parts of cage dust are collected at the position of the cage dust enrichment membrane by adopting 400 10ml sterile collection tubes, and the sampling amount of each tube is 2 ml. The collected cage frame dust is hermetically transferred into a biological safety cabinet, 200 samples are used for detecting staphylococcus aureus, and 10% sodium chloride tryptone soybean broth is added for enrichment culture; and (4) adding selenite enrichment fluid to the remaining 200 samples for detecting the Klebsiella pneumoniae so as to enrich the bacteria. Wherein the addition amount of the 10% sodium chloride trypticase soybean broth is 6 ml/tube, and the addition amount of the selenite enrichment broth is 6 ml/tube.
The formula of the sodium chloride trypticase soybean broth comprises the following components: tryptone 17.0g/L, sodium chloride 100.0 g/L, soybean peptone 3.0 g/L, dipotassium hydrogen phosphate 2.5 g/L, glucose 2.5 g/L, and sodium pyruvate 10.0 g/L.
The formula of the selenite enrichment broth comprises: 5.0 g/L of peptone, 4.0 g/L of lactose, 4.0 g/L of sodium hydrogen selenite, 6.5 g/L of disodium hydrogen phosphate and 3.5 g/L of sodium dihydrogen phosphate.
After the enrichment of the bacteria for 24 hours, 12000r/min, and centrifuging for 5min to collect bacterial colony precipitates. Digestion was carried out overnight at 56 ℃ using proteinase K + Tail Buffer. Proteinase K was added at 10mg/ml in an amount of 7.5. mu.L/sample and Tail Buffer was added at 500. mu.L/sample.
After centrifugation, the supernatant is extracted by isopropanol and absolute ethyl alcohol, 300 mu L of isopropanol (analytically pure) and 100 mu L of absolute ethyl alcohol are used for DNA detection, and the extracted DNA is respectively used for the subsequent qPCR detection of staphylococcus aureus and klebsiella pneumoniae.
Example 2
The nucleic acid composition for detecting staphylococcus aureus in cage dust provided by the embodiment comprises: a first nucleic acid combination comprising: the probe comprises a first primer pair and a first probe, wherein the sequences of the first primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the first probe is shown as SEQ ID NO. 5. HEX is labeled at the 5 'end of the first probe, and MGB is labeled at the 3' end of the first probe.
This example provides a qPCR and PCR detection/assay for Staphylococcus aureus in cage dust. Because the dust itself is a non-biological sample and the uncertainty of the microorganism contained therein, no reference gene is set.
(1) Fluorescent quantitative PCR amplification:
mu.L of 200 DNA templates obtained from example 1 were mixed with the combination of Vazyme AceQ U + Probe Mix and the first nucleic acid, and each sample was tested 3 times, mixed and amplified in a fluorescent quantitative PCR instrument. Vazyme AceQ U + Probe Mix was purchased from Nanjing Novozam Biotech GmbH, and the amplification reaction system is shown in Table 1 below:
TABLE 1 amplification reaction System for Staphylococcus aureus.
The amplification reaction procedure was: at 37 ℃ for 3 min; at 95 ℃ for 6 min; the preparation is circulated for 40 times at 95 deg.C, 10s, 60 deg.C, 31s, and stored at 4 deg.C.
The length of the target fragment amplified by the first primer pair is 186bp, and the sequence of the target fragment is as follows:
ttgtcggtacacgatattcttcacgactaaataaacgctcattcgcgattttataaatgaatgttgataacaatgtgccgtcttgataatctttagtagtaccgaagctggtcatacgagagttatattttccagccaaaacgatatttttataatcattacgtgaaaaaggtttcccttcattat。
(2) conventional PCR amplification:
mu.L of the DNA template obtained by extraction in example 1 was mixed with PCR Mix and the first primer pair, mixed well and amplified in a PCR instrument. PCR Mix was purchased from Biotech GmbH of Nanjing NuoWei Zan, Cat.P 112-P1-AA, and the amplification reaction system is shown in Table 1 below:
TABLE 2 amplification reaction System for Staphylococcus aureus.
The amplification reaction procedure was: 95 ℃ for 5 min; at 95 ℃ for 30 s; at 58 ℃ for 30 s; circulating for 32 times at 72 ℃ for 20 s; 72 ℃ for 10 min; storing at 4 ℃.
Example 3
The nucleic acid composition for detecting klebsiella pneumoniae in cage dust provided in this example includes: a second nucleic acid combination comprising: the sequences of the second primer pair are shown as SEQ ID NO.3 and SEQ ID NO.4, and the sequence of the second probe is shown as SEQ ID NO. 6. HEX was labeled at the 5 'end of the second probe, and MGB was labeled at the 3' end of the second probe.
This example provides a method for detection/assay of klebsiella pneumoniae qPCR and PCR in cage dust.
(1) Fluorescent quantitative PCR amplification:
mu.L of 200 DNA templates obtained from example 1 were mixed with the combination of Vazyme AceQ U + Probe Mix and the second nucleic acid, and each sample was tested 3 times, mixed and amplified in a fluorescent quantitative PCR instrument. Vazyme AceQ U + Probe Mix was purchased from Nanjing Novozam Biotech GmbH, and the amplification reaction system is shown in Table 3 below:
TABLE 3 amplification reaction system for Klebsiella pneumoniae.
The amplification reaction procedure was: at 37 ℃ for 3 min; at 95 ℃ for 6 min; the preparation is circulated for 40 times at 95 deg.C, 10s, 60 deg.C, 31s, and stored at 4 deg.C.
The length of the target fragment amplified by the second primer pair is 172bp, and the sequence of the target fragment is as follows:
aaagcgcagaactttgaagcggtggcgcagtatcagttcgacttcggtctgcgtccgtccctcggctatgtgctgtcgaaagggaaggatatcgaaggggtggggagtgaagatctggttaactacatcgacgtgggcctgacctactacttcaacaaaaacatgaacgcct。
(2) conventional PCR amplification:
mu.L of the DNA template obtained by extraction in example 1 was mixed with PCR Mix and the second primer pair, mixed well and amplified in a PCR instrument. PCR Mix was purchased from Biotech, Inc. of Kinzoka, N.J., under the product number P112-P1-AA. The amplification reaction system is shown in table 4 below:
TABLE 4 amplification reaction system for Klebsiella pneumoniae.
The amplification reaction procedure was: 95 ℃ for 5 min; at 95 ℃ for 30 s; at 58 ℃ for 30 s; circulating for 32 times at 72 ℃ for 30 s; 72 ℃ for 10 min; storing at 4 ℃.
Example 4
This example provides a graph of amplification of a standard sample prepared by extracting DNA from a pure culture of Staphylococcus aureus, the concentration of the DNA template of the standard sample was 52.2 ng/. mu.L, and the standard samples were diluted to 52.2 ng/. mu.L, 5.22 ng/. mu.L, and 5.22 × 10-1ng/μL、5.22×10-2ng/μL、5.22×10-3ng/μL、5.22×10-4ng/μL、5.22×10-5ng/μL、5.22×10-6ng/muL (corresponding to curves 1-8 in sequence), then carrying out fluorescence quantitative PCR according to the fluorescence quantitative reaction system and the reaction program shown in the embodiment 2, repeating each standard for 3 times, recording corresponding Ct and Rn values, wherein a fluorescence quantitative amplification curve chart is shown in figure 1, a standard curve is shown in figure 2, and as can be seen from figure 1, the detection sensitivity of the staphylococcus aureus detection method provided by the invention can reach 5.22 × 10-5ng/uL. search Genbank database, upload longest Staphylococcus aureus whole genome total length 2902619bp, calculate 5.22 × 10 from this data-5The concentration of DNA at ng/. mu.L is approximately equal to 16 copies/. mu.L. As is clear from the sensitivity data, the nucleic acid composition provided by the present invention has the advantages of high sensitivity and low detection limit. Table 5 shows Ct values for the standards at different dilution times.
Table 5 Ct values for standards at different dilution.
Referring to FIG. 3, the electrophoresis chart of the conventional PCR amplification of Staphylococcus aureus in example 2 shows that the concentrations of the DNA templates in lanes 1-8 are 52.2 ng/. mu.L, 5.22 ng/. mu.L, and 5.22 × 10-1ng/μL、5.22×10-2ng/μL、5.22×10-3ng/μL、5.22×10-4ng/μL、5.22×10-5ng/μL、5.22×10-6ng/. mu.L, as can be seen from FIG. 3, the lowest concentration of DNA template for detecting Staphylococcus aureus by conventional PCR was 5.22 × 10-2ng/. mu.L, i.e., the sensitivity of conventional PCR amplification is worse than that of fluorescent quantitative PCR.
Example 5
This example provides amplification profiles of standard samples obtained by extracting DNA from pure cultures of Klebsiella pneumoniae DNA template at an initial concentration of 92.8 ng/. mu.L, and diluting the standard samples to 92.8 ng/. mu.L, 9.28 ng/. mu.L, and 9.28 × 10 respectively-1ng/μL、9.28×10-2ng/μL、9.28×10-3ng/μL、9.28×10-4ng/μL、9.28×10-5ng/μL、9.28×10-6ng/. mu.L, then carrying out fluorescent quantitative PCR according to the fluorescent quantitative reaction system and the reaction program shown in the embodiment 3, repeating each standard for 3 times, recording corresponding Ct and Rn values, referring to the fluorescent quantitative standard curve as shown in FIG. 5, referring to the fluorescent quantitative amplification curve as shown in FIG. 4, and referring to FIG. 4, the lowest detection limit of the Klebsiella pneumoniae qPCR detection method provided by the invention is 9.28 × 10-5ng/μL。
Table 6 Ct values corresponding to qPCR for klebsiella pneumoniae standards at different dilution times.
Referring to FIG. 6, the electrophoresis chart of the conventional PCR amplification of Klebsiella pneumoniae in example 3 shows that the concentrations of the DNA templates in lanes 1-8 are 92.8 ng/. mu.L, 9.28 ng/. mu.L, and 9.28 × 10-1ng/μL、9.28×10-2ng/μL、9.28×10-3ng/μL、9.28×10-4ng/μL、9.28×10-5ng/μL、9.28×10-6ng/. mu.L As can be seen from FIG. 6, the lowest concentration of DNA template for detecting Klebsiella pneumoniae by conventional PCR was 9.28 × 10-2ng/. mu.L, i.e., the sensitivity of conventional PCR amplification is worse than that of fluorescent quantitative PCR.
Comparative example 1
200 parts of the dust of example 1 were tested for Staphylococcus aureus using conventional bacterial culture methods. The bacterial culture method refers to the test method of staphylococcus aureus which is an experimental animal in GB/T14926.14-2001.
Comparative example 2
The detection of Klebsiella pneumoniae was carried out on 200 parts of the dust obtained in example 1 by a conventional bacterial culture method. The bacterial culture method refers to the detection method of Klebsiella pneumoniae of experimental animals in GB/T14926.13-2001.
Comparative example 3
The comparative example provides a pair of control primers for detecting staphylococcus aureus, the sequences of the control primers are shown as SEQ ID No.7 and SEQ ID No.8, the sequence of the probe is shown as SEQ ID No.9, the amplification reaction system is the same as that in the table 1, and the reaction procedure is the same as that of the fluorescent quantitative PCR reaction in the example 2.
Comparative example 4
The comparative example provides a pair of control primers for detecting klebsiella pneumoniae, the sequences of the control primers are shown as SEQ ID No.10 and SEQ ID No.11, the sequence of the probe is shown as SEQ ID No.12, the amplification reaction system is the same as that in Table 3, and the reaction procedure is the same as that of the fluorescent quantitative PCR reaction in example 3.
Experimental example 1
(1) Staphylococcus aureus assay of 30 fecal samples.
30 parts of feces are collected from cages in the houses of the rats and the mice, qPCR detection and conventional PCR detection are respectively carried out by adopting the detection method provided by the embodiment 2, and staphylococcus aureus is detected by adopting the bacteria culture method provided by the comparative example 1.
Referring to FIG. 7, it can be seen from FIG. 7 that the number of positive samples detected from 30 stool samples was 11, the length of the PCR product was 279bp, and the numbers of the positive samples were 3, 5, 8, 10, 11, 14, 16, 20, 22, 25, and 27.
Referring to fig. 8, the qPCR detection amplification curves of 30 stool samples are shown, and referring to table 7 for CT values and Rn values, it is understood from fig. 8 that the number of positive samples detected from 30 stool samples is 15, and the positive sample numbers are 3, 5, 6, 8, 10, 11, 14, 16, 18, 20, 22, 25, 27, and 30.
Table 7 experimental example 1 stool sample staphylococcus aureus qPCR assay curves corresponding CT values, Rn values.
Compared with the PCR detection result, more positive samples can be detected by qPCR, which indicates that the sensitivity of the detection method of qPCR is higher.
(2) Staphylococcus aureus assay of 200 dust samples.
After DNA extraction from 200 dust samples collected in example 1, the bacterial culture detection, qPCR and PCR detection of Staphylococcus aureus in dust were performed according to the methods provided in comparative example 1 and example 2, respectively.
Referring to FIG. 9, it is seen from FIG. 9 that 200 dust samples were 5 positive samples and the band size was 279 bp. The positive sample numbers are 5, 17, 30, 117 and 195, respectively.
Referring to FIG. 10, the qPCR detection amplification curves of 200 dust samples are shown, and referring to tables 8, 9 and 10 for the CT value and Rn value, it is understood from FIG. 10 and tables 8 to 10 that the number of positive samples detected from 200 dust samples is 8, and the positive samples are numbered 5, 17, 30, 117, 120, 155, 178 and 195.
Table 8 CT values and Rn values corresponding to qPCR detection curves for staphylococcus aureus nos. 5, 17 and 30 of experimental example 1 dust sample.
Table 9 experimental example 1. qPCR detection curves for staphylococcus aureus nos. 117, 120, 155, 178 for dust sample CT and Rn values.
Table 10 experimental example 1. qPCR detection curve for staphylococcus aureus No. 195 of dust sample CT, Rn values.
Compared with the PCR detection result, more positive samples can be detected by qPCR, which indicates that the sensitivity of the detection method of qPCR is higher.
The positive detection rate is shown in Table 11.
Experimental example 1 bacterial culture assay, qPCR and PCR positive detection rates of Staphylococcus aureus in feces are shown in Table 11.
As can be seen from Table 11, the qPCR detection method for Staphylococcus aureus provided by the invention has a higher positive detection rate than the PCR method, and the positive detection rate is obviously higher than the bacterial culture result.
Table 11 positive detection rates.
| Results of bacterial culture | | PCR | |
| Dust | |||
| 6/200 | 8/200 | 5/200 | |
| Excrement and |
10/30 | 15/30 | 11/30 |
Experimental example 2
(1) Klebsiella pneumoniae assay in 30 stool samples.
30 parts of feces were collected from cages in the houses of rats and mice, and qPCR detection and conventional PCR detection were performed respectively by the detection method provided in example 3, and Klebsiella pneumoniae detection was performed by the bacterial culture method provided in comparative example 2.
Referring to FIG. 11, it can be seen from FIG. 11 that the number of positive samples detected from 30 stool samples was 13, the length of the PCR product was 368bp, and the positive sample numbers were 4,5, 8, 11, 12, 13, 17, 19, 21, 22, 24, 29, and 30.
Referring to fig. 12, the qPCR detection amplification curves of 30 stool samples are shown, and referring to table 12, the CT values and Rn values of the qPCR detection amplification curves of 30 stool samples are shown, and as can be seen from fig. 12 and table 12, the number of positive samples detected from 30 stool samples is 18, and the positive sample numbers are 1, 3, 4,5, 6, 8, 11, 12, 13, 17, 19, 21, 22, 24, 25, 27, 29, and 30.
Table 12 experimental example 2 stool sample klebsiella pneumoniae qPCR detection curve corresponding CT value, Rn value.
Compared with the PCR detection result, more positive samples can be detected by qPCR, which indicates that the sensitivity of the detection method of qPCR is higher.
(2) Klebsiella pneumoniae assay of 200 dust samples.
After DNA extraction from 200 dust samples collected in example 1, the bacterial culture detection, qPCR and PCR detection of Klebsiella pneumoniae in dust were carried out according to the methods provided in comparative example 2 and example 3, respectively.
Referring to FIG. 13, it is seen from FIG. 13 that the number of positive samples detected in 200 dust samples was 6, and the band size was 368 bp. The positive sample numbers are 56, 124, 140, 145, 175 and 188, respectively.
The qPCR detection amplification curves of 200 dust samples are shown in fig. 14, and the CT values and Rn values corresponding to the qPCR detection amplification curves of 200 dust samples are shown in table 13, table 14, and table 15. As is clear from FIG. 14 and tables 13 to 15, the number of positive specimens detected from 200 dust specimens was 8, and the numbers of the positive specimens were 14, 56, 78, 124, 140, 145, 175 and 188.
Table 13 experimental example 2 dust samples 14, 56 and 78 klebsiella pneumoniae qPCR detection curves corresponding to CT values, Rn values.
Table 14 experimental example 2 dust samples 124, 140, 145 and 175 klebsiella pneumoniae qPCR detection curves corresponding to CT values, Rn values.
Table 15 CT values, Rn values corresponding to the qPCR detection curve for klebsiella pneumoniae No. 188 dust sample of experimental example 2.
Compared with the PCR detection result, more positive samples can be detected by qPCR, which indicates that the sensitivity of the detection method of qPCR is higher.
The positive detection rates are shown in Table 16.
Experimental example 2 bacterial culture detection, qPCR and PCR positive detection rates of Klebsiella pneumoniae in feces are shown in Table 16.
As can be seen from Table 16, the positive detection rate of the Klebsiella pneumoniae qPCR detection method provided by the invention is higher than that of the PCR method, and the positive detection rate is obviously higher than that of a bacterial culture result.
Table 16 positive detection rate.
| Results of bacterial culture | | PCR | |
| Dust | |||
| 5/200 | 8/200 | 6/200 | |
| Excrement and |
14/30 | 18/30 | 13/30 |
Experimental example 3
The experimental example provides an amplification curve chart of a standard product prepared by extracting DNA from a pure culture of staphylococcus aureus, the concentration of the DNA template of the standard product is 52.2 ng/mu L, and the standard product is respectively diluted to 52.2 ng/mu L, 5.22 ng/mu L and 5.22 × 10-1ng/μL、5.22×10-2ng/μL、5.22×10-3ng/μL、5.22×10-4ng/μL、5.22×10-5ng/μL、5.22×10-6ng/. mu.L (in turn pair)Curve 1-8), then performing fluorescent quantitative PCR according to the fluorescent quantitative reaction system and reaction program shown in the comparative example 3, repeating each standard for 3 times, recording corresponding Ct and Rn values, referring to the graph of the fluorescent quantitative amplification as shown in FIG. 15, referring to the Table 17 for the Ct and Rn values corresponding to the fluorescent quantitative amplification curve, referring to the graph of the standard curve as shown in FIG. 16, it can be seen from FIG. 15 that the detection sensitivity of the comparative example 3 for detecting staphylococcus aureus by using the control primer can reach 5.22 × 10-3ng/. mu.L. From the sensitivity data, the nucleic acid composition provided by the invention has the advantages of higher sensitivity and lower detection limit compared with the control primer of the comparative example 3.
Table 17 CT values of the standards at different dilution concentrations.
Experimental example 4
This example provides an amplification curve of a standard product prepared by extracting DNA from a pure culture of Klebsiella pneumoniae, the concentration of the DNA template of the standard product is 52.2 ng/. mu.L, and the standard product is respectively diluted to 92.8 ng/. mu.L, 9.28 × 10-1ng/μL、9.28×10-2ng/μL、9.28×10-3ng/μL、9.28×10-4ng/μL、9.28×10-5ng/μL、9.28×10-6ng/muL (corresponding to curves 1-8 in sequence), then carrying out fluorescent quantitative PCR according to the fluorescent quantitative reaction system and the reaction program shown in the comparative example 4, repeating each standard for 3 times, recording the corresponding Ct and Rn values (shown in the reference table 18), referring to the fluorescent quantitative amplification curve chart shown in the reference table 17 and the standard curve shown in the reference table 18, and knowing from the reference table 17, the detection sensitivity of the control primer of the comparative example 4 for detecting Klebsiella pneumoniae can reach 9.28 × 10-4ng/. mu.L. From the sensitivity data, the nucleic acid composition provided by the invention has the advantages of higher sensitivity and lower detection limit compared with the control primer of the comparative example 4.
Table 18 CT values of the standards at different dilution concentrations.
Experimental example 5
In this experimental example, the control primers provided in comparative example 3 and the primers provided in example 2 were used to detect staphylococcus aureus standards (pure culture of staphylococcus aureus), respectively, and the amplification curves are shown in fig. 19, and fig. 19 shows that the detection specificity of the detection primers provided in example 2 and the control primers of comparative example 3 is good.
Experimental example 6
In this experimental example, the primers provided in comparative example 4 and the primers provided in example 3 were used to detect a Klebsiella pneumoniae standard (a pure culture of Klebsiella pneumoniae), respectively, and the amplification curves are shown in FIG. 20, and FIG. 20 shows that the detection specificity of the primers provided in example 3 and the control primers of comparative example 4 is good.
Experimental example 7
In this example, the control primers provided in comparative example 3 were used to perform qPCR detection on 200 dust samples from example 1. From 200 dust samples, 6 positive samples were detected, and the 6 positive samples were numbered 5, 17, 30, 117, 120, and 195, respectively. The detection rate of the positive sample was 6/200, and the amplification curve for detecting Staphylococcus aureus is shown in FIG. 21. The CT and Rn values corresponding to the amplification curves are shown in Table 19.
Table 19 experimental example 7 detected CT values and Rn values corresponding to amplification curves of staphylococcus aureus.
The positive rate of the control primer was significantly lower than that of Experimental example 1.
Experimental example 8
In this example, the control primers provided in comparative example 3 were used to perform qPCR on 30 stool samples collected in example 1. From 30 stool samples, 13 positive samples were detected, which were numbered 3, 5, 6, 10, 11, 14, 16, 18, 20, 22, 25, 27 and 30, respectively. The detection rate of the positive sample was 13/30, and the amplification curve for detecting Staphylococcus aureus is shown in FIG. 22. The CT and Rn values corresponding to the amplification curves are shown in Table 20.
Table 20 experimental example 8 the amplification curves for staphylococcus aureus were tested for CT and Rn values.
The positive rate of the control primer was significantly lower than that of Experimental example 1.
Experimental example 9
In this example, the control primers provided in comparative example 4 were used to perform qPCR detection on 200 dust samples from example 3. From 200 dust samples, 7 positive samples were detected, which were numbered 14, 56, 78, 124, 140, 145 and 175, respectively. The detection rate of the positive sample was 7/200, and the amplification curve for detecting Klebsiella pneumoniae was shown in FIG. 23. The CT value and Rn value corresponding to the amplification curve are shown in tables 21 and 22.
TABLE 21 EXPERIMENT EXAMPLE 8 CT and Rn values for the amplification curves for Klebsiella pneumoniae ( sample numbers 14, 56 and 78).
TABLE 22 Experimental example 8 detection of amplification curves for Klebsiella pneumoniae corresponds to CT and Rn values ( sample numbers 124, 140, 145 and 175).
Compared with the experimental example 2, the positive rate of the control primer is obviously lower.
Experimental example 10
In this example, the control primers provided in comparative example 4 were used to perform qPCR on 30 stool samples collected in example 2. From 30 stool samples, 16 positive samples were detected, numbered 1, 3, 4,5, 6, 8, 11, 12, 17, 19, 21, 22, 24, 25, 27 and 30, respectively. The detection rate of the positive sample was 16/30, and the amplification curve for detecting Staphylococcus aureus is shown in FIG. 24. The CT value and Rn value corresponding to the amplification curve are shown in Table 23.
TABLE 23 Experimental example 10 detection of CT and Rn values for amplification curves of Staphylococcus aureus.
The control primers showed significantly lower positive detection rates than in Experimental example 1 (positive detection rate 18/30).
The invention utilizes cage frame dust qPCR to monitor the infection conditions of staphylococcus aureus and klebsiella pneumoniae in experimental mice, and can replace the traditional sentinel mice to detect two pathogenic bacteria. The method has the following advantages:
(1) the detection result can represent the health condition of the whole cage, the usage amount of sentinel animals is reduced, and the animal cost is saved;
(2) the qPCR detection has the advantage of high flux, all the cage health conditions in the whole room/facility can be screened in one detection, and the detection period is within 48 h.
(3) The detection method provided by the invention has good application prospect, and the qPCR detection method for the two microorganisms has high sensitivity and strong specificity, and can be used for detecting pathogenic bacteria in various samples; aiming at different pathogenic bacteria in the dust, a enrichment medium and a detection method can be used in a targeted manner, and the cage dust is utilized more and more quickly for monitoring microorganisms.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Jiangsu Jiejiaokang Biotech limited
<120> nucleic acid composition, kit and method for detecting staphylococcus aureus and klebsiella pneumoniae
<160>12
<170>PatentIn version 3.5
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aggcgttcat gtttttgttg aag 23
<210>5
<211>18
<212>DNA
<213> Artificial sequence
<400>5
ctcattacgt tgcatcgg 18
<210>6
<211>23
<212>DNA
<213> Artificial sequence
<400>6
cctcggctat gtgctgtcga aag 23
<210>7
<211>27
<212>DNA
<213> Artificial sequence
<400>7
gtagattggg caattacatt ttggagg 27
<210>8
<211>25
<212>DNA
<213> Artificial sequence
<400>8
cgcatctgct ttgttatccc atgta 25
<210>9
<211>25
<212>DNA
<213> Artificial sequence
<400>9
cgctaggcgc attagcagtt gcatc 25
<210>10
<211>23
<212>DNA
<213> Artificial sequence
<400>10
cttcaacaaa aacatgaacg cct23
<210>11
<211>19
<212>DNA
<213> Artificial sequence
<400>11
<210>12
<211>25
<212>DNA
<213> Artificial sequence
<400>12
cgctaggcgc attagcagtt gcatc 25
Claims (10)
1. A nucleic acid composition for detecting staphylococcus aureus and klebsiella pneumoniae, comprising: a first nucleic acid combination for detecting staphylococcus aureus and a second nucleic acid combination for detecting klebsiella pneumoniae; the first nucleic acid combination comprises a first primer pair and a first probe, the second nucleic acid combination comprises a second primer pair and a second probe, the sequences of the first primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, the sequence of the first probe is shown as SEQ ID NO.5, the sequences of the second primer pair are shown as SEQ ID NO.3 and SEQ ID NO.4, and the sequence of the second probe is shown as SEQ ID NO. 6.
2. The nucleic acid composition of claim 1, wherein the first probe and the second probe are labeled with different fluorescent reporter groups at their 5 'ends and a fluorescent quencher group at their 3' ends.
3. The nucleic acid composition of claim 2, wherein the fluorescent reporter is HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, Texas Red, or VIC and the quencher is MGB or BHQ.
4. A kit for the detection of staphylococcus aureus and klebsiella pneumoniae, comprising the nucleic acid composition according to any one of claims 1 to 3.
5. The kit according to claim 4, further comprising a reaction mixture, wherein the reaction mixture is a fluorescent quantitative premix or a PCR premix; the reaction mixed solution is qPCR probe method fluorescent quantitative premixed solution; the PCR premix comprises PCR buffer and enzyme mixed liquor; the sample detected by the kit is mouse excrement or dust taken from facilities for feeding mice.
6. The kit according to claim 5, wherein the concentration of the first primer pair of the first nucleic acid combination in the kit is 0.1 to 20. mu.M, the concentration of the first probe is 0.1 to 20. mu.M, the concentration of the second primer pair of the second nucleic acid combination in the kit is 0.1 to 20. mu.M, and the concentration of the second probe is 0.1 to 20. mu.M.
7. A method for testing Staphylococcus aureus and Klebsiella pneumoniae, comprising: performing PCR amplification detection by using a nucleic acid sample to be detected as a template sequence and the nucleic acid composition of any one of claims 1 to 3 or the kit of any one of claims 4 to 6.
8. The assay method of claim 7, wherein the PCR amplification detection procedure comprises: 35-37 deg.C for 3-10 min; 95-96 deg.C for 6-8 min; circulating for 40-50 times at 94-96 deg.C for 5-10s, 55-60 deg.C for 25-32 s;
the PCR amplification detection system comprises: a reaction system for detecting staphylococcus aureus and a reaction system for detecting klebsiella pneumoniae; wherein the concentration of the first primer pair in the reaction system for detecting staphylococcus aureus is 0.1-20 mu M, and the concentration of the first probe is 0.1-20 mu M; the concentration of the second primer pair in the reaction system for detecting the Klebsiella pneumoniae is 0.1-20 mu M, and the concentration of the second probe is 0.1-20 mu M.
9. The assay of claim 8, wherein the test nucleic acid sample is extracted from mouse feces or dust from animal facilities feeding mice.
10. The test method according to claim 9, wherein the method for extracting a sample of nucleic acid to be tested from dust of an animal facility in which the mouse is kept comprises: culturing the dust in a enrichment culture medium, collecting thalli and extracting DNA; the enrichment culture medium of staphylococcus aureus is 10% sodium chloride trypticase soy broth, and the enrichment culture medium of klebsiella pneumoniae is selenite enrichment liquid.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1161060A (en) * | 1994-09-12 | 1997-10-01 | M·G·伯杰龙 | Specific and universal probe and amplification primer for rapidly detecting and identifying common bacterial pathogens and antibiotic resistance genes in clinical samples in routine diagnosis of microbiological laboratories |
-
2020
- 2020-08-17 CN CN202010822856.2A patent/CN111733269A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1161060A (en) * | 1994-09-12 | 1997-10-01 | M·G·伯杰龙 | Specific and universal probe and amplification primer for rapidly detecting and identifying common bacterial pathogens and antibiotic resistance genes in clinical samples in routine diagnosis of microbiological laboratories |
Non-Patent Citations (2)
| Title |
|---|
| 祝岩波等: "金黄色葡萄球菌、绿脓杆菌和肺炎克雷伯杆菌多重PCR方法的建立与初步应用", 《中国比较医学杂志》 * |
| 郑皓等: "脓毒症常见病原菌多重实时PCR检测方法的建立", 《疾病监测》 * |
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