CN116334254B - Novel Pasteurella multocida nucleic acid detection kit and non-diagnostic detection method thereof - Google Patents
Novel Pasteurella multocida nucleic acid detection kit and non-diagnostic detection method thereof Download PDFInfo
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Abstract
The invention discloses a novel pasteurella multocida nucleic acid detection kit and a non-diagnostic detection method thereof, wherein the kit comprises a reaction buffer solution, bstDNA polymerase, dNTPs, betaine, graphitized carbon nanotubes, polyethylene glycol methyl ether, SYBRGreenI and detection primers, and the sequences of the detection primers are shown as SEQ ID NO.1 and SEQ ID NO. 2. Compared with the traditional detection kit and detection method, the method has the advantages of simplicity, convenience, rapidness, strong specificity, high sensitivity and good repeatability, has obvious advantages in the aspects of detection time and detection cost, and is suitable for batch detection.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a pasteurella multocida nucleic acid detection kit and a non-diagnostic detection method thereof.
Background
Pasteurella multocida (Pasteurella multocida, pm) is an important zoonotic pathogen, belongs to the genus Pasteurella of the family Pasteurella in bacterial taxonomy, was obtained in 1880 by Pasteurella, a famous microbiologist in France, and was collectively named Pasteurella multocida in 1939. Pasteurella multocida can infect humans and various animals, cause various infectious diseases of livestock and poultry, such as cholera fowl, swine plague, bovine hemorrhagic septicemia, rabbit hemorrhagic septicemia and the like, and cause serious harm to the breeding industry. According to the Carter typing method and the Heddleston typing method, the pasteurella multocida is divided into 5 capsular serotypes (A, B, D, E, F) and 16 thallus serotypes (1-16) according to different capsular antigens and different lipopolysaccharide antigens, and the pasteurella multocida serotypes are complex, so that the cross protectiveness among the types is not strong, the vaccine prevention and control difficulty is high, and the occurrence and the epidemic of the disease can be effectively prevented only through pathogen monitoring, sanitary disinfection, biosafety and the like.
At present, the detection standard for the Pasteurella multocida is based on clinical diagnosis, serological detection and etiology detection (such as NYT 563-2002 fowl cholera (fowl Pasteurella) diagnosis technology, NYT 564-2002 pig Pasteurella) diagnosis technology, NYT 567-2002 rabbit hemorrhagic septicemia diagnosis technology, DB 63/T776-2009 cow hemorrhagic septicemia diagnosis technical specification and the like), and the related process of the method is very tedious and time-consuming, and can not reach the requirements of timely discovering pathogens, controlling transmission paths and eliminating epidemic disease prevention and control of pollution sources. With the development of technology, PCR, LAMP and other molecular biological methods are gradually applied to the rapid detection of Pasteurella multocida, but the methods still have a plurality of defects. For example, the sensitivity of the PCR method is relatively low, the detection time is relatively long, and detection after bacteria increase is usually required for improving the detection rate; although the LAMP method has greatly improved sensitivity and shortened detection time, aerosol pollution is very easy to occur, primer dimers are easy to cause due to more primers, and false positive results cannot be distinguished by agarose gel electrophoresis.
Based on the reasons, the invention provides a novel method for detecting the Pasteurella multocida nucleic acid, which can effectively solve the defects in the traditional PCR and LAMP detection and can achieve the advantages of simplicity, convenience, rapidness, strong specificity, high sensitivity, good repeatability and the like by using only one pair of primers.
Disclosure of Invention
Aiming at the defects of bacteriology detection and traditional nucleic acid detection technology in the detection of the Pasteurella multocida, in particular to how to improve the detection efficiency, sensitivity and specificity, the invention provides a novel nucleic acid detection kit for specifically detecting the Pasteurella multocida and a non-diagnostic detection method thereof.
The technical scheme of the invention is as follows:
the invention provides a novel nucleic acid detection kit for specifically detecting Pasteurella multocida and a non-diagnostic detection method thereof, wherein the nucleic acid detection kit for the Pasteurella multocida comprises a reaction buffer solution, DNA polymerase, dNTPs, a detection primer, betaine, graphitized carbon nano-tubes, polyethylene glycol methyl ether, positive control and negative control; the detection primer is a combination of a primer of SEQ ID NO.1 and a primer of SEQ ID NO. 2.
The invention can realize the advantages of strong specificity and high sensitivity by using a pair of specially designed detection primers. In particular, the amplification strip of the kit of the present invention after adding graphitized carbon nanotubes has a single target-specific strip (as shown in FIG. 1). The present invention design is improved on the basis of strand-exchange amplification (isothermal amplification using a pair of primers) (still using a pair of primers, amplification in two steps). It was found that under the condition that graphitized carbon nanotubes are not added, non-specific amplification phenomena (such as primer dimer appearance and unclear specific bands) similar to strand exchange amplification can occur (as shown in fig. 1), so that the reaction specificity can be improved to the greatest extent by adding novel nano materials such as graphitized carbon nanotubes. The mechanism of graphitized carbon nanotubes for gene amplification is unknown, and it is presumed that a relatively stable structure can be formed with single-stranded DNA due to the surface charge property, while double-stranded DNA is more stable due to the hydrogen bonding force between double strands, so that graphitized carbon nanotubes are not bonded. Therefore, the graphitized carbon nano tube can adsorb redundant primer single chains which do not participate in the reaction, so that the generation of primer dimers is prevented, and the reaction specificity is effectively improved.
The betaine is added into the kit of the invention to help the DNA polymerase to pass through some complex secondary structures of the DNA smoothly and prevent the DNA polymerase from dissociating from the template DNA. The localized region of DNA, which contains multiple complex bases (Py-G-C), promotes the arrest of the DNA polymerase, ultimately resulting in the cessation of efficient extension of the DNA polymerase. And betaine can improve the hydration of guanine and cytosine in the regions rich in guanine and cytosine in the minor groove of the DNA, influence the molecular structure of the DNA, change the flexibility of the DNA and help the DNA polymerase extend along the DNA template. Second, betaine can eliminate the base dependence of denaturation temperature. The concentration dependence reduces the Tm of high GC content sequences and brings the Tm of different primers close, eventually reducing the DNATm value. In addition, betaine solutions stabilize the DNA-protein complex.
Further, the positive control is the genomic DNA of Pasteurella multocida CVCC44801, and the negative control is sterile physiological saline.
Further, the components of the kit detection system are as follows: each 25. Mu.L of the reaction solution contained 2.5. Mu.L of 10 Xreaction buffer, 1. Mu.L of Bst DNA polymerase, 3.5. Mu.L of dNTPs, 2.5. Mu.L of 10 XSYBR Green I, 1. Mu.L of betaine (10 mM), 1. Mu.L of polyethylene glycol methyl ether, 1. Mu.L of graphitized carbon nanotube solution (10 ng/. Mu.L), 1.5. Mu.L of each of PM-F and PM-R, 3. Mu.L of DNA to be examined, and the balance of sterilized physiological saline.
Further, the reaction flow of the method for detecting the Pasteurella multocida is as follows: 73℃for 1 second, 63℃for 1 second, the total number of cycles being 45.
Further, the 10X reaction buffer contains 200mM Tris-HCl，100mM(NH4) 2 SO 4 ，500mM KCl，20mM MgSO 4 ，1％TritonX-100，pH 8.0～8.8。
Furthermore, the primer sequences of the method for detecting the Pasteurella multocida are shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the dNTPs included dGTP, dCTP, dATP, dTTP, each component concentration was 2.5mM.
Still further, a method for non-diagnostic testing using a pasteurella multocida test kit comprising the steps of:
s1: bacterial genomic DNA was extracted using a boiling method or commercial kit to obtain a detection template.
S2: adding 10X reaction buffer solution, dNTPs, taqDNA polymerase, detection primers and a DNA template into a sterilization reaction tube, adding sterilized normal saline to the total volume of 25 mu l, setting positive and negative controls, and performing amplification reaction by using a fluorescent quantitative PCR instrument; the amplified products were electrophoretically detected by 3% agarose gel electrophoresis and analyzed for the results.
Compared with the prior art, the invention has the beneficial effects that:
the primer is designed according to the specific gene sequence of the Pasteurella multocida screened by comparative genomics, and has the advantages of high sensitivity and strong specificity. Meanwhile, the method can be used for rapidly detecting the Pasteurella multocida through one-time reaction, has great advantages in detection time and detection cost compared with the traditional detection method combining bacteriology with serology typing, and is suitable for batch detection.
Drawings
FIG. 1 shows the result of agarose gel electrophoresis in example 1. In the figure: m is Takara 20bp DNALader (Dye Plus), lane 1 is a graphitized carbon nanotube system-containing test Pasteurella multocida CVCC44801, lane 2 is a graphitized carbon nanotube-free test Pasteurella multocida CVCC44801, lane 3 is a negative control;
FIG. 2 shows the test results of the anti-interference test in example 3. In the figure: 1 is a pasteurella multocida bacteria increasing liquid diluted 10000 times; 2 is a mixture of the bacteria-increasing liquid, namely, the bacteria-increasing liquid of the pasteurella multocida, the escherichia coli, the salmonella, the shigella flexneri and the klebsiella pneumoniae is diluted 10000 times and then mixed according to the proportion of 1:1:1:1:1; 3 is a negative control;
FIG. 3 shows the detection results of the sensitivity evaluation in example 4. In the figure: 1 is 8.5X10 2 COPIES/. Mu.l, 2 is 8.5X10 1 COPIES/. Mu.l, 3 is 8.5X10 0 COPIES/. Mu.l, 4 is 8.5X10 -1 COPIES/. Mu.l, 5 is 8.5X10 -2 COPIES/. Mu.l, 6 is a negative control.
Detailed Description
The present invention is further described below with reference to the examples and drawings, which are given by way of illustration only, and not by way of limitation, of the preferred embodiments of the present invention, and any person skilled in the art may make modifications to the equivalent embodiments using the technical matters disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
EXAMPLE 1 establishment of a method for detecting Pasteurella multocida nucleic acid
Designing a primer: based on all the DNA sequences of Pasteurella multocida and the DNA sequences of other species known in GenBank database, the universal nucleotide sequences of Pasteurella multocida are selected after analysis and comparison, and the primers for specifically detecting the Pasteurella multocida are designed and preferably obtained on the basis of the universal nucleotide sequences, as shown in the following table.
TABLE 1 Papanicolaou multocida nucleic acid detection primer sequences
Template preparation: culturing Pasteurella multocida CVCC44801 in brain heart infusion broth culture medium, and extracting bacterial genome DNA (deoxyribonucleic acid) by using a commercial bacterial genome DNA extraction kit to serve as a template to be detected.
Preparing a detection reagent: 10 Xreaction buffer (its composition includes 200mM Tris-HCl,100mM (NH 4) 2SO4, 500mM KCl,20mM MgSO4,1%Triton X-100, pH 8.0-8.8.), dNTPs (containing dGTP, dCTP, dATP, dTTP, each component concentration of 2.5 mM), bst DNA polymerase (8000U/. Mu.l), 10. Mu.M detection primer, 0.5. Mu.L polyethylene glycol methyl ether, betaine (10 mM), graphitized carbon nanotube solution (10 ng/. Mu.L), 10 XSYBR Green I. In addition, a graphitized carbon nanotube default control group was set.
Detection system and amplification procedure: the detection system is 25 mu L, and specifically comprises: 2.5. Mu.L of 10 Xreaction buffer, 1. Mu.L of Bst DNA polymerase, 3.5. Mu.L of dNTPs, 2.5. Mu.L of 10 XSYBR Green I, 1. Mu.L of betaine (10 mM), 1. Mu.L of polyethylene glycol methyl ether, 1. Mu.L of graphitized carbon nanotube solution (10 ng/. Mu.L), 1.5. Mu.L of each of PM-F and PM-R, 3. Mu.L of sample DNA to be detected, and the balance of sterilized physiological saline. The amplification procedure steps included 73℃for 1 second, 63℃for 1 second, and a total number of cycles of 45.
And (3) judging a detection result: when the reaction is finished, the fluorescence curve is positive when the peak value (fluorescence value is more than 0.0001); when the fluorescence curve does not peak (fluorescence value is 0.0001 or less), it is judged as negative. Or mixing 5 μl of amplified product with 1 μl of 6×loading buffer, spotting in 3% agarose gel electrophoresis plate hole, electrophoresis at 100V voltage for 40min, photographing under gel imager, and judging that clear band of about 50bp is visible, but default graphitized carbon nanotube control group band is not obvious, and obvious primer dimer appears (as shown in FIG. 1).
Example 2 specificity evaluation experiment
The specificity of the detection method of the present invention was evaluated by the method of example 1, 24 reference strains were cultured in brain heart infusion broth, the amplified solution was diluted 10000 times, and bacterial genomic DNA was extracted using a commercial bacterial genomic DNA extraction kit, and the detection was performed according to the method described in example 1, and the results are shown in Table 2, and only the detection results of Pasteurella multocida (SEQ ID NO: 1-12) were positive, and the other species were negative.
TABLE 2 results of the inventive specificity evaluation test
Example 3 anti-interference evaluation experiment
Sensitivity evaluation of the detection method of the present invention was performed by the method of example 1. Pasteurella multocida CVCC44801, escherichia coli ATCC25922, salmonella ATCC13076, shigella flexneri CMCC51572 and klebsiella pneumoniae ATCC700603 are inoculated into brain heart infusion broth culture medium for enrichment culture, and after the enrichment culture solutions are diluted 10000 times respectively, bacterial genome is extracted by using a commercial bacterial genome DNA extraction kit. The extracted gene component is 2 parts, the sample No.1 only contains the Pasteurella multocida CVCC44801, the sample No.2 is the genome of the Pasteurella multocida, the Escherichia coli, the salmonella, the shigella and the Klebsiella pneumoniae, and the sample No. 3 is a negative control. The test is carried out according to the method described in the example 1, and the results are shown in fig. 2, wherein the sample No.1 and the sample No.2 show obvious fluorescence curves, and the sample No. 3 shows no fluorescence curve, so that the method has better anti-interference capability.
Example 4 sensitivity evaluation experiment
Sensitivity evaluation of the detection method of the present invention was performed by the method of example 1. Culturing Pasteurella multocida CVCC44801 in brain heart infusion broth culture medium, extracting genome with commercial bacterial genome DNA extraction kit, measuring the original concentration, performing 10-time gradient dilution, and amplifying bacterial genome DNA with different dilution gradients. As can be seen from FIG. 3, the method of the present invention for detecting Pasteurella multocida has a minimum lower limit of up to a single copy (8.5X10 -1 cobies/. Mu.l), has higher sensitivity and application value.
Example 5 stability evaluation experiment
The reproducibility of the detection method of the present invention was evaluated by the method of example 1. 10 amplification tests were performed with the Pasteurella multocida CVCC44801 genomic DNA as template. The results are shown in Table 3, and the fluorescence signal time threshold (Tt) is relatively stable (the variation coefficient is about 3.4%), and the negative control has no fluorescence signal time threshold, so that the method has better stability.
TABLE 3 stability test results
Example 6 Assembly of detection kit
Synthesizing a specific detection primer of the Pasteurella multocida according to the sequences in the table of the example 1, diluting the specific detection primer to a concentration of 10 mu M by using sterilized normal saline, and mixing the specific detection primer in an equal volume to obtain a detection primer; extracting salmonella ATCC13076 genomic DNA as a positive control using a commercial bacterial genomic DNA extraction kit; nucleic acid amplification reagents: 10 Xreaction buffer (its composition comprises 200mM Tris-HCl,100mM (NH 4) 2 SO 4 ，500mM KCl，20mM MgSO 4 1% Triton X-100, pH 8.6-8.8), dNTPs (containing dGTP, dCTP, dATP, dTTP, each component concentration of 2.5 mM), bst DNA polymerase (8000U/. Mu.l), betaine (10 mM), polyethylene glycol methyl ether, graphitized carbon nanotube solution (10 ng/. Mu.L) detection primers, positive control, and negative control (sterile physiological saline).
The reagent and the product are packaged together, and then the product using instruction (comprising the product preservation condition, the reaction program, the result judging method and the like) is matched, so that the novel pasteurella multocida nucleic acid detection kit is assembled.
Example 7 sample collection and detection
Detection of the clinically isolated 124 strains of bacteria was performed as in example 1, with simultaneous identification of the pasteurella multocida isolates with reference to the method in the pasteurella multocida test standard (NYT 563-2002). The results are shown in Table 4, and in 124 clinical isolates, 15 Pasteurella multocida isolates are detected by the detection method provided by the invention and the national standard detection method, and the coincidence rate of 2 detection methods reaches 100%, which indicates that the detection method established by the kit has better specificity. In addition, the test standard for Pasteurella multocida (NYT 563-2002) requires several days for the identification of Pasteurella multocida, and only about half an hour for the detection using the kit of the invention.
Table 4 clinical sample validation test results
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A pasteurella multocida nucleic acid detection kit, characterized in that the kit comprises: 1) A reaction buffer; 2) And (3) detecting a primer: the forward primer PM-F and the reverse primer PM-R have primer sequences shown in SEQ ID NO.1 and SEQ ID NO. 2; 3) Bst DNA polymerase; 4) dNTPs; 5) SYBR Green I; 6) Betaine; 7) Polyethylene glycol methyl ether; 8) Graphitized carbon nanotubes.
2. The pasteurella multocida nucleic acid detection kit of claim 1, further comprising: 9) Positive control: pasteurella multocida CVCC44801 genomic DNA;10 Negative control: sterilizing physiological saline.
3. The pasteurella multocida nucleic acid detection kit of claim 1, wherein the reaction buffer comprises Tris-HCl, potassium chloride, ammonium sulfate, magnesium sulfate, and Triton X-100.
4. The pasteurella multocida nucleic acid detection kit of claim 1, wherein the concentration of both the forward primer PM-F and the reverse primer PM-R is 10 pmol/. Mu.l.
5. The pasteurella multocida nucleic acid detection kit according to claim 1, wherein the detection reaction system of the kit is: every 25 mu L of reaction solution comprises 2.5 mu L of 10 Xreaction buffer solution, 1 mu L of Bst DNA polymerase, 3.5 mu L of dNTPs, 2.5 mu L of 10 XSYBR Green I, 1 mu L of 10mM betaine, 1 mu L of polyethylene glycol methyl ether, 1 mu L of 10 ng/mu L graphitized carbon nanotube solution, 1.5 mu L of PM-F and PM-R, 3 mu L of sample DNA to be detected and the balance of sterilized normal saline.
6. Use of the pasteurella multocida nucleic acid detection kit according to any one of claims 1-5 for the preparation of a reagent for detecting pasteurella multocida.
7. A method of non-diagnostic testing using the pasteurella multocida nucleic acid testing kit of any one of claims 1-5, comprising the steps of:
s1: extracting bacterial genome DNA by using a boiling method or a commercial kit to obtain a detection template;
s2: adding buffer solution, bst DNA polymerase, dNTPs, SYBR Green I, betaine, polyethylene glycol methyl ether, graphitized carbon nanotube solution, detection primers and a DNA template into a sterilization reaction tube, adding sterilized normal saline to a total volume of 25 mu l, setting positive and negative controls, and performing amplification reaction by using a fluorescent quantitative PCR instrument;
s3: when the reaction is finished, the fluorescence curve is peaked, namely the fluorescence value is more than 0.001, and the positive result is judged; when the fluorescence curve does not peak, namely the fluorescence value is less than or equal to 0.001, the fluorescence curve is judged to be negative; or electrophoresis detection is carried out on the amplified products by adopting 3% agarose gel electrophoresis, and the result is analyzed.
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