CN111719007A - Listeria monocytogenes nucleic acid detection plasmid DNA reference sample, preparation method and application thereof - Google Patents
Listeria monocytogenes nucleic acid detection plasmid DNA reference sample, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a Listeria monocytogenes nucleic acid detection plasmid DNA reference sample, a preparation method and application thereof, belonging to the field of microbial detection, wherein the reference sample is formed by inserting fragments containing hlyA, plcB and inlA genes derived from different Listeria monocytogenes strains into a plasmid, the genes are connected in series in a ratio of 1:1, and the genes are separated by unrelated gene fragments; the reference product comprises three specific detection genes of hlyA, plcB and inlA, has clear sequence sources, is stored in plasmids in a single copy mode, can be used for qualitative detection, and can be used as a reference product with a traceable quantity value for carrying out reagent performance identification and capability evaluation between laboratories.
Description
Technical Field
The invention relates to the field of microbial detection, in particular to a Listeria monocytogenes nucleic acid detection plasmid DNA reference sample, a preparation method and application thereof.
Background
Listeria Monocytogenes (L Monocytogenes), abbreviated as Listeria Monocytogenes, belongs to Listeria (Listeria) and is recognized as an important zoonosis pathogenic bacterium worldwide. Listeria monocytogenes has strong pathogenicity, and can mainly cause clinical manifestations such as mononucleosis, septicemia, meningitis, pregnant woman abortion, dead fetus and the like. The listeria monocytogenes still has strong growth and reproduction capability in a low-temperature environment of 4 ℃, and foods frozen and stored in a refrigerator for a long time in daily life are very easy to be polluted by the listeria monocytogenes. The listeria monocytogenes is easy to cause infection and attack of people through meat and dairy products, so the control of the listeria monocytogenes has been gradually emphasized by the related health departments of multiple countries in the world.
At present, two methods for detecting listeria monocytogenes are mainly used in laboratories and detection mechanisms. The first is the traditional culture and improvement method depending on biochemical and morphological characteristics, which is characterized in that: the detection period is long, the experimental operation is complex, the result accuracy is poor, and false negative detection results are easy to occur. The second is a Polymerase Chain Reaction (PCR) method, including a general PCR method and a real-time fluorescent quantitative PCR method. The principle of the PCR method is to establish PCR and real-time fluorescence PCR by selecting specific sequences from a plurality of target genes of the listeria monocytogenes which are different from other bacteria in the listeria. The method has the characteristics of rapidness, sensitivity and specificity, and has become an effective detection means for the food-borne pathogenic microorganisms. However, the nucleic acid reference used in PCR detection is currently very lacking. The genomic DNA is used as a reference product, and the traceability quantitative reference is difficult to provide for a plurality of targets at the same time. Small fragments of nucleic acid do not provide a comparable reference between the various detection reagents and methods. Therefore, novel traceable nucleic acid reference materials for various detection target scalar values are of great importance for the development of nucleic acid detection of listeria.
The plasmid DNA standard (pDNA) is a new biological standard and has the characteristics of high purity, good stability and low development cost. By means of a developed synthesis platform, double-stranded DNA fragments of various lengths can be easily designed and prepared from biotech companies and applied to the preparation of new plasmid DNA standard samples. It can be used not only as a qualitative positive control for nucleic acid detection, but also as a quantitative standard by constructing a standard curve for quantitative analysis. Therefore, the plasmid DNA standard product can also play a key role in the traceability process of nucleic acid detection.
Disclosure of Invention
The invention aims to provide a Listeria monocytogenes nucleic acid detection plasmid DNA reference sample, a preparation method and application thereof, aiming at solving the problems in the prior art, aiming at synthesizing plasmids containing hlyA, plcB and inlA genes detected by Listeria monocytogenes as qPCR reference samples, evaluating the performances of uniformity, stability and the like, discussing the feasibility of replacing the traditional genome reference samples, and solving the contradiction between the rapid development of pathogenic nucleic acid detection capability and the lack of reference materials by the traceable and effective reference material.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a listeria monocytogenes nucleic acid detection plasmid DNA reference sample, which is formed by inserting fragments containing hlyA, plcB and inlA full-length genes derived from different listeria monocytogenes strains into a plasmid;
the nucleotide sequence of the listeria monocytogenes nucleic acid detection plasmid DNA reference sample is shown in SEQ ID No. 1;
the hlyA, the plcB and the inlA genes are connected in series in equal proportion;
the hlyA, plcB and inlA genes are separated by an unrelated gene fragment.
Further, the ratio of the optical density values at 260nm and 280nm is between 1.8 and 2.0.
Further, the nucleotide sequence of the unrelated gene fragment is aagtcg.
The invention also provides a preparation method of the listeria monocytogenes nucleic acid detection plasmid DNA reference sample, which comprises the following steps:
artificially synthesizing listeria monocytogenes genes hlyA, plcB and inlA, and spacing the genes by using the unrelated gene segments;
cloning and inserting the synthesized plasmid into the plasmid, and extracting and purifying the plasmid after the listeria monocytogenes is amplified;
measuring the concentration and subpackaging; pre-freezing at-70 ℃, sublimating and drying again to obtain freeze-dried powder, namely the listeria monocytogenes nucleic acid detection plasmid DNA reference sample.
Further, the plasmid was a pUC57 vector plasmid.
Further, the method also comprises the step of qualitatively identifying the nucleic acid standard sample, and carrying out PCR amplification or sequencing through specific primers for qualitative analysis so as to confirm that the prepared plasmid reference product contains complete and accurate target DNA.
The invention also provides an application of the listeria monocytogenes nucleic acid detection plasmid DNA reference sample in detecting listeria monocytogenes.
Further, when the listeria monocytogenes is detected, the listeria monocytogenes nucleic acid detection plasmid DNA reference sample is used as a reference substance or a reference substance.
The invention discloses the following technical effects:
(1) the reference substance for detecting the listeria monocytogenes provides a unified reference substance for detecting three detection genes of listeria monocytogenes hlyA, plcB and inlA in biological product finished products, intermediate products and raw materials thereof.
(2) Compared with the existing Listeria monocytogenes genome reference product, all genetic information is only from a single strain, the single strain often cannot contain all targets to be detected, and the contained detection targets cannot be subjected to quantitative analysis.
(3) The reference substance for detecting the listeria monocytogenes is subjected to sequencing detection to detect the gene sequence, the species is determined to be the listeria monocytogenes, the magnitude of the reference substance is determined through cooperative calibration of more than three laboratories, and the information such as the uniformity, the stability and the like of the reference substance is researched; the information such as the sequence, uniformity stability and the like of the reference substance has definite information.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a map of plasmid DNA standard substance pDNA Listeria;
FIG. 2 is a real-time fluorescence PCR standard curve established by plasmid DNA standard substance pDNA Listeria.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Invention history
The traditional gene detection standard substance is usually a pathogen genome nucleic acid extract, but because a target sequence to be detected cannot exist in a uniform and magnitude-traceable form in a genome, the target sequence and the magnitude are difficult to trace in quality evaluation, and detection results of various laboratories are difficult to compare. Therefore, the development of a novel nucleic acid reference material is urgently needed.
The artificial DNA synthesis technology provides new possibility for preparing reference standard substances, can obtain complete gene segments through artificial synthesis, and even connects a plurality of target genes in series on the same plasmid vector through a genetic engineering technology, thereby realizing the quality reference of the detection of a plurality of detection target genes at the same time.
In this study, the present inventors constructed pDNA Listeria as a reference for Listeria qualitative detection and detection performance evaluation, as required for Listeria detection. One plasmid covers all 3 detection targets of hlyA, plcB and inlA. The detection sequence is artificially synthesized according to hlyA, plcB and inlA3 sequences published by NCBI, and the concentration of the plasmid is confirmed by joint fixed values in different laboratories, so that stable guarantee can be provided on the accuracy of the sequence and the traceability of the value. Meanwhile, through analyzing the uniformity and stability of the pDNA Listeria, the pDNA Listeria is shown to have good purity, and the uniformity and the stability meet the relevant requirements of JJJG 1006-94 'first-class standard substance'. Meanwhile, the application of pDNAListeria in Listeria qPCR detection is analyzed through qPCR, and the result shows that the standard curve prepared by using pDNAListeria has good linearity, and can replace Listeria monocytogenes genome DNA for quality reference of the PCR detection of hlyA, plcB and inlA3 genes.
Therefore, the listeria monocytogenes hlyA, plcB and inlA gene detection plasmid DNA reference substance developed by the invention has correct sequence, good purity, uniformity and stability meeting the requirements of 'first-level standard substance technical specification', and can be used for replacing listeria monocytogenes genome DNA for PCR amplification of hlyA, plcB and inlA genes. The development of the plasmid DNA reference substance provides a quantity value traceability basis for PCR related detection of listeria monocytogenes hlyA, plcB and inlA genes, and provides a powerful guarantee for quality control of listeria monocytogenes detection laboratories. The plasmid DNA standard substance has the characteristics of large-scale replication in a short period, economy, high efficiency, accurate and reliable measurement value and the like. The research of the invention hopes that the standard substance can be well popularized and applied in the Listeria monocytogenes inspection laboratory in China, can well improve the detection level of related projects of the Listeria monocytogenes inspection laboratory, ensures the comparability and the accuracy of measurement results of various laboratories, lays a good foundation for the mutual recognition and the sharing of the inspection results, and provides technical reserve for preventing large-scale emergent public security incidents caused by Listeria monocytogenes.
EXAMPLE 1 Synthesis of plasmid, fragment design and cloning
1. Fragment design
Three sections of genes are artificially synthesized according to hlyA (GenBank: KC770796.1), plcB (GenBank: JF712529.1) and inlA (GenBank: EF445938.1) gene sequences detected by Listeria monocytogenes as detection target fragments. The nucleic acid sequences of individual genes can be downloaded at the NCBI website (https:// www.ncbi.nlm.nih.gov /) and sequence alignment ensures that the selected genes have clear representativeness and universality.
2. Construction of plasmids
Three genes were expressed as 1:1 (separated by an unrelated gene fragment aagtcg), synthesized by a whole-gene artificial synthesis method (performed by Shanghai Jima Biotech engineering Co., Ltd.) and cloned into pUC57 to form a recombinant plasmid (the length of the chemically synthesized double-stranded DNA fragment is 1561bp, the length of the whole plasmid is determined by enzyme digestion and sequencing, and the recombinant plasmid is amplified and purified for further experiments, as shown in FIG. 1.
3. Identification of the plasmid:
after plasmid extraction and purification (plasmid mass extraction kit (OMEGA), the nucleotide sequence is shown as SEQID No.1 through sequencing verification, the sequence accuracy of the inserted fragment in the recombinant plasmid is 100 percent, and the plasmid meets expectations, and is named as pDNAllieria, so that the high-purity plasmid DNA standard substance is obtained.
4. Purification of plasmids
The purity of the plasmid DNA standard substance is identified by an ultraviolet spectrophotometry: the obtained plasmid DNA standard substance pDNAlisteria was subjected to ultraviolet spectrum scanning while A of pDNA was observed260/A280The ratio is 1.863 + -0.234, greater than 1.8 and less than 2.0, A260/A230A ratio of (A) to (B) of (B) above 2.0 indicates that no ethanol, protein or RNA contamination is present in the pDNA solution.
Example 2 measurement of the degree of homogeneity, the value of the measurement and the shelf life
Determination of homogeneity of pDNA
Homogeneity is a fundamental property of a standard substance that is used to characterize the spatial distribution of the characteristics of the standard substance. The plasmid DNA standard substance with good uniformity has a value which is not influenced by factors such as subpackaging and the like, and the reliability of a detection result can be ensured. In order to meet the requirements of the national standard material technical specification, the present invention analyzes the bottle-to-bottle uniformity and the bottle-to-bottle uniformity of pNDA using the uv method (minimum sampling amount is 1 μ L), and analyzes the data by the F-test.
1) The uniformity sampling method between bottles is as follows: for the inter-vial homogeneity test, 10 tubes of pDNA were randomly selected and 1 μ L of sample extracted from each tube was repeatedly measured 3 times with UV and averaged.
2) In-bottle uniformity sampling method: 9 tubes of pDNA were randomly selected and 1. mu.L of test samples were extracted from the upper, middle and lower layers of each tube sample according to analysis of variance (F-test).
3) The detection method comprises the following steps: ultraviolet spectrophotometry for quantifying DNA content in sample
4) And (3) detection results: the measurement results were analyzed by analysis of variance (F-test method) and judged. The statistical analysis result shows that at a 95% confidence level, on the basis of the F test, the property value of the pDNA is judged to have no significant difference in the homogeneity between bottles, and the pDNA meets the reference material of qPCR. Specific statistics are shown in table 1.
TABLE 1 pDNA Listeria homogeneity statistical analysis results
Stability of pDNA
One of the most common long-term stable storage conditions is the specific storage condition at a temperature of-20 ℃ as a nucleic acid reference substance. Thus, pDNA Listeria samples were monitored for 12 months under storage conditions for uv spectroscopy.
1. For stability studies, the following bulk DNA formulations were prepared:
pDNA (in TE buffer: 10mM Tris-HCl [ pH 8.0] and 1mM ethylenediaminetetraacetic acid [ EDTA ]) was stored at-20 ℃ for long-term storage. Each DNA sample contained sufficient DNA at a concentration of 30. mu.g/ml (as determined by UV absorbance at 260 nm). All DNA preparations were quantitatively analyzed using absorbance at 260nm and possible contaminants were characterized using absorbance ratios at 260/280 and 260/230nm, confirming the absence of RNA and protein contamination.
2. Sampling: 3 bottles of the prepared plasmid DNA standard substance pDNA Listeria (stored at-20 ℃) were randomly drawn within one year after completion of preparation of the plasmid DNA standard substance pDNA Listeria prepared in example 1 once a month, and the measurement was repeated 3 times per sample, averaged, and subjected to long-term stability examination.
3. Statistical analysis: this data was used to evaluate the stability over long storage times according to ISO guide 35 (https:// www.iso.org/standard/60281.ht ml). The slope of the line can be calculated using the following equation:
in the formula: xi-an ith time point; y isi-an observed value at an ith time point;
-average of all time points;
-average of all observations.
The intercept can be calculated by:
the standard deviation of each point on the line can be calculated by:
in the formula: xi-an ith time point; y isiObserved value of the ith time point β1，β0Regression coefficient, n-measurement coefficient β1The standard deviation of (d) is given by:
based on β1The standard deviation of (A) can be determined by t-test as if | β1|<t0.95,n-2·s(β1) Then the slope is not significant and no instability is observed.
Is divided by statisticsAnalysis shows that the product is | β1|＜t0.95，n－2·S(β1) The results showed that the difference in slope was not significant, as shown in table 2, indicating that the plasmid DNA standard substance pDNA Listeria did not have a significant tendency to rise or fall within a prescribed time, and the variation ranges were all within the range of the characteristic quantity values and their uncertainty.
TABLE 2 pDNA Listeria Long-term storage stability analysis results
Fixed value of pDNA
1) Co-assaying pDNA Listeria values with the UV method in 8 laboratories;
2) no outliers and significant differences were confirmed for each group of data by statistical examination: summarizing all original data, and checking by a skewness coefficient and a peak state coefficient method to obtain that all the original data conform to normal distribution;
3) the average value was calculated again from the 8 average values, and the total average value of the plasmid DNA standard substance pDNA L isteria was found to be 29.85 μ g/ml, which was the standard value (unit: μ g/ml).
4) Plasmid DNA standards were calculated based on the molecular weight of each plasmid molecule, and the following formula was used to calculate the copy number per microliter of pDNA Listeria.
Copy number of pDNA Listeria of 6.37 × 109copies/μl。
Uncertainty analysis of pDNA
The uncertainty of pDNA includes uncertainty introduced from packaging (uh), uncertainty introduced by long-term storage stability (us), and uncertainty introduced at constant value (uq).
1) Calculation of split charging introduction uncertainty (uh): calculating uncertainty introduced by split charging by using the uniformity data among bottles;
the uncertainty (uh) caused by the split of pDNA Listeria was calculated according to the following equation:
q1 (sum of variance between groups) is 23.845; q2 (sum of variance within group) is 25.833; v1 is a degree of freedom between groups 9; v2 is a degree of freedom within the 20 groups;
the final calculated dispensed introduction uncertainty was 0.67 μ g/mL with a relative uncertainty of 2.24%.
2) Uncertainty (us) introduced by long term storage stability uncertainty (us) caused by long term stability of pDNA listeria over 12 months was calculated according to the following formula:
us＝S(β1)×N；
S(β1) Is the standard deviation of the slope in the stability Linear model, S (β)1) 0.117; n is the time of stability assessment, N ═ 12 months;
the final calculated stability uncertainty was 1.40. mu.g/mL with a relative uncertainty of 4.69%.
3) Calculation of the uncertainty introduced at fixed value (uq):
the uncertainty of pDNA is evaluated with reference to ISO guidelines 35. The uncertainty (uq) introduced in the fixed value is calculated according to the following formula:
s is the standard deviation of the total mean of 0.63. mu.g/mL; p is the experimental number 8.
The final statistical method calculated the uncertainty to be 0.22. mu.g/mL, relative uncertainty to be 0.73%.
4) Calculating the comprehensive uncertainty:
through statistical calculation, the relative uncertainty component of the plasmid DNA reference material is as follows: homogeneity (u)rh) Relative uncertainty 2.24% due to variability over the life and relative uncertainty (u) due to variability over the lifers) 4.69%, relative uncertainty introduced by the quantification procedure (u)rq) 0.73%, then according toThe standard uncertainty (Ucrm) of the reference substance was calculated according to the following formula:
when calculating the extended uncertainty, the standard uncertainty should be multiplied by an inclusion factor (k-2). Therefore, the extended relative uncertainty is calculated by the following equation:
U＝Ucrm*k
the results show that: the standard uncertainty was 1.57. mu.g/ml and the expanded uncertainty was 3.14. mu.g/ml.
Example 3 preparation of a fluorescent quantitation Standard Curve
1. Fold dilution of pDNA
By A260The pDNA reference was extracted and quantified, and the copy number of pDNA Li steria was estimated based on 4271 bp.
After quantification, a 10-fold dilution series using pDNA Listeria was prepared to make 2 × 106、2×105、2×104、2×103、2×102And 2 × 101copies/ml diluted sample.
qPCR reaction conditions
A five point standard curve for qPCR was generated using 10-fold serial dilutions of 1. mu.l sample extracted from pDNA. Primer sequences and amplicon sizes are shown in table 3. PCR analysis Using ABI SYBR FASTqPCR Master Mix (2X) (NEWEngland Biolabs, UK), PCR reactions were performed in 25. mu.l volumes in eight tubes, each containing 10pM primer. The PCR protocol was as follows: 40 cycles of 94 ℃ for 10 minutes and 94 ℃ for 15s and 55 ℃ for 45s, respectively.
TABLE 3 Listeria monocytogenes PCR detection primers
3. Establishment of a Standard Curve
Each reaction was repeated 3 times, and a standard curve was established based on the relationship between Ct value and template concentration to estimate the limit of detection (LOD), amplification efficiency (e) and slope (K), as shown in FIG. 2. The linear correlation coefficient of each standard curve is shown in table 4. In the invention, the result shows that the correlation coefficients of the standard curves reach about 0.99, the linearity is good, and the pDNALTERIA is used as the reliability of a qPCR reference product and can be used as a positive standard material for Q PCR amplification for detecting a target gene sequence of Listeria monocytogenes.
TABLE 4 slope of standard curve, correlation coefficient, amplification efficiency and lower detection limit
Example 4 alternative Studies of pDNAlisteria and genomic DNA (gDNA)
The invention refers to a research method of a European Union certification reference substance MON810, and compares the amplification efficiency generated by two reference substances (a plasmid DNA reference sample and a genome DNA) and the slope of a standard curve to perform substitution research. Therefore, the invention uses the genome standard and the plasmid DNA reference sample to be diluted in a gradient way as templates to draw standard curves, and each standard curve is repeated for 6 times. The amplification efficiency and slope of each standard curve were statistically analyzed (t-test). The results demonstrate that by assessing slope and amplification efficiency, there was no significant difference P <0.05 between the standard curve established for genomic DNA and the standard curve established for pDNA Listeria at 95% confidence as shown in table 5. Therefore, the present invention recognizes that Listeria monocytogenes can be detected using pDNA Listeria instead of Listeria genomic DNA.
TABLE 5 pDNA Listeria and alternative comparison of genomic DNA
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> southern medical university
<120> listeria monocytogenes nucleic acid detection plasmid DNA reference sample, preparation method and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1561
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
taccaaagta aaagctgctt ttgacgctgc cgtaagtggg aaatctgtct caggtgatgt 60
agaactgaca aatatcatca aaaattcttc cttcaaagcc gtaatttacg gtggctccgc 120
aaaagatgaa gttcaaatca tcgacggtaa cctcggagac ttacgagata ttttgaaaaa 180
aggtgctact tttaaccggg aaacaccagg agttcccatt gcctatacaa caaacttctt 240
aaaagacaat gaattagctg ttattaaaaa caactcagaa tatattgaaa caacttcaaa 300
agcttataca gatggaaaaa tcaacatcga tcactctgga ggatacgttg caaaagtcgc 360
taggtatgtg cttgaccgca agtgttctag tctttcctgt aacgataaaa gcaagcgcct 420
gttgtgatga atatctaaag cccccagcag ctccgcatga tatcgacagc aaattaccac 480
ataaacttag ttggtccgcg gataacccgacaaatactga cgtaaataca cactattggc 540
tttttaaaca agcggaaaag atattagcta aagatgtaga tcatatgcga gctaatttaa 600
tgaatgaact taaaaatttc gataaacaaa ttgctcaagg aatatatgat gcggatcata 660
agaatccata ttatgatact agtacgtttt tatctcattt ttataatcct gataaagata 720
atacttattt gccgggattt gctaatgcga aaataacagg agctaaatat ttcaatcaat 780
cggtggctga ttaccgagaa gggaaatttg acacagcatt ttataaatta ggcttagcaa 840
tccattatta tacggatatt agtcaaccta tgcacgccaa taattttacc gcaatatcat 900
acccgccagg ctaccactgt gcatatgaaa attacgtaga taccattaaa cacaattatc 960
aagcaacaga agacatggta gtaaaaagat tttgctcaaa tgacgtgaaa gactggcttt 1020
atgaaaatgc gaaaagggcg aaagcggact acccgaaaat agtcaatgcg aaaactaaaa 1080
aatcatactt agtaggaaat tccgaatgga aaaaggatac agtggaacct actggagcta 1140
gactaagaga ttcacagcaa actttggcag gttttttaga gttttggtct aaaaaaacaa 1200
atgaaaagtc ggtgagaaga aaacgatatg tatggttgaa aagtatacta gtagcaatat 1260
tagtacttgg aagtggagta tggattaaca cgagtaacgg gaccaatgct caggcagcta 1320
caattacaca agatactcct attaatcaga tttttacaga tacagctcta gcggaaaaaa 1380
tgaagacggt cttaggaaaa acgaatgtaa cagacacggt ctcgcaaaca gatctagacc 1440
aagttacaac gcttcaggcg gatagattag ggataaaatc tatcgatgga ttggaatact 1500
tgaacaattt aacacaaata aatttcagca ataatcaact gacggatata acgccactta 1560
a 1561
Claims (8)
1. A Listeria monocytogenes nucleic acid detection plasmid DNA reference sample is characterized in that the reference sample is formed by inserting a fragment containing hlyA, plcB and inlA full-length genes derived from different Listeria monocytogenes strains into a plasmid;
the nucleotide sequence of the listeria monocytogenes nucleic acid detection plasmid DNA reference sample is shown in SEQ ID No. 1;
the hlyA, the plcB and the inlA genes are connected in series in equal proportion;
the hlyA, plcB and inlA genes are separated by an unrelated gene fragment.
2. The listeria monocytogenes nucleic acid detection plasmid DNA reference sample of claim 1, wherein the ratio of optical density values at 260nm and 280nm is between 1.8-2.0.
3. The listeria monocytogenes nucleic acid detection plasmid DNA reference sample of claim 1, wherein the nucleotide sequence of the unrelated gene fragment is aagtcg.
4. A method for preparing a listeria monocytogenes nucleic acid detection plasmid DNA reference sample of any one of claims 1-3, comprising:
artificially synthesizing listeria monocytogenes genes hlyA, plcB and inlA, and spacing the genes by using the unrelated gene segments;
cloning and inserting the synthesized plasmid into the plasmid, and extracting and purifying the plasmid after the listeria monocytogenes is amplified;
measuring the concentration and subpackaging; pre-freezing at-70 ℃, sublimating and drying again to obtain freeze-dried powder, namely the listeria monocytogenes nucleic acid detection plasmid DNA reference sample.
5. The method of claim 4, wherein the plasmid is a pUC57 vector plasmid.
6. The method of claim 4, further comprising the step of performing qualitative identification of the nucleic acid standard sample, and performing qualitative analysis by PCR amplification or sequencing using specific primers to confirm that the prepared plasmid reference contains the complete and accurate DNA of interest.
7. Use of the listeria monocytogenes nucleic acid detection plasmid DNA reference sample of any one of claims 1-3 for detecting listeria monocytogenes.
8. The use of claim 7, wherein the listeria monocytogenes nucleic acid detection plasmid DNA reference sample is used as a reference or control in performing listeria monocytogenes detection.
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