CN104928300A - Polynucleotide, method and kit for real-time fluorescence quantitative PCR (Polymerase Chain Reaction) detection of vibrio cholerae - Google Patents

Abstract

The invention discloses polynucleotide, a method and a kit for real-time fluorescence quantitative PCR (Polymerase Chain Reaction) detection of vibrio cholerae, and particularly provides the polynucleotide, usable for vibrio cholerae PCR detection, and a primer pair matched with the polynucleotide. A polynucleotide sequence is prepared into a standard plasmid molecule PLW07 by using an appropriate framework plasmid, and real-time fluorescence PCR detection is carried out by virtue of the primer pair; the specificity and the sensitivity are extremely high, and the stability is high.

Description

A polynucleotide, method and kit for real-time fluorescent quantitative PCR detection of Vibrio cholerae

technical field

The invention relates to a plasmid molecule in the technical field of bioengineering, in particular to a plasmid standard molecule suitable for real-time fluorescent quantitative PCR detection of Vibrio cholerae and its construction method, quantitative method and application.

Background technique

Vibrio cholerae (Vibrio cholerae) is a pathogenic bacterium that can cause a severe infectious disease with diarrhea as the main symptom. Rapid and accurate identification of Vibrio cholerae plays an important role in the prevention and treatment of the disease. It has been found for a long time that Vibrio cholerae group 01 can cause the epidemic of cholera. Group 0139 cholera, as an emerging infectious disease, caused a cholera pandemic in India and Bangladesh in 1992. Since 1993, outbreaks caused by Vibrio cholerae of group 0139 have also occurred to varying degrees in many areas of my country. In recent years, the proportion of outbreaks of group 0139 cholera is still rising. The pathogenicity of Vibrio cholerae O1 group and O139 group depends on the coding products related to the toxins of the strains, such as Vibrio cholerae toxin co-regulated pilus (TCP), toxin expression regulatory protein (toxR) and so on. Cholera detection methods include biochemical culture, serological test and phage typing, etc. These methods have the disadvantages of low detection rate and long inspection time, which often fail to meet the needs of epidemic control. The fluorescent quantitative PCR method can quickly and sensitively detect the specific gene sequence of Vibrio cholerae, and can complete the detection of the strain within a few hours. Plasmid DNA standard substance is a recombinant plasmid molecule containing a specific fragment of the target gene. It can be used as a positive control in PCR qualitative detection, and can be used as a standard for quantitative analysis in PCR quantitative analysis to construct a standard curve for quantitative analysis. At present, plasmid DNA molecules have been more and more in-depth research and application as standard materials for gene detection, but the plasmid DNA standard materials for the real-time fluorescent PCR detection method of Vibrio cholerae are still blank. In the actual real-time fluorescent PCR of Vibrio cholerae, most of the units use plasmid DNA molecules designed and constructed by themselves as standard products, and the specific fragments of the target genes are different, and there is no uniform value determination method. The accuracy of the value is poor, resulting in great differences in the test results between laboratories and lack of comparability.

Contents of the invention

The object of the present invention is to provide a polynucleotide, method and kit for real-time fluorescent quantitative PCR detection of Vibrio cholerae.

The first aspect of the present invention provides an isolated polynucleotide, which comprises the gene sequence of Vibrio cholerae virulence regulator toxR.

In another preferred example, the gene sequence of the Vibrio cholerae virulence regulator toxR is selected from the following group:

(a) the polynucleotide sequence whose sequence is shown in SEQ ID NO.:1;

(b) a polynucleotide sequence whose nucleotide sequence is ≥95% (preferably ≥98%) homologous to the sequence shown in SEQ ID NO.:1;

(c) a polynucleotide sequence whose sequence completely matches or is completely complementary to the polynucleotide shown in SEQ ID NO.: 1 and has a length of 100bp-654bp (preferably 150-645bp, more preferably 200-500bp);

(d) A polynucleotide sequence complementary to the polynucleotide sequence described in any one of (a)-(c).

In another preferred example, the sequence of the polynucleotide is selected from the following group:

(a) the polynucleotide sequence whose sequence is shown in SEQ ID NO.:2 or SEQ ID NO.:1;

(b) a polynucleotide sequence whose nucleotide sequence is ≥95% (preferably ≥98%) homologous to the sequence shown in SEQ ID NO.:2 or SEQ ID NO.:1;

(c) A polynucleotide sequence complementary to the polynucleotide sequence described in any one of (a)-(b).

The second aspect of the present invention provides an isolated DNA construct, which comprises the polynucleotide described in the first aspect of the present invention, and optionally a tag sequence, a restriction sequence, a promoter sequence and/or vector sequence. In a preferred embodiment of the present invention, linear restriction sites are provided at both ends of the polynucleotide described in the first aspect of the present invention.

In another preferred example, the DNA construct is a linear DNA construct or a circular DNA construct.

In another preferred example, the DNA construct is a plasmid or an expression vector.

In another preferred example, the plasmid or expression vector is used as a standard molecule (plasmid standard molecule) for detection of Vibrio cholerae.

In another preference, the DNA construct is a plasmid or an expression vector, and the backbone plasmid of the plasmid or expression vector is selected from the group consisting of pUC19, pUC18, pUC118, pUC119, pBlueScriptII SK and pGEM.

In another preferred example, the sequence of the plasmid is shown in SEQ ID NO: 2.

The third aspect of the present invention provides a kit comprising the polynucleotide described in the first aspect of the present invention or the DNA construct described in the second aspect of the present invention.

In another preferred example, the kit also includes primer sequences selected from the following group:

(1) primer sequences shown in SEQ ID NO.:3 and SEQ ID NO.:4;

(2) primer sequences shown in SEQ ID NO.:5 and SEQ ID NO.:6;

(3) primer sequences shown in SEQ ID NO.:7 and SEQ ID NO.:8; and

(4) The primer sequences shown in SEQ ID NO.:9 and SEQ ID NO.:10.

In another preferred example, the kit also includes the probe sequence shown in SEQ ID NO.:11.

The fourth aspect of the present invention provides the use of the polynucleotide as described in the first aspect of the present invention, the DNA construct as described in the second aspect of the present invention, or the kit as described in the third aspect of the present invention for cholera Vibrio detection.

In another preferred example, the detection is for non-diagnostic or therapeutic purposes.

In another preferred example, the detection is fluorescent quantitative PCR detection.

The fifth aspect of the present invention provides a real-time fluorescent quantitative PCR detection method for Vibrio cholerae, the standard substance used is the polynucleotide described in the first aspect of the present invention or the DNA construct described in the second aspect of the present invention .

In another preference, the primer pair used in the method is selected from the following group:

(1) A pair of primers composed of sequences shown in SEQ ID NO.:3 and SEQ ID NO.:4;

(2) a pair of primers composed of sequences shown in SEQ ID NO.:5 and SEQ ID NO.:6; and

(3) A pair of primers composed of the sequences shown in SEQ ID NO.:7 and SEQ ID NO.:8.

In another preferred example, the probe sequence used in the method is shown in SEQ ID NO.:11.

The sixth aspect of the present invention provides a polynucleotide product, said product comprising:

(i) Vibrio cholerae standard product, the standard product is selected from: the polynucleotide described in the first aspect of the present invention and the DNA construct described in the second aspect of the present invention;

(ii) a pair of primers for specifically amplifying the Vibrio cholerae sequence, said primer pair being selected from the group consisting of:

(1) A pair of primers composed of sequences shown in SEQ ID NO.:3 and SEQ ID NO.:4;

(2) a pair of primers composed of sequences shown in SEQ ID NO.:5 and SEQ ID NO.:6; and

(3) A pair of primers composed of the sequences shown in SEQ ID NO.:7 and SEQ ID NO.:8.

In another preferred embodiment, the product is a combination of polynucleotides, preferably the components (i) and (ii) are independent.

In another preferred example, the product is in the form of a kit.

In another preferred example, the product also includes the probe sequence shown in SEQ ID NO.:11.

The seventh aspect of the present invention provides a method for preparing a plasmid standard molecule of Vibrio cholerae, characterized in that it comprises the following steps:

① Artificially synthesizing the virulence regulator toxR gene sequence of Vibrio cholerae, the expression gene sequence of the virulence regulator toxR is shown in SEQ ID NO: 1 in the sequence listing;

② Cloning the virulence regulator toxR expression gene sequence of Vibrio cholerae obtained in step ① into a cloning vector to obtain a plasmid standard molecule of Vibrio cholerae.

In another preferred example, the cloning vector in step ② is plasmid pUC19, pUC18, pUC118, pUC119, pBlueScript II SK or pGEM; preferably, the cloning vector in step ② is pUC19.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Fig. 1 is a map of the plasmid standard molecule PLW07 of the present invention.

Fig. 2 is a graph showing the stability detection results of the plasmid standard molecule PLW07 obtained in the present invention.

Fig. 3 is a real-time fluorescent PCR standard curve established by using the plasmid standard molecule of the present invention.

Detailed ways

Through extensive and in-depth research, the inventor obtained a polynucleotide sequence that can be used for PCR detection of Vibrio cholerae and a pair of primers that match it. The experimental results showed that the polynucleotide sequence Prepared as a standard plasmid molecule, combined with the primer pair of the present invention for real-time fluorescent PCR detection, it has excellent specificity and sensitivity, and good stability.

The technical problem to be solved by the present invention is to provide a plasmid standard suitable for real-time fluorescent PCR detection of Vibrio cholerae in order to overcome the lack of positive standard and positive standard configuration in the existing Vibrio cholerae real-time fluorescent PCR detection method Molecule and the construction method, quantitative method and application of the plasmid standard molecule.

The principle of real-time fluorescent PCR detection of Vibrio cholerae

Real-time fluorescent PCR technology can be used to specifically amplify the virulence regulator toxR gene sequence in the Vibrio cholerae genome DNA, and design primers for the above target genes and fluorescent probes labeled at both ends to amplify the test sample DNA. Real-time fluorescent PCR can monitor PCR products in real time by detecting the increase in fluorescent signal. At the same time, positive standard substances (or positive standard molecules) of known concentration are amplified with the same primers, probes and conditions. In the PCR method, the positive standard substance (or positive standard molecule) can be used as a positive control; in real-time fluorescent PCR, the positive standard substance (or positive standard molecule) can construct a stable standard curve, and the corresponding gene in the sample can be calculated according to the standard curve. Absolute content (copy number or concentration).

standard material

A reference material is a material or substance having one or more sufficiently homogeneous and well-defined property values for calibrating equipment, evaluating measurement methods, or assigning values to materials.

Plasmid standard molecule

In the present invention, the specific sequence of Vibrio cholerae is a part of the toxR gene of Vibrio cholerae virulence regulator, and the length is 885bp.

In order to solve the above-mentioned technical problems, one of the technical solutions adopted by the present invention is: a plasmid standard molecule of Vibrio cholerae, the plasmid standard molecule comprises the toxR gene sequence of the Vibrio cholerae virulence regulator, and the Vibrio cholerae virulence regulator The sub-toxR gene sequence is shown in SEQ ID NO: 1 in the sequence listing:

The plasmid standard molecules of the present invention preferably respectively contain the target gene of Vibrio cholerae virulence regulator toxR gene sequence detected by PCR of Vibrio cholerae.

The vibrio cholerae virulence regulator toxR gene described in the present invention plays a role in the synthesis of DNA, RNA and protein, and it can be used as a kind of target gene detected by vibrio cholerae, and the vibrio cholerae virulence regulator The sequence of the toxR gene is preferably shown in SEQ ID NO: 1.

The sequence of the plasmid standard molecule of the present invention is preferably as shown in SEQ ID NO: 2, wherein the 423rd to the 1307th are the toxR gene sequence:

In order to solve the above-mentioned technical problems, the second technical solution adopted by the present invention is: a quantitative method for the plasmid standard molecule of Vibrio cholerae as described above, which comprises the following steps:

① Extract plasmid standard molecules;

2. According to the base composition and sequence length of the plasmid standard molecule obtained in step 1., calculate the content of phosphorus element in the plasmid standard molecule;

③ Prepare a phosphorus standard solution with gradient concentration, and use this standard solution to make a standard curve for high-resolution inductively coupled plasma emission mass spectrometry;

④ High-resolution inductively coupled plasma emission mass spectrometry detects the plasmid standard molecular solution, and obtains the phosphorus content of the plasmid standard molecular solution according to the standard curve obtained in step ③;

⑤ Calculate the concentration of the plasmid standard molecule according to the phosphorus content of the plasmid standard molecule solution obtained in step ④ and the phosphorus element content in the plasmid standard molecule obtained in step ②.

The conditions for the detection of high-resolution inductively coupled plasma emission mass spectrometry as described in the steps are as follows: the cooling air flow is preferably 10L/min to 25L/min, the best is 16.86L/min, and the auxiliary air flow is preferably 0.5 L/min～3.0L/min, the best is 0.88L/min, the atomizing gas flow rate is preferably 0.5L/min～2.43L/min, more preferably 1.123L/min, the power is better It is 1200W～1400W, preferably 1350W.

The quantification method of the plasmid standard molecule in the present invention preferably includes ultraviolet spectrophotometry and high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS).

Specific implementation instructions:

The quantification method of the plasmid standard molecule in the present invention preferably includes ultraviolet spectrophotometry and high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS).

Wherein the ultraviolet spectrophotometry preferably comprises the following steps to the plasmid standard molecular quantitative method:

① Extract plasmid standard molecules;

② Calibrate the UV spectrophotometer to zero with TE buffer;

③Take an appropriate amount of DNA (according to the needs of the instrument) into the cuvette (nanodrop is directly placed on the detection area), and record the reading of the instrument.

④ If the DNA concentration can be read directly, record it directly; if not, record the optical density of the sample at 260nm and 280nm. The concentration of the DNA sample is OD260×nucleic acid dilution factor×50, and the concentration unit is ng/μL.

(2) HR-ICP-MS preferably comprises the following steps to plasmid standard molecule quantitative method:

① Extract plasmid standard molecules;

②According to the base composition and sequence length of the plasmid standard molecules, calculate the phosphorus content of each plasmid standard molecule;

③ Prepare phosphorus standard solution with gradient concentration, and use this standard solution to make a standard curve for HR-ICP-MS.

④HR-ICP-MS detects the plasmid standard molecule, and obtains the phosphorus content of the plasmid standard molecule according to the standard curve.

⑤ Calculate the concentration of the plasmid standard molecule according to the phosphorus content of the plasmid standard molecule.

In order to solve the above-mentioned technical problems, the third technical solution adopted by the present invention is: a real-time fluorescent quantitative PCR detection method for Vibrio cholerae, the standard substance used in it is the plasmid standard molecule as described above.

In order to solve the above-mentioned technical problems, the fourth technical solution adopted by the present invention is: a method for preparing a plasmid standard molecule of Vibrio cholerae as described above, comprising the following steps:

① Artificially synthesizing the virulence regulator toxR gene sequence of Vibrio cholerae, the virulence regulator toxR gene sequence is shown in SEQ ID NO: 1 in the sequence listing;

② Cloning the virulence regulator toxR gene sequence of Vibrio cholerae obtained in step ① into a cloning vector to obtain a plasmid standard molecule of Vibrio cholerae.

The artificial synthesis method described in step ① is preferably: the sequence is obtained by whole gene synthesis or PCR primer amplification.

The plasmid standard molecular construction method of the present invention preferably comprises the following steps:

① Query the virulence regulator toxR gene of Vibrio cholerae in Genbank of NCBI (National Center for Biotechnology Information);

②Analyze the above sequence, select a suitable sequence and a suitable restriction site, and add the restriction site to the 5' end and 3' end of the selected sequence.

③Provide the whole gene artificial synthesis service for the processed sequence, including the synthesis of single-stranded Oligo DNA, splicing of DNA fragments, etc., and clone the full-length gene into a plasmid vector to obtain a plasmid standard molecule.

The plasmid vector can be any conventional vector, preferably a cloning vector, more preferably a cloning vector that can propagate in Escherichia coli, and the cloning vector is preferably: pUC19, pUC18, pUC118, pUC119, pBlueScript II SK or pGEM series vectors, preferably pUC19 cloning vectors.

④ Sequencing to verify the sequence of the plasmid standard molecule.

⑤Validation of real-time fluorescent PCR detection method for plasmid standard molecules.

The verification of the described real-time fluorescent PCR detection method refers to the characteristics such as the specificity and the ability to construct a standard curve of the detection plasmid standard molecule when performing real-time fluorescent PCR analysis, so as to identify the plasmid standard molecule as the real-time fluorescent PCR method to detect Vibrio cholerae. The capacity of the standard substance.

The present invention also provides a kit for the above method, which contains the polynucleotide or DNA construct of the present invention, and a primer sequence selected from the following group:

(1) primer sequences shown in SEQ ID NO.:3 and SEQ ID NO.:4;

(2) primer sequences shown in SEQ ID NO.:5 and SEQ ID NO.:6;

(3) primer sequences shown in SEQ ID NO.:7 and SEQ ID NO.:8;

(4) the primer sequence shown in SEQ ID NO.:9 and SEQ ID NO.:10; and optionally the probe sequence shown in SEQ ID NO.:11.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The main advantages of the present invention are:

(1) The plasmid standard molecule comprising the polynucleotide sequence of the present invention has the advantages of strong uniformity and high stability. At the same time, the present invention solves the problem of lack of standard substances in the real-time fluorescent PCR detection of Vibrio cholerae, ensuring real-time fluorescence of Vibrio cholerae The comparability of PCR method detection results provides quality control for the detection of Vibrio cholerae real-time fluorescent PCR method;

(2) The product prepared by using the plasmid standard molecule of the present invention combined with the primer pair of the present invention has strong specificity, high sensitivity and stable linearity when used for real-time fluorescent PCR detection of Vibrio cholerae.

Below in conjunction with specific embodiment, further state the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate detailed conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions. Percentages and parts are by weight unless otherwise indicated.

The construction of embodiment 1 plasmid standard molecule

Experimental reagents and experimental equipment:

A large number of plasmid extraction kits (OMEGA), other biochemical reagents are imported aliquots or domestic analytically pure biochemical reagents; experimental equipment includes centrifuges, constant temperature water baths, constant temperature culture shakers, pipette guns, etc.

The experimental method includes the following steps:

1. Search the gene virulence regulator toxR gene sequence of Vibrio cholerae in GenBank,

2. Analyze the above sequence, select a suitable sequence and a suitable restriction site, the length of the gene virulence regulator toxR gene sequence is 885bp, and BamHI restriction sites are added at both ends;

3. Send the processed sequence to Treasure Bioengineering (Dalian) Co., Ltd., which will be responsible for the artificial synthesis of the whole gene, including the synthesis of single-stranded Oligo DNA, splicing of DNA fragments, etc., and the obtained gene virulence regulator toxR gene The sequence is shown in SEQ ID NO: 1 in the sequence listing, and the resulting full-length gene is cloned into the plasmid vector pUC19 (purchased from TAKARA Company), and the plasmid standard molecule PLW07 comprising the toxR gene sequence is constructed (its sequence is shown in SEQ ID NO in the sequence listing). ID NO: 2), the plasmid map of the constructed plasmid standard molecule PLW07 is shown in Figure 1.

4. Large-scale culture of recombinant Escherichia coli containing the obtained plasmid standard molecule PLW07, using a large amount of plasmid extraction kit (OMEGA) to extract the plasmid, and obtaining high-purity plasmid standard molecule PLW07. Purity analysis is carried out by ultraviolet spectrophotometer and electrophoresis, and then the plasmid DNA standard molecule is stored at -20°C. The method for extracting the plasmid includes the following steps:

a. Centrifuge 100-200 mL overnight cultured bacteria at room temperature at 5000×g for 10 minutes, discard the supernatant;

b. Add 12mL Solution Ⅰ (containing RNase A), shake and mix thoroughly;

c. Add 12mL Solution Ⅱ, mix gently up and down, and place at room temperature for 2 minutes to fully lyse the bacteria;

d. Add 16mL SolutionⅢ, and immediately mix up and down thoroughly several times until a uniform white precipitate is formed;

e. ≥12000×g, centrifuge at 4°C for 10 minutes;

f. Take 20mL of the supernatant and carefully transfer it to a clean HiBind Maxi adsorption column (in a 50mL collection tube), centrifuge at 5000×g for 5 minutes at room temperature;

g. Discard the liquid in the collection tube, add 10mL Buffer HB to wash the adsorption column, and centrifuge at room temperature for 5 minutes at 5000×g;

h. Discard the liquid in the collection tube, add 15mL DNA Wash Buffer (diluted with absolute ethanol) to wash the adsorption column;

i. Repeat the above cleaning steps;

j. Centrifuge at 6000×g for 15 minutes to dry the adsorption column;

k. Put the adsorption column in a clean 50mL tube, add 2mL~3Ml TE buffer, let it stand at room temperature for 1~2 minutes, centrifuge at 8000×g for 2 minutes to elute the DNA, and the resulting DNA solution is the extracted plasmid standard molecule solution, stored at -20°C.

5. Sequencing verification plasmid standard molecule PLW07

The extracted plasmid standard molecules were sent to eight sequencing companies for sequence verification. The eight sequencing companies were Beijing Liuhe Huada Gene Technology Co., Ltd., Shanghai Boshang Biotechnology Co., Ltd., and Yingwei Jieji (Shanghai) Trading Co., Ltd. , Shanghai Jieli Biotechnology Co., Ltd., Shanghai Meiji Biotechnology Co., Ltd., Shanghai Sangong Biotechnology Co., Ltd., Suzhou Jinweizhi Biotechnology Co., Ltd., Bao Biological Engineering Co., Ltd. For plasmid DNA reference materials, the sequence length is directly related to the quantification, and sequence determination by 8 different sequencing companies is a requirement for definite value in the development of reference materials. For details, see ISO Guide 35, the specific requirements of the part of the standard substance valuation.

Sequence inspection formula: sequence accuracy rate = number of correct bases/total number of bases

Sequencing results proved that all units involved in the sequence verification obtained a 100% correct sequence rate, and the 423rd to 1307th positions in the plasmid sequence were the gene virulence regulator toxR gene.

The results of the experiment are as follows: After the steps of sequence selection, whole gene artificial synthesis, and large-scale extraction of plasmids, a high-purity plasmid standard molecule PLW07 was obtained. After sequencing verification, the insert sequence of the plasmid standard molecule was completely consistent with the design.

The homogeneity test of embodiment 2 plasmid standard molecule PLW07

Homogeneity is the consistent state of structure or composition that characterizes one or more properties in a substance. By measuring samples of specified size taken from different packaging units (such as bottles, bags, etc.) or from different locations of the same packaging unit, if the measurement results fall within the specified uncertainty range, it can be considered that the reference material has a certain effect on the specified characteristic quantity. is even. Homogeneity is a basic property of reference materials, which is used to describe the spatial distribution characteristics of reference materials. Uniformity evaluation must be carried out during the development (production) of reference materials to prove that they have good uniformity. The value of plasmid standard molecules with good uniformity will not be affected by factors such as aliquoting, and there is little difference in the value of each bottle, thus ensuring the reliability of the test results.

Experimental reagent TE buffer (pH7.5). Experimental instrument Nanodrops ND-2000.

experimental method:

1. In accordance with the uniform sampling requirements of "JJG 1006-1994 Technical Specifications for Primary Standard Substances", 15 bottles were randomly selected from each plasmid DNA standard substance.

2. Sampling each sample 3 times, each sampling volume is 1 μL. Each sample amount was measured three times by UV spectrophotometry, and the average value was taken.

3. Use the F test method to make statistics on the measurement results, and judge the uniformity test results. The specific method is: extract m samples, and measure m groups of equal-precision measurement data under the same conditions. If there is no significant difference in the measurement variance, the statistical requirements of the following formula should be met.

$\mathrm{<span\; class="notranslate">\; f</span>}<span\; class="notranslate">\; =</span>\frac{\frac{{\mathrm{<span\; class="notranslate">\; Q</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{{\mathrm{<span\; class="notranslate">\; v</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}}{\frac{{\mathrm{<span\; class="notranslate">\; Q</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}{{\mathrm{<span\; class="notranslate">\; v</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}}<span\; class="notranslate">\; \&le;</span>{\mathrm{<span\; class="notranslate">\; f</span>}}_{\mathrm{<span\; class="notranslate">\; \&alpha;</span>}}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; v</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; ,</span>{\mathrm{<span\; class="notranslate">\; v</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; )</span>$

The formula for calculating the difference between groups is:

${\mathrm{<span\; class="notranslate">\; Q</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; =</span>\underset{\mathrm{<span\; class="notranslate">\; i</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\overset{\mathrm{<span\; class="notranslate">\; m</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}}{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}{<span\; class="notranslate">\; (</span>{\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; x</span>}}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; -</span>\stackrel{<span\; class="notranslate">\; =</span>}{\mathrm{<span\; class="notranslate">\; x</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}$

The formula for calculating the square sum of intragroup variance is:

${\mathrm{<span\; class="notranslate">\; Q</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; =</span>\underset{\mathrm{<span\; class="notranslate">\; i</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\overset{\mathrm{<span\; class="notranslate">\; m</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}}\underset{\mathrm{<span\; class="notranslate">\; j</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\overset{\mathrm{<span\; class="notranslate">\; nj</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}}{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; x</span>}}_{\mathrm{<span\; class="notranslate">\; ij</span>}}<span\; class="notranslate">\; -</span>{\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; x</span>}}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}$

ν ₁ = m-1 (degrees of freedom between groups)

ν ₂ =Nm (within degrees of freedom).

Experimental results:

1. The homogeneity test results of the plasmid standard molecule PLW07 are shown in Table 1

Table 1 Plasmid standard molecule pLW07 homogeneity test data (unit: ng/μL)

sample repeat 1 repeat 2 repeat 3 average value 1 94.9 95.4 94.4 94.90 2 94.9 95 94.1 94.67 4 94.1 95.4 93.1 94.20 5 93.3 95.1 93 93.80 6 93.5 93.7 93.9 93.70 7 93.3 94.9 93.2 93.80 8 93.6 92.3 95.8 93.90 9 92.6 94 92.9 93.17 10 93.8 94 93.6 93.80 11 92.6 93 92.4 92.67 12 92.7 95.4 92.7 93.60 13 93.5 94.4 92.2 93.37 14 93.3 94 93.8 93.70 15 94.1 93.7 91.8 93.20

Table 2 Statistical results of uniformity of plasmid standard molecule pLW07

Plasmid DNA Standard Material Q ₁ Q ₂ F value F _0.05 (14, 30) pLW07 13.72 27.31 1.08 2.04

The statistical results of homogeneity are shown in Table 2. At the 95% confidence level, the F value is less than F _0.05 (14, 30), which proves that the plasmid standard molecule PLW07 is uniform and conforms to the homogeneity test of "JJG1006-94 Technical Specifications for Primary Standard Substances" Eligibility requirements.

The stability investigation of embodiment 3 plasmid standard molecule PLW07

Stability refers to the ability of the characteristic values of the standard substance to remain within the specified range under specific time intervals and storage conditions. Stability is a basic attribute of standard substances, which is used to describe the characteristics of standard substances changing with time, that is, to describe the time distribution characteristics of standard substances. Stability assessment must be performed during the development of reference materials. Stability assessment not only evaluates the measurement uncertainties related to material stability, but also identifies suitable storage and transport conditions. Plasmid standard molecules with good stability, their characteristic values will not change over time under appropriate storage and transportation conditions, and the test results will not be affected by their instability, which ensures the reliability of the test results.

Experimental reagent TE buffer (pH7.5). Experimental instrument Nanodrops ND-2000.

experimental method:

The stability of plasmid standards was investigated using classical stability studies, where samples prepared at the same time were measured over time under the same conditions.

1. At 0 month, 0.5 month, 1 month, 2 months, 4 months, and 6 months after the preparation of the plasmid standard molecule, randomly select 3 bottles of the prepared plasmid standard molecule (stored at -20°C) , each sample was repeatedly measured 3 times, and the average value was taken for long-term stability investigation. In this study, ultraviolet spectrophotometry was used to track plasmid standard molecules.

2. Evaluate the stability test data and judge the stability test results. The specific inspection method is as follows:

The slope of a straight line can be calculated with the following formula:

${\mathrm{<span\; class="notranslate">\; \&beta;</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; =</span>\frac{\underset{\mathrm{<span\; class="notranslate">\; i</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\overset{\mathrm{<span\; class="notranslate">\; no</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; x</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; -</span>\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; x</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; Y</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; -</span>\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; Y</span>}}<span\; class="notranslate">\; )</span>}{\underset{\mathrm{<span\; class="notranslate">\; i</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\overset{\mathrm{<span\; class="notranslate">\; no</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}}{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; x</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; -</span>\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; x</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}$

In the formula: X _i - the i-th time point; Y _i - the observed value of the i-th time point; - the average value of all time points; - the average value of all observations.

The intercept can be calculated by the following formula:

${\mathrm{<span\; class="notranslate">\; \&beta;</span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}<span\; class="notranslate">\; =</span>\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; Y</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; \&beta;</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; x</span>}}$

The standard deviation of each point on the line can be calculated by the following formula:

${\mathrm{<span\; class="notranslate">\; the\; s</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; =</span>\frac{\underset{\mathrm{<span\; class="notranslate">\; i</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\overset{\mathrm{<span\; class="notranslate">\; no</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}}{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; Y</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; \&beta;</span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; \&beta;</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; x</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}{\mathrm{<span\; class="notranslate">\; no</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 2</span>}}$

In the formula: Xi _i - the i-th time point; Y _i - the observed value of the i-th time point; β ₁ , β ₀ - the regression coefficient; n - the measurement coefficient.

_The standard deviation of β1 is given by:

$\mathrm{<span\; class="notranslate">\; the\; s</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; \&beta;</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; the\; s</span>}}{\sqrt{\underset{\mathrm{<span\; class="notranslate">\; i</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\overset{\mathrm{<span\; class="notranslate">\; no</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}}{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; x</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; -</span>\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; x</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}}$

Based on the standard deviation of β ₁ , the t-test can be used to make the following judgment: that is, if |β ₁ |<t _0.95,n-2 ·s(β ₁ ), it indicates that the slope is not significant and no instability is observed.

Experimental results:

1. The stability test results of the plasmid standard molecule PLW07 are shown in Table 3 and Figure 2:

Table 3 Stability monitoring data of plasmid standard molecule PLW07 (unit: ng/μL)

time (month) repeat 1 repeat 2 repeat 3 average value SD 0 91.25 91.1 92.45 91.60 0.74 0.5 92.3 91.2 93.3 92.27 1.05 1 90.9 91.2 89.7 90.6 0.80 2 90.7 89.1 89.5 89.77 0.83 4 90.5 89 89.9 89.80 0.75 6 91.3 89.5 92.1 90.97 1.33

The statistical results are shown in Table 4.

Table 4 Stability statistics of plasmid standard molecule PLW07

Plasmid DNA Standard Material beta ₁ β ₀ s(β ₁ ) t _0.95,n-2 ·s(β ₁ ) PLW07 0.17 91.26 0.19 0.52

It can be seen from the statistical results that the plasmid standard molecule PLW07 is stable when stored at -20°C for 6 months.

Experimental Example 4 Quantification of plasmid standard molecule PLW07 by UV spectrophotometry

Experimental reagent TE buffer (pH7.5). Experimental instrument Nanodrops ND-2000.

The experimental method includes the following steps:

1. Calibrate the UV spectrophotometer to zero with TE buffer;

2. Take 1 μL of the DNA solution prepared in the example, directly point it on the detection area, and record the reading of the instrument;

3. Read out the DNA concentration directly, and the concentration unit is ng/μL.

4. Each sample is tested eight times, and the average value is taken.

The results of the experiment are as follows: After UV spectrophotometry, the concentration of the plasmid standard molecule PLW07 is shown in Table 5:

Table 5 UV spectrophotometric value results (unit: ng/μL)

plasmid repeat 1 2 3 4 5 6 7 8 average value SD PLW07 94.9 94.9 94.1 93.3 93.5 93.3 93.6 92.6 93.78 0.81

Example 5 Quantification of the plasmid standard molecule PLW07 by HR-ICP-MS

The experimental reagent is P standard solution (NIST, SRM3139a). The experimental instrument is: inductively coupled plasma emission mass spectrometer (Thermofisher element2).

The experimental method includes the following steps:

1. According to the base composition and sequence length of the plasmid standard molecule PLW07, calculate the content of phosphorus in it;

2. The experimental parameters of HR-ICP-MS are: cooling air flow rate 16.86L/min, atomizing air flow rate 1.123L/min, auxiliary air flow rate 0.99L/min, power 1350W.

3. Prepare phosphorus standard solutions (0, 1, 2, 3, 4, 5g/L) with gradient concentrations, and use this standard solution to make a standard curve for HR-ICP-MS;

4. Dilute the plasmid standard molecule PLW07 by 3000 times, detect the diluted plasmid standard molecule by HR-ICP-MS, and obtain the phosphorus content of the diluted plasmid standard molecule according to the standard curve;

5. Calculate the concentration of the plasmid standard molecule PLW07 according to the measured phosphorus content and dilution factor;

6. Each sample is tested eight times, and the average value is taken.

The experimental results are as follows: after calculation, the phosphorus content of the plasmid standard molecule PLW07 is 10.22%. The phosphorus content data of plasmid standard molecules obtained through HR-ICP-MS determination are shown in Table 6, and the dilution factor is 3000, and the mass concentration of plasmid molecules is converted, as shown in Table 7.

Table 6 Phosphorus content of plasmid standard molecule PLW07 (unit: μg/L)

plasmid repeat 1 2 3 4 5 6 7 8 average value SD PLW07 2.38 2.28 2.20 2.24 2.39 2.33 2.34 2.36 2.32 0.07

Table 7 Mass concentration results of plasmid standard molecule PLW07 (unit: ng/μL)

Application of the plasmid standard molecule PLW07 developed in Example 6 in real-time fluorescent PCR detection

Experimental reagents: The inventors designed dozens of pairs of primers by conventional methods, and handed over the designed primers and probes to Youbao Bioengineering (Dalian) Co., Ltd. for detection after synthesis. Master Mix was purchased from Lifetechnology Company. Experimental equipment includes: real-time fluorescent quantitative PCR amplification instrument (Life technology) centrifuge, constant temperature water bath, incubator, balance, etc.

Application of plasmid standard molecule PLW07 in real-time fluorescent PCR detection:

Primers, probes, PCR detection system

PCR loading system:

Reaction conditions PCR program:

95°C for 10 minutes;

95°C for 15 seconds, 60°C for 1 minute, 40 cycles.

Dilute the plasmid standard molecule PLW07 prepared in Example 1 into different concentrations of the plasmid standard molecule PLW07 (2.6×10 ⁶ copies/μL, 2.6×10 ⁵ copies/μL, 2.6×10 ⁴ copies/μL, 2.6×10 ³ copies/μL, /μL, 2.6×10 ² copies/μL, 2.6×10 ¹ copies/μL, 2.6×10 ⁰ copies/μL) Different concentrations of plasmid standard molecule PLW07 were used as templates, and real-time fluorescent PCR amplification was performed according to the methods in the literature . Each reaction was repeated three times, and a standard curve was established based on the relationship between the Ct value of different concentration template amplification and the concentration.

Experimental results

1. Four pairs of primers that can effectively amplify the target sequence were selected from dozens of pairs of primers, as shown in Table 8, among which primer pair 1 had the best amplification effect,

The primers and probe sequences used in the experiment are listed in Table 8.

Primer and probe sequences used in table 8 experiments

Primer pair 1 has the best detection effect, strong specificity, no non-specific bands after amplification, the highest detection sensitivity, and the lowest detectable target sequence of 2.6 copies/μL; primer pair 2 and 3 have lower sensitivity and can 2.6×10 ² copies/μL of the target sequence were detected; primer pair 4 could detect 2.6×10 ¹ copies/μL of the target sequence, but the specificity was poor and non-specific bands appeared.

2. Real-time fluorescent PCR detection using plasmid standard molecule PLW07 and primer pair 1

Using the above PCR detection system and reaction conditions, different concentrations of plasmid standard molecule PLW07 (such as: 2.6×10 ⁶ copies/μL, 2.6×10 ⁵ copies/μL, 2.6×10 ⁴ copies/μL, 2.6×10 ³ copies/μL, /μL, 2.6×10 ² copies/μL, 2.6×10 ¹ copies/μL, 2.6×10 ⁰ copies/μL) were used as standards to establish a standard curve for real-time fluorescent PCR. The established standard curve is shown in Figure 3, the correlation coefficient reaches 0.9927, the linearity is good, the blank control has no amplification peak, and the target sequence can be detected at a minimum of 2.6 copies/μL, indicating that the plasmid standard molecule PLW07 is suitable for real-time fluorescent PCR detection and can be used as a real-time PCR assay. Fluorescence PCR amplifies the positive standard substance of Vibrio cholerae.

In the actual detection of Vibrio cholerae, the standard curve with good linearity can be used to detect the copy number of the specific gene of Vibrio cholerae in the sample to be tested by the experimental fluorescent PCR method, and the copy number of the specific gene of Vibrio cholerae can be compared with the bacteria The linear relationship between the total numbers was converted to the specific number of Vibrio cholerae. Since the research and development of standard materials for the real-time fluorescent PCR detection method of Vibrio cholerae is still blank, in the actual real-time PCR detection of Vibrio cholerae, most units use plasmid DNA molecules designed and constructed by themselves as standard products. Gene-specific fragments are different, and at the same time, there is a lack of a unified valuation method, and the accuracy of plasmid DNA molecular valuation is poor, resulting in great differences in test results between laboratories, lacking comparability, effectiveness and reliability. Therefore, this plasmid standard molecule can solve the problem of lack of standard substances in the detection of Vibrio cholerae by real-time fluorescent PCR, and is suitable for a variety of real-time fluorescent PCR methods for amplifying the toxR gene of the virulence regulator of Vibrio cholerae, ensuring the comparability of the detection results. Provide biometric technical support for the detection of Vibrio cholerae real-time fluorescent PCR method.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. an isolated polynucleotide, characterized in that, said polynucleotide comprises the gene sequence of vibrio cholerae virulence regulator toxR; said isolated polynucleotide is selected from the group consisting of: (a) the polynucleotide sequence whose sequence is shown in SEQ ID NO.:2 or SEQ ID NO.:1; (b) a polynucleotide sequence whose nucleotide sequence is ≥95% (preferably ≥98%) homologous to the sequence shown in SEQ ID NO.:2 or SEQ ID NO.:1; (c) A polynucleotide sequence complementary to the polynucleotide sequence described in any one of (a)-(b). 2. An isolated DNA construct, characterized in that, the DNA construct comprises the gene sequence of Vibrio cholerae virulence regulator toxR, and optional tag sequence, enzyme cutting sequence, promoter sequence and/or vector sequence. 3. DNA construct as claimed in claim 2, is characterized in that, described DNA construct is plasmid or expression vector, and the backbone plasmid of described plasmid or expression vector is selected from the group: pUC19, pUC18, pUC118, pUC119 , pBlueScript II SK and pGEM; Preferably, the sequence of the plasmid is as shown in SEQID NO:2. 4. A kit, characterized in that the kit comprises the polynucleotide according to claim 1 or the DNA construct according to claim 2. 5. The use of the polynucleotide according to claim 1, the DNA construct according to claim 2 or the kit according to claim 4, characterized in that it is used for the detection of Vibrio cholerae. 6. A real-time fluorescent quantitative PCR detection method for Vibrio cholerae, characterized in that the standard substance used is the polynucleotide as claimed in claim 1 or the DNA construct as claimed in claim 2. 7. A polynucleotide product, characterized in that the product comprises: (i) Vibrio cholerae standard item, described standard item is selected from: polynucleotide described in claim 1 and DNA construct described in claim 2; And (ii) a pair of primers for specifically amplifying the Vibrio cholerae sequence, said primer pair being selected from the group consisting of: (1) A pair of primers composed of sequences shown in SEQ ID NO.:3 and SEQ ID NO.:4; (2) a pair of primers composed of sequences shown in SEQ ID NO.:5 and SEQ ID NO.:6; and (3) A pair of primers composed of the sequences shown in SEQ ID NO.:7 and SEQ ID NO.:8. 8. A preparation method of a plasmid standard molecule of Vibrio cholerae, characterized in that, comprising the following steps: ① Artificially synthesizing the virulence regulator toxR gene sequence of Vibrio cholerae, the expression gene sequence of the virulence regulator toxR is shown in SEQ ID NO: 1 in the sequence listing; ② Cloning the virulence regulator toxR expression gene sequence of Vibrio cholerae obtained in step ① into a cloning vector to obtain a plasmid standard molecule of Vibrio cholerae. 9. the preparation method of the plasmid standard molecule of vibrio cholerae as claimed in claim 8, is characterized in that, step 2. described cloning carrier is pUC19, pUC18, pUC118, pUC119, pBlueScript II SK or pGEM. 10. The preparation method of the plasmid standard molecule of Vibrio cholerae as claimed in claim 8, characterized in that, the cloning vector described in step 2. is pUC19.