CN117051142A - Brucella TaqMan real-time fluorescent quantitative RT-PCR detection primer and application thereof - Google Patents
Brucella TaqMan real-time fluorescent quantitative RT-PCR detection primer and application thereof Download PDFInfo
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Abstract
The invention discloses a Brucella TaqMan real-time fluorescence quantitative RT-PCR detection primer and application thereof, and relates to the technical field of bacterial detection. The detection limit of the gene copy number of the TaqMan real-time fluorescence quantitative PCR method established by using the primer and the probe is 3.8 copies, and the TaqMan real-time fluorescence quantitative PCR method does not cross react with other clinical isolated strains. The method has the advantages of strong specificity, high sensitivity, good repeatability, rapid detection, simple and convenient operation, low cost and the like, can be used for rapid identification of brucella and absolute quantitative detection in tissue samples infected by the brucella, and provides a reliable method for high-throughput rapid detection of brucella infection.
Description
Technical Field
The invention relates to the technical field of bacterial detection, in particular to a Brucella TaqMan real-time fluorescent quantitative RT-PCR detection primer and application thereof.
Background
Brucellosis, also known as Malta fever or wavy fever, is a zoonosis caused by brucellosis in which various animals, mainly domestic animals, are the infectious sources of each other. Brucella can infect humans, various domestic animals and wild animals, causing similar clinical symptoms and pathological lesions such as fever, abortion and infertility, chronic arthritis and nerve damage. Thus causing a great economic loss and causing serious public health problems. The literature reports that about 50 tens of thousands of human brucellosis cases are reported annually worldwide, with an economic loss of about $30 billion per year due to the disease.
The rapid and accurate identification of brucella is critical to effective control and prevention of disease. Currently, etiology examination, i.e., bacterial isolation and culture, relies on the ability of bacteria to grow and multiply on laboratory media and form visible colonies, and the method is reliable, low cost, and is a "gold standard" for diagnosing brucella. However, the above methods are very time consuming, require up to one week to confirm, and require specialized equipment and expertise, nor are serological tests very specific in areas where the disease is highly prevalent. Thus, there is a need for a rapid, accurate method of identifying brucella that overcomes the limitations of culture-based assays.
With the development of technology, brucella can be clearly distinguished from each other by highly differentiated molecular techniques such as multiplex polymerase chain reaction, multi-site sequencing typing or multiple site variable number tandem repeat analysis. TaqMan PCR is a widely used method for nucleic acid detection and quantification. The method is based on the use of specific primers and probes, binding to the target DNA sequence and generating a fluorescent signal during the amplification process. Compared with the real-time fluorescent quantitative PCR directly added with fluorescent dye, the specificity is more excellent, and the method has the potential of developing high-flux and multiple gene detection. TaqMan PCR has been used to detect a variety of pathogenic bacteria such as Brucella. The research aims to establish a TaqMan fluorescent PCR detection method with high sensitivity and high specificity, which is used for early detection and screening of Brucella infection, so as to eliminate potential pathogenic risks in time and ensure public health safety.
Disclosure of Invention
The invention aims to establish a fluorescent quantitative PCR method for rapidly and accurately detecting Brucella infection, and utilizes Brucella IS711 gene sequence to design a specific primer and a probe, and evaluates the specificity and sensitivity of the method so as to solve the problems of complex operation, long time consumption, low sensitivity and the like of the existing Brucella detection technology.
In order to solve the technical problems, the invention adopts the following technical scheme:
the first object of the invention is to provide a primer and a probe for real-time fluorescent quantitative PCR of Brucella specificity TaqMan.
The primers comprise an upstream primer IS711-F and a downstream primer IS711-R, and the Probe IS IS711-Probe. The sequence of the upstream primer IS711-F IS: 5'-AAAGCGATTTCATGCGTCGTG-3'; the downstream primer IS711-R:5'-TTATCGAAAAGGATGGCAAGCG-3', the probe sequence is: IS711-Probe: the 5' -FAM-TCCGCCGGGTCCAGCCAATCCA-BHQ1-3' end of the probe is marked with a fluorescence report group FAM, and the 3' end of the probe is marked with a quenching group BHQ1.
The second object of the invention is to provide a real-time fluorescent quantitative PCR detection method for the specific TaqMan of the bacteria of the genus Mannheimia based on the primer and the probe of the invention, which comprises the following steps:
extracting bacterial genome DNA of a detection sample as a template;
carrying out real-time fluorescence quantitative PCR reaction by using the primer and the probe;
and after the reaction is finished, judging whether the Brucella exists in the detection sample according to the amplification curve and the Ct value of the detection sample.
Further:
(1) 20. Mu.L of optimal reaction system: premix Ex Taq (2X) 10. Mu.L, concentration of 10. Mu.M upstream and downstream primers each 0.5. Mu.L, concentration of 10. Mu.M probe 0.5. Mu.L, template 2. Mu.L, and 20. Mu.L of the system were supplemented with sterile water.
(2) Optimal reaction conditions: 95℃for 5sec, 55℃for 5sec (10 cycles), 95℃for 2min (1 cycle), 95℃for 5sec, 50℃for 30sec (40 cycles);
the standard curve equation is: y= -3.6945logx+35.997, r 2 ＝0.999 (1)
In formula (1): y is the Ct value of the amplified test sample, and X is the initial copy number of the DNA of the test sample.
Substituting the Ct value of the detection sample into the equation (1) to calculate the initial copy number of the Brucella DNA in the detection sample, and calculating the absolute content of the Brucella in the detection sample according to the quality of the detection sample used for extracting the DNA, thereby realizing the absolute quantitative detection of the Brucella in the detection sample.
(3) The qualitative detection result judgment standard is as follows: the amplification curve of the fluorescence quantitative PCR reaction of the detection sample is positive, which indicates that the Brucella DNA exists in the detection sample; a Ct value of greater than 35 for the test sample is considered negative, indicating the absence of Brucella DNA in the test sample.
The real-time fluorescent quantitative PCR detection method for the Brucella specificity TaqMan not only can be applied to rapid identification of the Brucella, but also can be used for absolute quantitative detection of the Brucella in a detection sample according to the cycle number Ct value and a standard curve equation of the detection sample.
The third object of the invention is to provide a method for identifying Brucella with high detection speed, wherein the method only needs 1 hour, and the total time is only 3-4 hours after RNA extraction and reverse transcription preparation; high throughput sample detection can be performed simultaneously.
By adopting the technical scheme, compared with the prior art, the invention has the following technical progress:
1. the invention provides a Brucella TaqMan real-time fluorescent quantitative RT-PCR detection primer and application thereof, which have the advantages of strong specificity, high sensitivity, good repeatability, quick detection, simple and convenient operation, low cost and the like, can be used for quick identification of Brucella and absolute quantitative detection in a Brucella infected tissue sample, and provides a reliable method for high-throughput quick detection of Brucella infection.
2. The invention provides a Brucella TaqMan real-time fluorescent quantitative RT-PCR detection primer and application thereof, the established TaqMan real-time fluorescent quantitative PCR detection method can carry out specific detection on Brucella genome, has no specific amplification curve on other bacteria, has a variation coefficient of 0.20-2.01% in batches and a variation coefficient of 0.71-2.57% in batches, has high repeatability, and can rapidly and accurately detect early infection of Brucella and carry out quantitative analysis.
Drawings
FIG. 1 is a schematic diagram of a standard curve of TaqMan real-time fluorescent quantitative PCR of the present invention;
FIG. 2 is a schematic diagram of the specificity evaluation result of TaqMan real-time fluorescence quantitative PCR of the present invention;
FIG. 3 is a schematic diagram of the reproducibility evaluation results of TaqMan real-time fluorescent quantitative PCR of the present invention;
FIG. 4 is a Wen chart showing the positive rate of Brucella detected at different parts of the same patient by TaqMan real-time fluorescent quantitative PCR.
Wherein: FIG. 1 a shows an amplification curve; b, recombinant plasmid standard curve; c, agarose gel electrophoresis result; FIG. 1c-M DL 600DNA Marker; 0:3.8X10 7 copies/μL；1:3.8×10 6 copies/μL；2:3.8×10 5 copies/μL；3:3.8×10 4 copies/μL；4:3.8×10 3 copies/μL；5:3.8×10 2 copies/μL；6:3.8×10 1 copies/μL；7:3.8×10 0 cobies/. Mu.L; 8, nuclease-free water.
FIG. 2 a shows amplification curves for positive plasmids, other clinical isolates and negative controls; b, agarose gel electrophoresis result; FIG. 2b-M DL 600DNA Marker; brucella (Brucella) is 0; 1, colibacillus; staphylococcus aureus; 3, streptococcus pneumoniae; 4, lactococcus lactis; gardnerella vaginalis; helicobacter pylori; 7, lactobacillus acidophilus; acinetobacter baumannii; 9, nuclease-free water.
FIG. 3 a shows amplification curves; b, recombinant plasmid standard curve; c, agarose gel electrophoresis result; FIG. 3c-M DL 600DNA Marker; 0:3.8X10 7 copies/μL；1:3.8×10 6 copies/μL；2:3.8×10 5 copies/μL；3:3.8×10 4 copies/μL；4:3.8×10 3 copies/μL；5:3.8×10 2 copies/μL；6:3.8×10 1 copies/μL；7:3.8×10 0 cobies/. Mu.L; 8, nuclease-free water.
Detailed Description
The invention is further illustrated by the following examples:
in a first aspect, the invention provides a Brucella TaqMan real-time fluorescent quantitative RT-PCR detection primer and a probe, wherein the primer is as follows: f:5'-AAAGCGATTTCATGCGTCGTG-3' and R:5'-TTATCGAAAAGGATGGCAAGCG-3' the probe is P:5'-FAM-TCCGCCGGGTCCAGCCAATCCA-BHQ1-3'.
In a second aspect, the invention also provides a method for detecting the real-time fluorescent quantitative PCR of the specific TaqMan of the Brucella, wherein the primers and the probes used for the real-time fluorescent quantitative PCR detection of the TaqMan are as in claim 1; the detection method comprises the following steps:
step one, extracting bacterial genome DNA of a detection sample as a template;
step two, performing real-time fluorescence quantitative PCR reaction by using the primer and the probe;
and step three, judging whether Brucella exists in the detection sample according to the amplification curve and the Ct value of the detection sample after the reaction is finished.
Wherein the real-time fluorescent quantitative PCR reaction comprises:
the PCR reaction system is as follows: premix Ex Taq (2X) 10. Mu.L, concentration of 10. Mu.M upstream and downstream primers each 0.5. Mu.L, concentration of 10. Mu.M probe 0.5. Mu.L, template 2. Mu.L, and 20. Mu.L of sterile water make-up system;
the PCR reaction conditions were: 95℃for 5sec, 55℃for 5sec (10 cycles), 95℃for 2min (1 cycle), 95℃for 5sec, 50℃for 30sec (40 cycles);
the standard curve equation is: y= -3.6945logx+35.997, r 2 ＝0.999 (1)
In formula (1): y is the Ct value of the amplified test sample, and X is the initial copy number of the DNA of the test sample.
In addition, the Ct value of the detection sample is substituted into the equation (1) to calculate the initial copy number of the Brucella DNA in the detection sample, and then the absolute content of the Brucella in the detection sample is calculated according to the mass of the detection sample used for extracting the DNA, so that the absolute quantitative detection of the Brucella in the detection sample is realized.
Wherein, judging whether the bacteria of the genus mannheimia exist in the detection sample belongs to qualitative detection, and judging standards of qualitative detection results are as follows: the amplification curve of the fluorescence quantitative PCR reaction of the detection sample is positive, which indicates that the Brucella DNA exists in the detection sample; a Ct value of greater than 35 for the test sample is considered negative, indicating the absence of Brucella DNA in the test sample.
Example 1 primer design and Synthesis
The method specifically comprises the following steps:
the sequences of the 16S gene (NR_ 043003.1) and IS711 genes (NC_ 003317.1) of Brucella were searched from GenBank, primers for 16S gene identification and primers and TaqMan probes for quantitative qPCR detection were designed using Beacon designer7 (PREMIER Biosoft International, palo Alto, CA, USA) software, the 5 'end of the probes were labeled with FAM fluorescent groups, the 3' end was labeled with BHQ quenching groups, and the primers and probes were synthesized by Shanghai Biotechnology Co Ltd, the primers were: f:5'-AAAGCGATTTCATGCGTCGTG-3' and R:5'-TTATCGAAAAGGATGGCAAGCG-3', the probe is P:5' -FAM-TCCGCCGGGTCCAGCCAATCCA-BHQ 1-3.
Example 2 optimization of TaqMan real-time fluorescent quantitative PCR method and establishment of Standard Curve
The method specifically comprises the following steps:
1. strain
After the blood enrichment is adopted in a microorganism room of a fourth people hospital in Ningxia, the blood agar plate is subjected to split-purity culture to obtain a pure Brucella strain, and the pure Brucella strain is identified as Brucella by a Mei Liai full-automatic bacteria identifier VITEK-2 (Mei Liai biological diagnostic company, france).
2. Extraction and reverse transcription of bacterial genomic DNA
Brucella is prepared into bacterial liquid with turbidity OD value of 2.0, inactivated in a water bath at 80 ℃ for 15 minutes, and Brucella DNA is extracted by using a fully automatic nucleic acid extractor SSNP-2000B and a matched reagent (Jiangsu Shuo Biotech Co., ltd.) and detected by using a Nanodrop100 ultra-micro spectrophotometer (Thermo Scientific, USA).
3. Construction of Standard plasmid
The preparation method comprises the steps of taking pUC19 as a skeleton vector, amplifying an IS711 gene of Brucella with high-fidelity enzyme, purifying a PCR product, connecting an IS711 gene fragment to the skeleton vector, transferring into escherichia coli DH5a for amplification culture and identification, extracting recombinant positive plasmid DNA, quantifying by using an ultra-micro spectrophotometer, and calculating the copy number of the recombinant plasmid by using the following formula:
TaqMan real-time fluorescence quantitative PCR reaction system
Full-length amplification of 16S gene 50uL system was used: usingGXL DNA Polymerase (Takiday doctor technologies Co., ltd., japan), 5× PrimeSTAR GXL Buffer 10uL,dNTP Mixture 4uL, 1.0uL each of the upstream and downstream primers 10uM, and the template was inactivated bacteria solution 2uL,PrimeSTAR GXL DNA Polymerase 1.0uL, and after the completion of the sample addition, the mixture was homogenized at 98℃for 2min (1 cycle), 98℃for 10sec 56℃for 15sec 72℃for 1min30sec (35 cycles), 72℃for 5min (1 cycle), and the 5uLPCR product was detected by 1.2% agarose gel electrophoresis and sequenced.
qPCR assay used a 20uL system: fluorescent quantitative PCR was performed using ABI7500 fluorescent quantitative detection System (Sesameiser technology, USA), and Premix Ex Taq was used as a quantitative reagent TM (Probe qPCR) (Takara doctor materials technology Co., ltd., japan). The reaction system is as follows: premix Ex Taq (2X) 10. Mu.L, upstream and downstream primer 10. Mu.M 0.5. Mu.L, probe 10. Mu.M 0.5. Mu.L, template 2. Mu.L, and sterile water make up system 20. Mu.L. The qPCR procedure was run as follows: fluorescence was collected at 95℃for 5sec 55℃for 5sec (10 cycles), 95℃for 2min (1 cycle), 95℃for 5sec 50℃for 30sec (40 cycles), and Ct value of more than 35 was regarded as negative.
5. Establishment of a Standard Curve
Serial dilutions of standard plasmids were performed at 10-fold ratio with TE buffer at 3.8×10 7 Up to 3.8X10 0 Plasmid DNA was used as a template to establish a standard curve for the IS711 gene (as shown in FIG. 1). The results show that the real-time fluorescence quantitative PCR marked by the probe can successfully detect the copy number of 3.8X10 0 And the standard curve has good linearity, the correlation coefficient R 2 Standard curve equation y= -3.6945log x+35.997, where Y is Ct and X is the initial copy number of the test sample DNA, indicating that the primer and RT-qPCR system can detect brucella efficiently and reliably.
6. Detection result judgment standard
In qualitative detection: the appearance of an amplification curve (the peak of the curve) is positive, which indicates that the Brucella DNA exists in the detection sample; no amplification curve (curve does not peak, approximately horizontal straight line) or Ct value greater than 35 is negative, indicating no presence of brucella DNA in the test sample.
In the relative quantitative detection: the smaller the Ct value of the test sample, the higher the concentration (or content) of Brucella DNA in the test sample.
In absolute quantitative detection: substituting the Ct value of the detection sample into a standard curve equation Y= -3.6945log X+35.997, wherein Y is Ct, X is the initial copy number of the DNA of the detection sample, and then calculating the copy number concentration of the DNA of the Brucella in the DNA sample, and calculating the absolute content of the Brucella in the detection sample according to the quality of the detection sample used for extracting the DNA.
Example 3 evaluation of Performance of TaqMan real-time fluorescent quantitative PCR method
The method specifically comprises the following steps:
1. specificity evaluation
Standard plasmid DNA and laboratory stored inactivated strain DNA, including Escherichia coli, staphylococcus aureus, streptococcus pneumoniae, lactococcus lactis, gardnerella vaginalis, helicobacter pylori, lactobacillus acidophilus and Acinetobacter baumannii are used as amplified templates, and water without nuclease is used as a negative control. The results showed that positive plasmid bacteria detected positive, while the other 8 clinically pathogenic bacteria had no amplified signal, which was consistent with the negative control (as shown in fig. 2), thus demonstrating the high specificity of the primers and probes in the RT-qPCR system.
2. Sensitivity evaluation
Serial dilutions of standard plasmids were made with TE buffer at 10-fold ratio at 3.8X10 7 Up to 3.8X10 -1 Plasmid DNA was detected using 1.0uL of each as a template, and nuclease-free water as a template was used as a negative control. The results showed 3.8X10 0 CT value of 34.65,3.8 ×10 -1 No amplified signal was shown, consistent with the negative control (as shown in table 1). The lowest detection line of the RT-qPCR system is 3.8 copies.
TABLE 1 sensitivity evaluation results of TaqMan real-time fluorescent quantitative PCR
3. Repeatability evaluation
Serial dilutions of standard plasmids were made with TE buffer at 10-fold ratio at 3.8X10 7 Up to 3.8X10 0 Plasmid DNA was used as a template for detecting the coefficient of variation of real-time PCR, and nuclease-free water was used as a template as a negative control. For intra-batch reproducibility, all samples were replicated three times. To determine the reproducibility between batches, the measurements were repeated three times at different locations, respectively (as shown in FIG. 3). The results show that the intra-batch variation coefficient of the reaction system is 0.20-1.99%, and the inter-batch variation coefficient is 0.71-2.57% (shown in Table 2), which shows that the RT-qPCR system has good repeatability and high stability.
TABLE 2 analysis of Ct values for reproducibility evaluation of TaqMan real-time fluorescent quantitative PCR
Example 4 application of TaqMan real-time fluorescent quantitative PCR method in rapid identification of Brucella
The method specifically comprises the following steps:
1. extraction of strains and genomic DNA
180 samples were taken from different departments of the Ningxia fourth people hospital, 60 samples of blood, urine and faeces each, between 2019 and 2022. After all samples are subjected to inactivation treatment, centrifuging at 1500 Xg and 4 ℃ for 20 minutes, collecting precipitate, adding 5mL of lysate into the precipitate, extracting genome DNA by vortex oscillation, centrifuging 1mL of supernatant at 8000g, taking the supernatant as a template, and preserving at-20 ℃ for later use.
TaqMan real-time fluorescent quantitative PCR (polymerase chain reaction) rapid identification of Brucella
The total of 180 blood, urine and fecal samples of 60 suspected Brucella infected patients were collected clinically and analyzed using the probe real-time fluorescence quantification system described above. The results showed that the blood and fecal samples had the highest detection positive rates of 76.67% and 51.67%, respectively, the urine detection rate of 40.00%, and the brucella positive from 180 total samples was 101 and the detection rate was 56.11% (as shown in table 3).
TABLE 3 Positive Rate detected in clinical samples
The ratio of simultaneous detection of Brucella positives was 18.33%, the ratio of simultaneous detection of urine and blood positives was 36.67%, the ratio of simultaneous detection of blood and feces positives was 41.67%, and the ratio of simultaneous detection of urine and feces positives was 18.33% among three samples (urine, blood and feces) of the same suspected infected person (as shown in FIG. 4).
In summary, the invention aims to establish a fluorescent quantitative PCR method for rapidly and accurately detecting Brucella infection, designs a specific primer and a probe by using Brucella IS711 gene sequence, and evaluates the specificity and sensitivity of the method. The standard curve of the established TaqMan real-time fluorescence quantitative PCR detection method is Y= -3.6945log X+35.997 (R2=0.999, Y is Ct value of amplified detection sample, X is detection sample)Initial copy number of product DNA) at standard concentration of 3.8X10 7 copies/μL-3.8×10 0 The method has good linear relation in the range of copies/mu L, the initial copy number of DNA of the bacteria of the genus Manchurian in the detection sample can be calculated by substituting the Ct value of the detection sample into an equation, and the absolute content of Brucella in the detection sample can be calculated according to the mass (unit is g) of the detection sample used for extracting the DNA, so that the absolute quantitative detection of the Brucella in the detection sample is realized. The established TaqMan real-time fluorescent quantitative PCR detection method can carry out specific detection on the Brucella genome and has no specific amplification curve on other bacteria. The variation coefficient in the batch is 0.20-2.01%, the variation coefficient between batches is 0.71-2.57%, and the repeatability is high. The TaqMan real-time fluorescent quantitative PCR detection method is applied to 180 collected blood, urine and feces samples. The results show that the copy number of blood and fecal samples is higher than that of urine samples during the same time period. The fluorescent quantitative PCR detection method established by the invention can rapidly and accurately detect early infection of brucella and carry out quantitative analysis.
The foregoing invention has been generally described in great detail, but it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, it is intended to cover modifications or improvements within the spirit of the inventive concepts.
Claims (5)
1. A Brucella TaqMan real-time fluorescent quantitative RT-PCR detection primer and probe are characterized in that: the primer is as follows: f:5'-AAAGCGATTTCATGCGTCGTG-3' and R:5'-TTATCGAAAAGGATGGCAAGCG-3', the probe is P:5'-FAM-TCCGCCGGGTCCAGCCAATCCA-BHQ1-3'.
2. A real-time fluorescent quantitative PCR detection method for Brucella specificity TaqMan is characterized in that: the primers and the probes used for the TaqMan real-time fluorescence quantitative PCR detection are as described in claim 1; the detection method comprises the following steps:
step one, extracting bacterial genome DNA of a detection sample as a template;
step two, performing real-time fluorescence quantitative PCR reaction by using the primer and the probe;
and step three, judging whether Brucella exists in the detection sample according to the amplification curve and the Ct value of the detection sample after the reaction is finished.
3. The method for real-time fluorescent quantitative PCR detection of Brucella specificity TaqMan according to claim 2, wherein the method comprises the following steps of: the real-time fluorescent quantitative PCR reaction comprises the following steps:
the PCR reaction system is as follows: premix Ex Taq (2X) 10. Mu.L, concentration of 10. Mu.M upstream and downstream primers each 0.5. Mu.L, concentration of 10. Mu.M probe 0.5. Mu.L, template 2. Mu.L, and 20. Mu.L of sterile water make-up system;
the PCR reaction conditions were: 95℃for 5sec, 55℃for 5sec (10 cycles), 95℃for 2min (1 cycle), 95℃for 5sec, 50℃for 30sec (40 cycles);
the standard curve equation is: y= -3.6945logx+35.997, r 2 ＝0.999(1)
In formula (1): y is the Ct value of the amplified test sample, and X is the initial copy number of the DNA of the test sample.
4. The method for real-time fluorescent quantitative PCR detection of Brucella specificity TaqMan according to claim 3, wherein the method comprises the following steps of: substituting the Ct value of the detection sample into the equation (1) to calculate the initial copy number of the Brucella DNA in the detection sample, and calculating the absolute content of the Brucella in the detection sample according to the quality of the detection sample used for extracting the DNA, thereby realizing the absolute quantitative detection of the Brucella in the detection sample.
5. The method for real-time fluorescent quantitative PCR detection of Brucella specificity TaqMan according to claim 2, wherein the method comprises the following steps of: the judging and detecting whether the bacteria of the genus mannheimia exist in the sample belongs to qualitative detection, and the judging standard of the qualitative detection result is as follows: the amplification curve of the fluorescence quantitative PCR reaction of the detection sample is positive, which indicates that the Brucella DNA exists in the detection sample; a Ct value of greater than 35 for the test sample is considered negative, indicating the absence of Brucella DNA in the test sample.
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