CN111705147A - Improved method for detecting Brucella nucleic acid DNA by fluorescence quantitative PCR - Google Patents
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Abstract
An improved method for detecting Brucella nucleic acid DNA in a specimen by fluorescence quantitative PCR relates to the technical field of molecular biology. According to the technology, the fluorescence quantitative PCR is combined with the ordinary PCR to detect the brucella nucleic acid DNA in the blood sample or other samples, the sensitivity is far higher than that of the ordinary PCR and a single-weight fluorescence quantitative PCR method, the copy number of the brucella nucleic acid DNA can be detected, the result is obvious, the operation is simple, and the detection can be completed by people with the ordinary PCR and fluorescence quantitative PCR technologies.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a PCR (polymerase chain reaction) technical method for brucella nucleic acid DNA.
Background
Brucellosis (Brucellosis), also known as geothermy moderate-sea atony, malachite fever or wave heat, is commonly called as 'lazy-han disease', is a zoonotic infectious-allergic disease caused by intracellular parasitic Brucella bacteria, and is widely distributed around the world.
At present, over 60 kinds of domestic animals, domestic fowls and wild animals are known as hosts of Brucella. The human-related sources of infection are mainly sheep, cattle and pigs, and secondly dogs. The secretion, excretion, flow product and milk of the sick livestock contain a large amount of germs, which are spread among animals to cause bacteria carrying or disease attack. Humans are infected with brucella via transmission modes such as exposure to brucella infected animals, consumption of milk products contaminated with brucella, and laboratory exposure.
The brucella is invaded into the body, reaches lymph nodes along with lymph fluid, and is phagocytized by phagocytes. If the bacteria are not killed by phagocytes, they grow and reproduce in cells to form local primary focus. The incubation period is 7-60 days, the average 15 days, and a few patients can reach months or more than 1 year. The mass propagation of bacteria in phagocytes leads to the rupture of phagocytes, and then a large amount of bacteria enter lymph and blood circulation to form bacteremia. Multiple lesions are formed in liver, spleen, lymph nodes, bone marrow and the like, and cause clinical manifestations of bacteremia, septicemia and toxemia. After a certain period of time, the bacteria of the infected focus grow and reproduce to enter the blood again, resulting in the recurrence of the disease. This is repeated as a chronic infection, which is characterized by chronic invasion of the spine and joints, with symptoms of long-term fever (including low grade fever), hyperhidrosis, arthralgia, or enlargement of the liver, spleen, lymph nodes and testis.
The conventional detection method for Brucella mainly comprises a culture method and immunological methods such as tiger red plate agglutination test (RBPT), Serum Agglutination Test (SAT), Complement Fixation Test (CFT), enzyme-linked immunosorbent assay (ELISA), and the like. The brucella isolated in the culture is still the 'gold standard' for brucellosis diagnosis, but the sensitivity of the brucella isolation culture is low, the time and the labor are consumed, and the biological safety hazard is larger. Immunological methods are currently used in brucellosis monitoring and epidemiological investigation studies, but false negative results are obtained because the body does not produce related antibodies or the produced antibodies do not reach the limit of detection in the early stage of infection; and the brucella and certain bacteria have cross reaction, so that a false positive result exists, and the specificity of detection is influenced.
With the development of molecular biology techniques, PCR methods (including general PCR and fluorescent quantitative PCR) have been used in the aided diagnosis of brucellosis and the detection of pathogens. For trace nucleic acid DNA in a sample to be detected, common PCR and multiplex PCR methods of a single primer are difficult to detect; (ii) a The fluorescent quantitative PCR technology is a nucleic acid qualitative and quantitative detection technology integrating PCR technology, fluorescent signal detection and data analysis. The method has the characteristics of high specificity, sensitivity, accuracy, low false positive rate and the like on pure bacterium DNA, and can detect nucleic acid DNA with 2 copy numbers, but in the current fluorescent quantitative PCR technology, due to numerous influencing factors in samples to be detected such as blood, the false negative rate is high, a correct detection result cannot be made, and the application of the method in brucella nucleic acid DNA detection is influenced.
The PCR technology of the invention uses a fluorescence quantitative PCR method to detect the brucella nucleic acid DNA in a sample such as a blood sample or other samples by combining a common PCR technical method, and the minimum detection limit is 1 brucella nucleic acid DNA copy number.
Disclosure of Invention
The invention aims to provide an improved fluorescent quantitative PCR method for detecting brucella nucleic acid DNA. The method of the invention is useful for non-disease diagnosis.
In order to achieve the purpose, the invention provides the following technical scheme:
an improved fluorescent quantitative PCR method for detecting brucella nucleic acid DNA comprises the following steps:
(1) various methods are available for extracting nucleic acid DNA of a sample to be detected, such as extraction of nucleic acid DNA of a sample.
(2) Designing a primer: the application software is used for respectively designing specific primers and probes of the Brucella, and the specific nucleotide sequences of the primers and the probes comprise the following sequences:
first common PCR amplification primers:
a forward primer: f1: 5'-CGAGATGGACGAAACCCATGAAT-3', SEQ ID NO: 1;
reverse primer: r1: 5'-AGTGGCGTTGATAACCGATTATTT-3', SEQ ID NO: 2;
primers and probes for the second fluorescent quantitative PCR amplification:
a forward primer: f2: 5'-GACAACAGCATGCAGCTTGGTCGTC-3', SEQ ID NO: 3;
reverse primer: r2: 5'-GCACCATATCGAAAGTCCACGCAGAT-3', SEQ ID NO: 4;
and (3) probe: probe: 5'-TCCACCGCGCGAGCGACCGATGCAGGCAGCTT-3', SEQ ID NO: 5
(3) Taking the nucleic acid DNA extracted in the step (1) as a template, and carrying out primary amplification on the template nucleic acid DNA through a common PCR reaction by using primers shown in SEQ ID NO. 1-2 to obtain a primary common PCR product; performing a second amplification of the DNA of the first PCR product by a fluorescent quantitative PCR reaction by using primers and probes shown in SEQ ID NO. 3-5;
(4) analyzing the result; after the fluorescent quantitative PCR reaction is finished, if the positive control and the negative control are established, the analysis of the detection specimen can be carried out, the CT value is less than 28, and the brucella nucleic acid DNA exists in the detection specimen.
As a further preferable scheme of the invention: the PCR reaction comprises two times of PCR, a first common PCR and a second fluorescent quantitative PCR;
as a further preferable scheme of the invention: the template nucleic acid DNA in the first common PCR system is the nucleic acid DNA directly extracted from the sample to be detected;
as a preferred embodiment of the present invention: the nucleic acid DNA to be detected in the second fluorescence quantitative PCR system is the nucleic acid DNA of the first PCR reaction product;
the PCR reaction system as a preferred embodiment of the present invention comprises: PCR reaction enzyme compound, primer, probe, double distilled water and template nucleic acid DNA to be detected.
As a preferred embodiment of the present invention: the common PCR method comprises the following steps: common PCR reaction enzyme complex 12.5 μ L (2. x. Ex Taq mastermix, kang century Biotechnology Co., Ltd.), forward primer F1 and reverse primer R1 each 0.4 μ L, template nucleic acid DNA 4 μ L, supplementary double distilled water to 25 μ L volume;
as a preferred embodiment of the present invention: the second fluorescent quantitative PCR reaction system comprises: fluorescent quantitative PCR enzyme complex 10 μ L (Premix Ex Taq TM (Probe qPCR, TaKaRa)), forward primer F2, reverse primer R2 and Probe Probe solution 0.4 μ L each, taking the first PCR amplification product DNA 2 μ L as template, supplementing double distilled water to 20 μ L volume;
as a further preferred embodiment of the present invention, the concentrations of the primer and probe solutions in the two PCR reaction systems are both 10. mu. mol/L.
As a further preferable scheme of the invention: the PCR method has the following conditions of the first common PCR reaction: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, 30 cycles; extension at 72 ℃ for 5 min.
As a further preferable scheme of the invention: the conditions of the second fluorescent quantitative PCR reaction of the fluorescent quantitative PCR method are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 5sec, annealing at 60 ℃ for 20sec, 40 cycles; fluorescence signals were collected during annealing.
Compared with the prior art, the invention has the beneficial effects that: can detect brucella micro nucleic acid DNA in a sample to be detected, such as blood and the like. The sensitivity is high, and the Brucella nucleic acid DNA can be detected as long as the PCR reaction system contains 1 copy of Brucella nucleic acid DNA; meanwhile, the result analysis can be standardized, the test operation is simple, and the personnel with common PCR and fluorescent quantitative PCR technologies can complete the analysis.
Drawings
FIG. 1 shows the result of the fluorescence quantitative PCR amplification directly performed on DNA extracted from the specimen according to the present invention;
FIG. 2 shows the result of fluorescent quantitative PCR amplification of 4. mu.L of DNA template extracted from the specimen of the present invention after the first ordinary PCR amplification, and 2. mu.L of the first ordinary PCR amplification product.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments, and are not to be taken as limitations of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
In the embodiment of the invention, an improved method for detecting brucella nucleic acid DNA in a blood sample by fluorescence quantitative PCR comprises the following steps:
fluorescent quantitative PCR enzyme complex: premix Ex Taq TM (Probe qPCR, TaKaRa). General PCR enzyme complex: 2 Ex Taq mastermix, kang, century biotechnology limited.
Primers and probes specific to Brucella are designed according to the gene sequences of Transposase orfA and Transposase orfB registered in GenBank by using software such as Primer 5.0 software.
The first common PCR amplification primer comprises:
a forward primer: f1: 5'-CGAGATGGACGAAACCCATGAAT-3', SEQ ID NO: 1;
reverse primer: r1: 5'-AGTGGCGTTGATAACCGATTATTT-3', SEQ ID NO: 2;
the primer and the probe for the second time of fluorescent quantitative PCR amplification comprise:
a forward primer: f2: 5'-GACAACAGCATGCAGCTTGGTCGTC-3', SEQ ID NO: 3;
reverse primer: r2: 5'-GCACCATATCGAAAGTCCACGCAGAT-3', SEQ ID NO: 4;
a probe T: 5'-TCCACCGCGCGAGCGACCGATGCAGGCAGCTT-3', SEQ ID NO: 5
Referring to fig. 1-2, the present invention is tested by the following steps:
1. primer specificity verification
1) The forward primer F1 and the reverse primer R1 are used for amplifying a nucleic acid DNA template of bacillus cereus, bacillus anthracis, escherichia coli O:157, salmonella, pseudomonas aeruginosa, yersinia enterocolitica O:9 serotype and vibrio cholerae N330 by common PCR.
2) The forward primer F2, the reverse primer R2 and the Probe Probe are used for amplifying a nucleic acid DNA template of bacillus cereus, bacillus anthracis, escherichia coli O157, salmonella, pseudomonas aeruginosa, yersinia enterocolitica O9 serotype and vibrio cholerae N330 by fluorescent quantitative PCR.
3) Amplifying a nucleic acid DNA template of bacillus cereus, bacillus anthracis, escherichia coli O:157, salmonella, pseudomonas aeruginosa, yersinia enterocolitica O:9 serotype and vibrio cholerae N330 by using a forward primer F1 and a reverse primer R1 for the first time; the forward primer F2, the reverse primer R2 and the Probe are applied for the second time to amplify the template of the first PCR amplification product.
4) The first conventional PCR reaction system consisted of 12.5. mu.L of conventional PCR enzyme complex, 0.4. mu.L (10. mu. mol/L) of each of forward primer F1 and reverse primer R1, 4. mu.L of nucleic acid DNA template, and double distilled water to a volume of 25. mu.L. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, 30 cycles: extension at 72 ℃ for 5 min.
(1) The second time fluorescent quantitative PCR reaction system comprises 10 mu L of fluorescent quantitative PCR enzyme compound, 0.4 mu L of each of forward primer F2 and reverse primer R2, 0.4 mu L of Probe Probe, 2 mu L of nucleic acid DNA of the first time PCR amplification product as a template, and double distilled water added to the volume of 20 mu L. Carrying out fluorescent quantitative PCR reaction conditions, and carrying out pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 5sec, annealing at 60 ℃ for 20sec, 40 cycles; fluorescence signals were collected during annealing.
2. Primer sensitivity verification
1) Carrying out first common PCR amplification, wherein a forward primer F1 and a reverse primer R1 are adopted, the template is Brucella melitensis strain 1 type strain 16M nucleic acid DNA, the concentration of the nucleic acid DNA diluted by times is respectively 3.350 fg/mu L,1.675 fg/mu L,0.837 fg/mu L,0.419 fg/mu L,0.209 fg/mu L,0.105 fg/mu L,0.053 fg/mu L,0.0265 fg/mu L and other 8 groups, and 1 mu L of Brucella melitensis nucleic acid DNA template is taken;
2) and performing second fluorescent quantitative PCR amplification by using a forward primer F2, a reverse primer R2 and a Probe, wherein the DNA template of the second fluorescent quantitative PCR amplification product is 2 mu L of the first PCR amplification product.
3) Reaction system and reaction conditions:
(1) a typical PCR reaction system included 12.5. mu.L of a typical PCR enzyme complex, 0.4. mu.L (10. mu. mol/L) of each of the forward and reverse primers, 1. mu.L of the nucleic acid DNA template, and a volume of 25. mu.L supplemented with double distilled water. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, 30 cycles; extension at 72 ℃ for 5 min.
(2) And performing secondary fluorescent quantitative PCR amplification, wherein 10 mu L of fluorescent quantitative PCR enzyme complex, 0.4 mu L of each of forward primer F2 and reverse primer R2, 0.4 mu L of Probe Probe, 2 mu L of nucleic acid DNA of a primary PCR amplification product as a template, and supplementing double distilled water to 20 mu L of volume. Carrying out fluorescent quantitative PCR reaction conditions, and carrying out pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 5sec, annealing at 60 ℃ for 20sec, 40 cycles; fluorescence signals were collected during annealing.
3. Results
1) Primer specificity: the PCR primers designed by the invention have higher specificity, and the non-Brucella bacteria are as follows: the strains such as bacillus cereus, bacillus anthracis, escherichia coli O157, salmonella, pseudomonas aeruginosa, yersinia enterocolitica O9 serotype, vibrio cholerae N330 and the like have no specific electrophoresis bands.
2) Sensitivity of the primers: the improved fluorescence quantitative PCR method designed by the invention has higher sensitivity. The PCR reaction system can detect the Brucella as long as 1 copy number of the Brucella nucleic acid DNA is available.
3) PCR detection result of nucleic acid DNA extracted from blood sample:
the invention has no electrophoretic band amplified by common PCR for nucleic acid DNA extracted from 96 blood samples, and diagnosis can not be made by using the result of the fluorescent quantitative PCR method singly, as shown in figure 1, the nucleic acid DNA to be detected is amplified by the common PCR method for the first time and then is subjected to the fluorescent quantitative PCR method, and the result is obvious and is easy to be interpreted, as shown in figure 2. The samples made in FIG. 1 and FIG. 2 are the same batch of samples.
4. Analysis of
The established method for detecting brucella nucleic acid DNA in the blood sample by using the improved fluorescent quantitative PCR has strong specificity, and other strains do not have specific amplification electrophoresis bands except the brucella nucleic acid DNA; the sensitivity is high, and only one copy number of the nucleic acid DNA can be detected. The invention is not only superior to the single PCR method, but also has sensitivity far higher than the single fluorescent quantitative PCR detection level.
The blood can be replaced by other liquid to be detected. Or even the liquid to be tested can be a method for detecting the infectivity of germs of blood products such as blood medicines and the like.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
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gcacc atatc gaaag tccac gcaga t
<210>5
<211>32
<212>DNA
<213> "Artificial sequence"
<400>5
tccac cgcgc gagcg accga tgcag gcagc tt
Claims (9)
1. An improved method for detecting brucella nucleic acid DNA in a specimen by fluorescence quantitative PCR is characterized by comprising the following steps:
(1) extracting nucleic acid DNA of a sample to be detected;
(2) designing a primer: the specific primers and probes of the Brucella are respectively designed by application software, and the specific nucleotide sequences comprise the following sequences:
first PCR amplification primer:
a forward primer: f1: 5'-CGAGATGGACGAAACCCATGAAT-3', SEQ ID NO: 1;
reverse primer: r1: 5'-AGTGGCGTTGATAACCGATTATTT-3', SEQ ID NO: 2;
primers and probes for the second fluorescent quantitative PCR amplification:
a forward primer: f2: 5'-GACAACAGCATGCAGCTTGGTCGTC-3', SEQ ID NO: 3;
reverse primer: r2: 5'-GCACCATATCGAAAGTCCACGCAGAT-3', SEQ ID NO: 4;
and (3) probe: probe: 5'-TCCACCGCGCGAGCGACCGATGCAGGCAGCTT-3', SEQ ID NO: 5
The probe sequence is as follows: probe: SEQ ID NO: 5, wherein, the 5 'end is marked by a fluorescent group FAM, and the 3' end is marked by a quenching group BHQ-1;
(3) taking the nucleic acid DNA extracted in the step (1) as a template, and carrying out first amplification on the nucleic acid DNA through PCR reaction by using primers shown in SEQ ID NO. 1-2 to obtain a first PCR product; performing second amplification by using primers and probes shown in SEQ ID NO. 3-5 through a fluorescent quantitative PCR reaction, wherein a template of the second amplification is a first PCR product;
(4) detecting and analyzing the PCR amplification product; only the result of the fluorescent quantitative PCR reaction needs to be analyzed, and after the reaction is finished, the positive control and the negative control are established so as to analyze the detection result of the sample; and as a result of fluorescent quantitative PCR, the CT value is less than 28, namely the brucella nucleic acid DNA exists in the sample.
2. The method for detecting brucella nucleic acid DNA in a specimen by improved fluorescence quantitative PCR according to claim 1, wherein the PCR amplification reaction: adding the nucleic acid DNA extracted from the blood sample into the PCR reaction mixed solution for PCR amplification; the PCR reaction system comprises: PCR reaction enzyme compound, primer, probe, double distilled water and nucleic acid DNA to be detected.
3. The method for detecting Brucella nucleic acid DNA in a specimen by improved fluorescence quantitative PCR according to claim 1,
the first PCR reaction system comprises: PCR enzyme complex 12.5. mu.L, forward primer F1 and reverse primer R1 each 0.4. mu.L, sample nucleic acid DNA to be detected 4. mu.L, and double distilled water was supplemented to a volume of 25. mu.L.
4. The improved method for detecting brucella nucleic acid DNA in a sample by fluorescence quantitative PCR as claimed in claim 1, wherein the second PCR reaction system comprises 10 μ L of fluorescence quantitative PCR enzyme complex, 0.4 μ L of each of forward primer F2 and reverse primer R2, 0.4 μ L of probe, 2 μ L of nucleic acid DNA as the first PCR amplification product, and double distilled water to 20 μ L volume.
5. The improved fluorescence quantitative PCR method for detecting Brucella nucleic acid DNA in specimen according to claim 3 or 4, wherein the concentration of all primer and probe solutions is 10 μmol/L.
6. The method for detecting brucella nucleic acid DNA in blood sample by improved fluorescence quantitative PCR as claimed in claim 1, wherein the PCR method, the conditions of the first PCR reaction are: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, 30 cycles; extension at 72 ℃ for 5 min.
7. The method for detecting brucella nucleic acid DNA in a specimen by using improved fluorescence quantitative PCR as claimed in claim 1, wherein the conditions of the PCR method and the second fluorescence quantitative PCR reaction are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 5sec, annealing at 60 ℃ for 20sec, 40 cycles; fluorescence signals were collected during annealing.
8. The improved method for detecting brucella nucleic acid DNA in a sample by fluorescence quantitative PCR as claimed in claim 1, wherein result analysis is carried out, positive control and negative control are established, and the Ct value of the fluorescence quantitative PCR of the sample to be detected is less than 28, so that the positive result can be judged.
9. The improved method for detecting brucella nucleic acid DNA in a specimen by fluorescence quantitative PCR as claimed in claim 3 or 4, wherein the fluorescence quantitative PCR enzyme complex: premix Ex Taq TM (Probe qPCR, TaKaRa). General PCR enzyme complex: 2 Ex Taq mastermix, kang, century biotechnology limited.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005021795A1 (en) * | 2002-08-01 | 2005-03-10 | The Regents Of The University Of California | Nucleotide sequences specific to brucella and methods for the detection of brucella |
| CN109306372A (en) * | 2018-09-14 | 2019-02-05 | 中国疾病预防控制中心传染病预防控制所 | A kind of method nest-type PRC detection or/and identify brucella |
-
2019
- 2019-10-09 CN CN201910955250.3A patent/CN111705147A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005021795A1 (en) * | 2002-08-01 | 2005-03-10 | The Regents Of The University Of California | Nucleotide sequences specific to brucella and methods for the detection of brucella |
| CN109306372A (en) * | 2018-09-14 | 2019-02-05 | 中国疾病预防控制中心传染病预防控制所 | A kind of method nest-type PRC detection or/and identify brucella |
Non-Patent Citations (4)
| Title |
|---|
| 崔玉花等: "基于样品检测的布鲁氏菌荧光定量PCR方法的建立", 《中国人兽共患病学报》 * |
| 张宏霞等: "实时荧光定量PCR检测布鲁杆菌方法的应用", 《中国地方病学杂志》 * |
| 聂丹文等: "巢式聚合酶链反应结合Ｒeal-time 聚合酶链反应用于检测", 《中国疫苗和免疫》 * |
| 高正琴等: "TaqMan MGB探针实时荧光定量PCR快速检测布鲁氏菌", 《中国人兽共患病学报》 * |
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