CN112301150A - Method for detecting respiratory viruses by direct fluorescence PCR - Google Patents
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Abstract
The invention provides a method for qualitatively detecting respiratory viruses by direct fluorescence polymerase chain reaction (fluorescence PCR), wherein a template used for the fluorescence PCR amplification is a clinically collected specimen containing the respiratory viruses. Belongs to the field of biotechnology and medicine. The method of the invention skips the process of sample nucleic acid treatment, so that the whole detection process is smooth and rapid, the chance of mutual pollution is reduced, and the obtained PCR detection result is accurate and reliable.
Description
The technical field is as follows:
the invention relates to the fields of biotechnology and medicine, in particular to a fluorescence PCR detection method of respiratory virus nucleic acid, which has the characteristics of safety, rapidness, sensitivity and accuracy.
Background art:
the fluorescence polymerase chain reaction (fluorescence PCR) is characterized in that a PCR product is labeled and tracked through a fluorescent dye or a fluorescence-labeled specific probe, the reaction process is monitored on line in real time, the product can be analyzed by combining with corresponding software, and the condition of nucleic acid in a detected sample can be judged qualitatively, quantitatively or semi-quantitatively in real time. The fluorescence PCR detection technology integrates the PCR technology, the fluorescence labeling technology, the laser technology and the digital visualization technology, and has the advantages of high detection sensitivity, good linear relation, wide linear range and the like.
The fluorescent PCR detection technology is developed for nearly 20 years, and the fluorescent PCR method is widely applied to the fields of pathogen detection, noninvasive prenatal diagnosis, drug efficacy detection, virus variation monitoring, food safety and medical molecular biology research by being matched with a fluorescent quantitative PCR instrument, and plays an irreplaceable role in the diagnosis and monitoring processes of a plurality of infectious disease microorganisms. Meanwhile, along with the aggravation of global warming, germs pass through extreme weather and climatic events, the epidemic situation of infectious diseases is expanded, and the harm to human health is increased.
Up to thousands of fluorescent PCR detection reagents and methods have been clinically used at present; the conventional method for detecting pathogen nucleic acid by fluorescence PCR generally requires two independent processes, namely preparation of pathogen DNA or RNA nucleic acid template and addition of the nucleic acid template into a reaction system for PCR amplification detection. The reagents and methods used and established for nucleic acid processing in various laboratories are not completely consistent, depending on the structure of the various pathogens. But the treatment process is basically consistent, and a plurality of steps such as digestion cracking, adsorption purification, washing and collection are required. The processing of a single sample often takes tens of minutes, and when a large number of samples need to be processed, it takes even longer than a PCR detection reaction. Due to the multiple operation steps, mutual pollution among samples cannot be avoided in the large sample treatment process; because of improper protection, it is also common for humans to become infected with pathogens during the treatment process.
The direct PCR technology adopts an optimized PCR buffer solution system and special DNA polymerase, reduces the interference of various inhibitors existing in a sample on PCR reaction, and has the characteristics of strong activity, high stability, strong tolerance and the like. The technology does not need to carry out an expensive and time-consuming nucleic acid extraction process, and can be directly used for PCR amplification identification by using an animal specimen or a pathogen preservation solution, thereby greatly shortening the detection time and simplifying the operation steps. The direct PCR technology is being applied to the research of various animal and plant genes, and the development of molecular biology research is actively promoted. In the field of medical detection of direct fluorescence PCR technology, a small amount of reports exist, and the reported application method does not need the step of nucleic acid purification, but generally needs to simply process a sample for PCR detection. The sample is directly used as a nucleic acid template for amplification detection, and no feasible report exists yet.
The invention content is as follows:
the invention aims to provide a method for qualitatively detecting respiratory viruses by direct fluorescent polymerase chain reaction (fluorescent PCR), which shortens PCR detection time and simplifies operation steps.
The invention provides a method for qualitatively detecting respiratory viruses by direct fluorescence polymerase chain reaction (fluorescence PCR), wherein a template used for the fluorescence PCR amplification is a clinically collected specimen containing the respiratory viruses.
The invention is realized as follows:
a method for qualitatively detecting pathogen nucleic acid by direct fluorescent polymerase chain reaction (fluorescent PCR) comprises the following detection steps:
uniformly mixing clinical samples containing the respiratory viruses, and adding 2-10 microliters of the clinical samples serving as amplification templates into the fluorescent PCR reaction solution;
the clinical sample containing the pathogen refers to pharyngeal swabs containing respiratory viruses, or sputum.
The fluorescent PCR may be reverse transcription fluorescent PCR using ribonucleic acid (RNA) as an initial amplification object or fluorescent PCR using deoxyribonucleic acid (DNA) as an initial amplification object according to the respiratory virus type.
Specifically, when the fluorescence PCR is reverse transcription fluorescence PCR using RNA as an initial amplification object, the reaction solution components comprise: dNTPs, specific primer, specific taqman probe, optimized DNA polymerase and matched reaction buffer solution, reverse transcriptase and Mg2+、ddH2O。
Specifically, when the fluorescent PCR uses DNA as an initial amplification object, the reaction solution components include: dNTPs, specific primer, specific taqman probe, optimized DNA polymerase, reaction buffer solution matched with the optimized DNA polymerase and Mg2+、ddH2O。
And (3) PCR reaction conditions: and a PCR amplification reaction stage containing 1-6 cycles is carried out between the DNA polymerase function starting stage and the detection signal collecting stage, and the detection signal is not collected in the amplification reaction at the stage.
The PCR reaction may be a single PCR reaction or a multiplex PCR reaction according to actual needs.
The invention establishes a method for qualitatively detecting pathogen nucleic acid by direct fluorescent polymerase chain reaction (fluorescent PCR), uses an innovative PCR reaction process, and has the advantages that: the method is simple, rapid and practical. The method skips the sample nucleic acid processing process, so that the whole detection process is smooth and rapid, the chance of mutual pollution is reduced, and the obtained PCR detection result is accurate and reliable. The chance of infecting pathogens in a sample by people is greatly reduced, and the social and economic benefits are higher.
Description of the drawings:
FIG. 1 shows the fluorescence PCR results of respiratory adenovirus in two specimens tested for oropharyngeal-ragvirus preservative fluid by the method of the present invention.
FIG. 2 shows the fluorescence PCR results of respiratory adenovirus in two specimens of oropharyngeal-ragvirus preservative fluid detected by conventional fluorescence PCR method.
FIG. 3 is the fluorescence PCR results of two pharyngeal-swab saline wash specimens tested for influenza A virus using the method of the invention.
FIG. 4 is the fluorescent PCR results for detection of influenza A virus in two pharyngeal-swab saline-wash specimens using a conventional fluorescent PCR method.
The specific implementation mode is as follows:
the test methods used in the following examples are conventional methods unless otherwise specified
The materials, reagents and the like used in the following examples are commercially available without specific reference
The 6# DNA polymerase and the reaction buffer solution matched with the same are purchased from Shanghai Qihai Biotechnology limited company
Example 1, direct fluorescence PCR detection of respiratory adenovirus (genotype 7) in a orofacial tissue virus preservative fluid specimen, which is characterized by the following detection steps:
first, primer design
Adenovirus genome sequence is analyzed, and specific primer probes are designed in hexon coding genes. Specific primer probes were designed using the primer probe in-line design tool http:// www.yeastgenome.org/cgi-bin/web-primer. Considering the specificity and the amplification efficiency comprehensively, a group of primer probe sequences are finally selected as follows:
designing a primer sequence as follows:
advF：catcgccggacaggatgcttc(SEQ ID No.1)；
advR：tacggtcggtggtcacatcgtg(SEQ ID No.2)；
the probe sequence was designed as follows: and (5) advP: catcgatgctgccccagtgggca (SEQ ID No.3), FAM fluorescent group and BHQ1 quenching group are marked at two ends of the probe
Second, sample preparation
The samples tested in this example were 2 samples of orofacial pappy virus preservative fluid, accession numbers ADV1 and ADV 2. Sequencing to determine that the samples all contain 7 type respiratory adenovirus. In order to compare with the traditional fluorescent PCR method, sucking 200 microliters of samples, using a virus DNA nucleic acid purification kit to extract and purify DNA nucleic acid, respectively obtaining 50 microliters of DNA solution with the number ADV1-D, ADV2-D, and storing at 4 ℃; preserving the residual pharynx paper virus preserving fluid specimen at 4 ℃;
thirdly, PCR detection is carried out
Direct fluorescence PCR detection system: 2. mu.L of 10mM dNTPs, 0.5. mu.L of each 10. mu.M primer, 0.2. mu.L of 10. mu.M probe, 1.5. mu.L of 6# DNA polymerase, 5. mu.L of matched reaction buffer, and 25mM MgCl 22 μ L, specimen 4 μ L, ddH2Make up to 25. mu.L of O.
Traditional fluorescent PCR detection systems: 2. mu.L of 10mM dNTPs, 0.5. mu.L of each 10. mu.M primer, 0.2. mu. L, DNA of polymerase with 10. mu.M probe, 2.5. mu.L of a reaction buffer matched with the probes, and 25mM MgCl 22 μ L, specimen 4 μ L, ddH2Make up to 25. mu.L of O.
And 4 mu L of the sample prepared in the second step is added into the reaction solution. Adding 4 mu L of normal saline washing solution into a direct fluorescence PCR system; mu.L of the DNA solution was added to a conventional fluorescent PCR system. During the sample application process, air bubbles should be avoided as much as possible so as not to affect the PCR reaction.
The reaction conditions of the direct fluorescence PCR are as follows: pre-denaturation at 95 ℃ for 10min → (denaturation at 95 ℃ 5S → annealing at 60 ℃ for 35S) × 3 cycles → (denaturation at 95 ℃ 10S → annealing at 58 ℃ for 40S) × 40 cycles, and the fluorescence collection point is the annealing temperature point at 58 ℃.
The reaction conditions of the traditional fluorescent PCR are as follows: pre-denaturation at 95 ℃ for 10min → (denaturation at 95 ℃ 5S → annealing at 60 ℃ for 35S) × 3 cycles → (denaturation at 95 ℃ 10S → annealing at 58 ℃ for 40S) × 40 cycles, and the fluorescence collection point is the annealing temperature point at 58 ℃.
And (3) detection results: the detection results of the two methods for the two samples show typical amplification, and the amplification is positive. Wherein Ct values of ADV1 and ADV2 detected by direct fluorescence PCR are 20.2 and 23.7 respectively; the Ct values of ADV1-D, ADV2-D detected by traditional fluorescent PCR are 20.5 and 23.4 respectively, and the detection results of the two methods are basically consistent. The method for detecting the respiratory tract DNA by the direct fluorescence PCR method is effective and feasible.
Example 2, direct fluorescence PCR detection of influenza a virus in a yangmei virus preservation fluid specimen is characterized by the following detection steps:
first, primer design
Analyzing the genome sequence of the influenza A virus, and selecting a conservative section to design a universal specific primer probe. Specific primer probes were designed using the primer probe in-line design tool http:// www.yeastgenome.org/cgi-bin/web-primer. Considering the specificity and the amplification efficiency comprehensively, a group of primer probe sequences are finally selected as follows:
designing a primer sequence as follows: IFAF: AAGACCRATYYTGTCACCTCTRACTAAG (SEQ ID NO. 4);
IFAR：CAAANCGTCTACGYTGCAGTCC(SEQ ID No.5)；
the probe sequence was designed as follows: IFAP: TCACTGGGCACGGTGAGYGTRAA (SEQ ID No.6), FAM fluorescent group and BHQ1 quenching group are marked at two ends of the probe
Second, sample preparation
The samples tested in this example were 2 samples of orofacial pappy virus preservative fluid, accession numbers IFA1 and IFA 2. Sequencing to determine that the samples all contain 7 type respiratory adenovirus. In order to compare with the traditional fluorescent PCR method, sucking 200 microliters of samples, using a viral RNA nucleic acid purification kit to extract and purify RNA nucleic acid, respectively obtaining 50 microliters of RNA solution with the number IFA1-R, IFA2-R, and storing at 4 ℃; preserving the residual pharynx paper virus preserving fluid specimen at 4 ℃;
thirdly, PCR detection is carried out
Direct fluorescence PCR detection system: 2. mu.L of 10mM dNTPs, 0.5. mu.L of each 10. mu.M primer, 0.2. mu.L of 10. mu.M probe, 1.5. mu.L of 6# DNA polymerase, 5. mu.L of matched reaction buffer, 0.3. mu.L of reverse transcriptase and 25mM MgCl 22 μ L, specimen 4 μ L, ddH2Make up to 25. mu.L of O.
Traditional fluorescent PCR detection systems: 2. mu.L of 10mM dNTPs, 0.5. mu.L of each 10. mu.M primer, 0.2. mu. L, DNA of polymerase, 0.5. mu.L of reaction buffer, 0.2. mu.L of reverse transcriptase, and 25mM MgCl 22 μ L, specimen 4 μ L, ddH2Make up to 25. mu.L of O.
And 4 mu L of the sample prepared in the second step is added into the reaction solution. Adding 4 mu L of normal saline washing solution into a direct fluorescence PCR system; mu.L of the RNA solution was added to a conventional fluorescent PCR system. During the sample application process, air bubbles should be avoided as much as possible so as not to affect the PCR reaction.
The reaction conditions of the direct fluorescence PCR are as follows: 45 ℃ pre-denaturation 15min → 95 ℃ pre-denaturation 10min → (95 ℃ denaturation 5S → 61 ℃ annealing 35S) × 3 cycle → (95 ℃ denaturation 10S → 59 ℃ annealing 40S) × 40 cycle, and the fluorescence collection point is the 59 ℃ annealing temperature point.
The reaction conditions of the traditional fluorescent PCR are as follows: 45 ℃ pre-denaturation 15min → 95 ℃ pre-denaturation 10min → (95 ℃ denaturation 5S → 61 ℃ annealing 35S) × 3 cycle → (95 ℃ denaturation 10S → 59 ℃ annealing 40S) × 40 cycle, and the fluorescence collection point is the 59 ℃ annealing temperature point.
And (3) detection results: the detection results of the two methods for the two samples show typical amplification, and the amplification is positive. Wherein Ct values of IFA1 and IFA2 detected by direct fluorescence PCR are 25.5 and 35.1 respectively; the Ct values of IFA1-R, IFA2-R detected by traditional fluorescent PCR are 25.2 and 34.8 respectively, and the detection results of the two methods are basically consistent. The method for detecting the respiratory RNA virus by the direct fluorescence PCR method is effective and feasible.
Sequence listing
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Claims (7)
1. A method for qualitatively detecting respiratory viruses by direct fluorescent polymerase chain reaction (fluorescent PCR) is characterized in that a nucleic acid processing step is not needed, and a clinically collected specimen containing viruses can be directly added into a fluorescent PCR detection reaction system as an amplification template of PCR for detection.
2. The method of claim 1, wherein the PCR reaction conditions used in the qualitative detection of respiratory viruses include a PCR amplification reaction stage comprising 1-6 cycles between the initiation stage of DNA polymerase function and the detection signal collection stage, and the detection signal is not collected during the amplification reaction of the PCR amplification reaction stage.
3. The method for qualitatively detecting respiratory viruses by direct fluorescence PCR as claimed in claim 1, wherein the nucleic acid treatment or extraction step is a process of extracting and purifying viral nucleic acid from the sample outside the PCR detection reaction system.
4. The method of claim 1, wherein the viral nucleic acid treatment and the PCR detection reaction are integrated into the same reaction system.
5. The method of claim 1, wherein the nucleic acids comprise ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
6. The method of claim 1, wherein the sample used as the template for PCR amplification comprises a pharyngeal swab lotion or sputum containing the respiratory virus.
7. The method of any one of claims 1 to 6, wherein the fluorescent PCR reaction is a single PCR reaction or a multiplex PCR reaction.
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