CN106222304A - A kind of used by RT PCR method five pairs of upstream and downstream primers simultaneously detecting five kinds of Respiroviruses - Google Patents

Abstract

The invention discloses five pairs of upstream and downstream primers used in an RT-PCR method for simultaneously detecting five respiratory viruses, belonging to the technical field of bioengineering. Combining the epidemic characteristics of common respiratory viruses in Wuxi area, the present invention aims to establish a method capable of simultaneously detecting adenovirus (ADV), human metapneumovirus (MPV), human bocavirus (BOV), respiratory syncytial virus (RSV) and human Rhinovirus (HRV) multiplex PCR method for five respiratory viruses, providing five pairs of upstream and downstream primers. The method can detect multiple viruses in one reaction system, simplifies the experimental process, shortens the experimental time, reduces the experimental cost, and improves the detection efficiency.

Description

Five pairs of upper and lower for a RT-PCR method for the simultaneous detection of five respiratory viruses Swimming primer

technical field

The invention discloses five pairs of upstream and downstream primers used in an RT-PCR method for simultaneously detecting five respiratory viruses, belonging to the technical field of bioengineering.

Background technique

Respiratory diseases include upper and lower respiratory tract acute and chronic infections, respiratory allergic diseases, etc. Among them, acute respiratory infection is the most common. Patients caused by acute respiratory infection often show symptoms such as cough, fever, sore limbs, runny nose, nasal congestion, and dyspnea. There are many types of pathogens that can cause acute respiratory infections, including bacteria, viruses, mycoplasma, chlamydia, and fungi. According to statistics, more than 80% of acute respiratory infections are viral infections.

At present, the laboratory diagnosis of respiratory viruses mainly includes virus isolation, serological diagnosis, direct fluorescent antibody detection and nucleic acid detection and other methods. Although virus isolation and culture is the "gold standard" for the diagnosis of respiratory virus infection, this method requires high equipment technology, is time-consuming and labor-intensive, and cannot be applied to early clinical diagnosis and rapid treatment of epidemics. Serological diagnosis requires the collection of blood samples from patients in the acute and convalescent stages, which can only be used for retrospective diagnosis, and cannot be applied to early clinical diagnosis and rapid treatment of epidemics. Although direct fluorescent antibody detection can obtain results in a relatively short period of time, its specificity and sensitivity are low, and the judgment has a certain degree of subjectivity and high labor costs. In recent years, with the continuous development of molecular biology technology, nucleic acid detection has been widely used in clinical etiological diagnosis, and a multiplex PCR nucleic acid detection method has been established on the basis of ordinary PCR, which can detect multiple viruses in one reaction system , which simplifies the experimental process, shortens the experimental time, reduces the experimental cost, and improves the detection efficiency.

The detection methods for RT-PCR products mainly include traditional agarose gel electrophoresis and capillary electrophoresis. Agarose gel electrophoresis is an electrophoresis method based on agar or agarose as the supporting medium. It requires manual preparation of the gel, which has poor repeatability, high requirements for operators and takes a long time. Capillary electrophoresis analysis has the advantages of high resolution, rapidity, less sample volume, and automation.

Based on this, the present invention combines the epidemic characteristics of common respiratory viruses in Wuxi to establish a method that can simultaneously detect adenovirus (ADV), human metapneumovirus (MPV), human bocavirus (BOV), and respiratory syncytial virus (RSV). and human rhinovirus (HRV) multiplex PCR method for five respiratory viruses.

Contents of the invention

The purpose of the present invention is to provide a RT-PCR method for simultaneously detecting five kinds of respiratory viruses, and five pairs of upstream and downstream primers used therein. A multiple PCR nucleic acid detection method has been established, which can detect multiple viruses in one reaction system, which simplifies the experimental process, shortens the experimental time, reduces the experimental cost, and improves the detection efficiency.

Technical scheme of the present invention: five pairs of upstream and downstream primers used in a RT-PCR method for simultaneous detection of five respiratory viruses,

Primer design: Design five pairs of primers for the conserved regions of ADV, BOV, MPV, RSV, and HRV genomes as follows:

Under the premise that the designed single primer can amplify the target fragments of the pathogen respectively, each pair of upstream and downstream primers (40µM) of the five primers was mixed at a volume ratio of 1:1, and then the primers ADV:BOV:MPV: RSV:HRV is mixed according to the volume ratio of 1:3:2:2:1, and the mixed primer is used as the primer used in the present invention. The primers were prepared by Shanghai Sangon Bioengineering Co., Ltd.

Extract total nucleic acid (DNA&RNA) from respiratory specimens;

The reaction system for PCR detection is 25 µL, including Enzyme Mix 1 µL, 5 X buffer 6 µL, dNTP 1 µL, mixed primer 4.5 µL, template to be tested 5 µL, water to 25 µL, template to be tested is total nucleic acid extracted from respiratory tract specimens , Enzyme Mix, dNTP and 5 X buffer are provided for QIAGEN OneStep RT-PCR Kit kit.

The reaction program of PCR detection was: 50°C for 30min; 95°C for 15min; 94°C for 30sec, 50°C for 30sec, 72°C for 1min, 35 cycles; 72°C for 10min.

Beneficial effects of the present invention: the present invention establishes a method capable of simultaneously detecting adenovirus (ADV), human metapneumovirus (MPV), human bocavirus (BOV), respiratory syncytial virus (RSV) and human rhinovirus (HRV). ) Multiplex PCR method for five respiratory viruses and five pairs of upstream and downstream primers used. The method can detect multiple viruses in one reaction system, simplifies the experimental process, shortens the experimental time, reduces the experimental cost, and improves the detection efficiency.

Description of drawings

Fig. 1 Electrophoretic patterns obtained by capillary electrophoresis system analysis of five kinds of viruses. M: Marker; 1: ADV+BOV+MPV+RSV; 2: ADV+BOV+MPV+HRV; 3: ADV+BOV+RSV+HRV; 4: ADV+MPV+RSV+HRV; 5: BOV+MPV+ RSV+HRV;

6: ADV+BOV+MPV+RSV+HRV; N: negative control.

Specific implementation method

Example 1:

1. Extract the total nucleic acid (DNA&RNA) of respiratory specimens;

2. Primer design: Design five pairs of primers for the conserved regions of ADV, MPV, BOV, RSV and HRV genomes as follows:

On the premise that the designed single primer can amplify the target fragments of the pathogen respectively, each pair of upstream and downstream primers (40µM) of the five primers were mixed at a volume ratio of 1:1, and then the primers ADV:BOV:MPV :RSV:HRV are mixed according to the volume ratio of 1:3:2:2:1, and the mixed primers are used as the primers used in the present invention.

The reaction system for PCR detection is 25 µL, including Enzyme Mix 1 µL, 5 X buffer 6 µL, dNTP 1 µL, mixed primer 4.5 µL, template to be tested 5 µL, water to 25 µL, template to be tested is total nucleic acid extracted from respiratory tract specimens , Enzyme Mix, dNTP and 5 X buffer are provided for QIAGEN OneStep RT-PCR Kit kit.

The reaction program of PCR detection is: 50°C 30min; 95°C 15min; 94°C denaturation 30sec, 50°C annealing 30sec, 72°C extension 1min, 35 cycles; 72°C 10min;

The obtained PCR product was analyzed by QIAxcel automatic capillary electrophoresis system, and the obtained electrophoresis pattern is shown in FIG. 1 .

ADV upstream primer: 5'-GACATGACTT TCGAGATCGA TCCCATGGA-3';

ADV downstream primer: 5'-CCGGCTGAGA AGGGTGTGCG CAGGTA-3';

BOV upstream primer: 5'-TATGGCCAAG GCAATCGTCC AAG-3';

BOV downstream primer: 5'-GCCGCGTGAA CATGAGAAAC AGA-3';

MPV upstream primer: 5'-GTGATGCACT CAAGAGATAC CC-3';

MPV downstream primer: 5'-CATTGTTTGA CCGGCCCCAT AA-3';

RSV upstream primer: 5'-TTAACCAGCAAAGTGTTAGA-3';

RSV downstream primer: 5'-TTTGTTATAG GCATATCATT G-3';

HRV upstream primer: 5'-CAAGCACTTC TGTTTCCC-3';

HRV downstream primer: 5'-CACGGACACC CCAAAGTAGT-3'.

Claims (1)

1. used by RT-PCR method the five pairs of upstream and downstream primers simultaneously detecting five kinds of Respiroviruses, it is characterised in that:

Design of primers: separately design five pairs of primers for ADV, BOV, MPV, RSV, HRV genome conserved region as follows:

ADV forward primer: 5'-GACATGACTT TCGAGATCGA TCCCATGGA-3 ',

ADV downstream primer: 5'-CCGGCTGAGA AGGGTGTGCG CAGGTA-3 ', product size: 139bp；

BOV forward primer: 5'-TATGGCCAAG GCAATCGTCC AAG-3 ',

BOV downstream primer: 5'-GCCGCGTGAA CATGAGAAAC AGA-3 ', product size: 291bp；

MPV forward primer: 5'-GTGATGCACT CAAGAGATAC CC-3 ',

MPV downstream primer: 5'-CATTGTTTGA CCGGCCCCAT AA-3 ', product size: 199bp；

RSV forward primer: 5'-TTAACCAGCA AAGTGTTAGA-3',

RSV downstream primer: 5'-TTTGTTATAG GCATATCATT G-3', product size: 250bp；

HRV forward primer: 5'-CAAGCACTTC TGTTTCCC-3 ',

HRV downstream primer: 5'-CACGGACACC CCAAAGTAGT-3 ', product size: 390bp；

The target fragment of cause of disease can be expanded out by the single primer of design respectively, respectively each right by the primer of five kinds of cause of diseases 11 mixing by volume of the upstream and downstream primer of 40 M, then by primer ADV BOV MPV RSV HRV by volume 13221 Mixing, mixed primer is as the use primer of the present invention；

Extract total nucleic acid DNA&RNA of respiratory tract specimens；

The reaction system of PCR detection is 25 L, and including Enzyme Mix 1 L, 5 X buffer 6 L, dNTP 1 L, after mixing Primer 4.5 L, masterplate 5 L to be checked, water is supplemented to 25 L, template to be checked be from respiratory tract specimens extract total nucleic acid, Enzyme Mix, dNTP and 5 X buffer provide for QIAGEN OneStep RT-PCR Kit test kit；

The response procedures of PCR detection is: 50 DEG C of 30min；95℃ 15min；94 DEG C of degeneration 30sec, 50 DEG C of annealing 30sec, 72 DEG C extend 1min, 35 circulations；72℃ 10min.