CN106854682A

CN106854682A - A kind of method that parting is carried out using gene infected by influenza

Info

Publication number: CN106854682A
Application number: CN201611226906.0A
Authority: CN
Inventors: 陈升; 丁方美; 孙子奎
Original assignee: SHANGHAI PERSONAL BIOTECHNOLOGY CO Ltd
Current assignee: SHANGHAI PERSONAL BIOTECHNOLOGY CO Ltd
Priority date: 2016-12-27
Filing date: 2016-12-27
Publication date: 2017-06-16

Abstract

A kind of method that parting is carried out using gene infected by influenza disclosed by the invention, is comprised the following steps：Step S1, viral RNA are extracted；Step S2, by viral RNA reverse transcription be cDNA；Step S3, entered using specific primer performing PCR amplification；Step S4, recovery pcr amplification product；Step S5, the recovery product is sequenced；Step S6, known array in sequencing result and public database is compared, unknown virus are divided to corresponding classification.The nucleotide sequence of the specific primer such as SEQ ID NO:1 to SEQ ID NO:Shown in 8.The method of the present invention is compared with traditional method based on Serologic detection, and the nucleic acid detection method detection efficiency of PCR-based principle is higher, more rapid and convenient, will be played a great role in the infectious disease emergency that happens suddenly.

Description

A kind of method that parting is carried out using gene infected by influenza

Technical field

The invention belongs to biology field, more particularly, to a kind of side that parting is carried out using gene infected by influenza Method.

Background technology

Influenza (influenza) is the Acute respiratory infectious disease caused by influenza virus, and infectiousness is stronger, clinically Based on the symptom such as hyperpyrexia, weak, headache, Muscular stiffness, often periodically trigger worldwide popular or be very popular.Stream Influenza Virus have three types：A type (A types) influenza infection mammal and birds；B-mode (Type B) influenza virus is only felt The dye mankind, the generation of disease is generally gentle compared with Alphavirus；Third type (c-type) influenza virus only infects the mankind, can't cause tight The disease of weight.From the point of view of infection scale, Flu-A often causes eruption and prevalence, even pandemics, occurs within about 2~3 years Small popular 1 time, according to the 4 pandemicities analysis for having occurred in the world, generation in general 10~15 years is once very popular.It is B-mode , in outburst or small prevalence, the third type is based on distributing for influenza.

The clinical symptoms of influenza are similar to common cold, but flu episode is rapid, and infectiousness is strong, and virus itself is to existing The drug resistance of medicine constantly strengthens, and vaccine protective effect is limited, therefore the quick of pathogen makes a definite diagnosis prevention of disease and early stage The influenza diagnostic criteria for treating the Ministry of Public Health's promulgation that is significant points out that antidiastole relies primarily on aetology, spy Heteronuclear acid is checked and serum antibody is determined.But rely on the classics sides such as virus purification culture, serodiagnosis and antigen detection Method wastes time and energy, it is difficult to adapt to during Epidemic outbreak of disease the need for quick diagnosis.And PCR detection method has, and sensitivity is high, specificity Good the advantages of, it is widely used in pathogen detection, is one of effective ways of pathogen antidiastole generally acknowledged at present.Accurate mirror Other influenza virus type, for monitoring influenza activity and fashion trend, and early warning is made in the variation of infected by influenza, for influenza Prevention and control be significant, while there is certain directive significance in diagnosis and treatment process to clinical line doctor.

The content of the invention

The technical problems to be solved by the invention are to be carried for the existing above-mentioned deficiency of existing influenza virus detection For a kind of method that parting is carried out using gene infected by influenza, the method can be sequenced to A type and B-mode by PCR and a generation Influenza virus carries out Genotyping.

The technical problems to be solved by the invention can be solved by the following technical programs:

A kind of method that parting is carried out using gene infected by influenza, is comprised the following steps：

Step S1, viral RNA are extracted；

Step S2, by viral RNA reverse transcription be cDNA；

Step S3, entered using specific primer performing PCR amplification；

Step S4, recovery pcr amplification product；

Step S5, the recovery product is sequenced；

Step S6, known array in sequencing result and public database is compared, unknown virus are divided to correspondence Classification.

In a preferred embodiment of the invention, the nucleotide sequence of the specific primer such as SEQ ID NO:1 to SEQ ID NO:Shown in 8.

The method of the present invention is compared with traditional method based on Serologic detection, the detection of nucleic acids side of PCR-based principle Method detection efficiency is higher, more rapid and convenient, will be played a great role in the infectious disease emergency that happens suddenly.

Brief description of the drawings

Fig. 1 is template-PCB products dosage schematic diagram of the present invention.

Fig. 2 is template of the present invention-copy number schematic diagram.

Fig. 3 and Fig. 4 is inventive samples type schematic diagram.

Specific embodiment

The present invention is described in further detail with reference to specific embodiments and the drawings.

1st, viral RNA is extracted

1.1st, virus stock solution used 500ul is added in the eppendorf pipes of 1.5ml, TRIzol 1ml are added, it is fully mixed Even, room temperature places 10min, it is ensured that virus coat is fully cracked；

1.2nd, the chloroform of 200ul is added, centrifugation lid is covered tightly, (solution is fully emulsified, into milky white firmly to shake centrifuge tube Shape, no phase separation phenomenon), room temperature places 10min, and (due to chloroform low boiling point, volatile, centrifuge tube may pop during vibration, small The heart)；

1.3rd, 12000g room temperatures centrifugation 15min, takes upper phase and moves into another pipe (never inhaling dynamic white interphase)；

1.4th, 500ul isopropanols are added, centrifuge tube is gently overturned and is fully mixed liquid, room temperature places 10min；

1.5th, 4 DEG C, 10000g centrifugation 10min, centrifugation finishes visible ttom of pipe white precipitate, and as RNA is carefully sucked with rifle All supernatants；

1.6th, the ethanol of 1ml 75% is added, allows RNA precipitate to hang, 4 DEG C, 7500g centrifugation 5min carefully suck institute with rifle There is supernatant, 5min is dried in super-clean bench；

1.7th, appropriate DEPC treatment water is added.

1.8th, RT is in suggestion immediately.To preserve, can be frozen in -70 DEG C after previous step adds ethanol, one can be preserved Year；Can only be preserved 1 month or so at -20 DEG C if adding after DEPC water.

2nd, it is cDNA by viral RNA reverse transcription

2.1 add following reactant mixture in the test tube of ice bath：

Template ribonucleic acid：Total serum IgE 1-3 μ g

Primer：Oligo(dT)(50uM) 1μl

dNTP Mix(10mmol/L) 1μl

The 2.2 plus μ l of RNase free dH2O to 10,65 DEG C of incubation 5min after mixing, rapid ice bath after terminating.

2.3 add following reactant mixture in the test tube of ice bath：

Plus after the μ of RNase free dH2O to 20 l are gently mixed.

2.4 42 DEG C of reactant mixture 30-60min.

2.5 heat 5min at 95 DEG C terminates reaction, and put carries out subsequent experimental or freezen protective on ice.

3rd, design of primers

This patent in order to improve detection efficiency, stromatin (M) gene and hemagglutinin (HA) gene according to influenza virus And neuraminidase (NA) separately designs primer and expanded, these primers are closed by upper Shanghai's style Sen Nuo bio tech ltd Into primer sequence is as follows：

4.PCR is expanded：Enter performing PCR amplification to viral cDNA, obtain genetic fragment to be measured

Amplification system and program are as follows：

Following component is added in 0.2ml centrifuge tubes：

Mixing is flicked, the drop on brief centrifugation collection tube wall to ttom of pipe, in the enterprising performing PCR reaction of PCR amplification instrument, reaction Parameter is as follows：

After the completion of reaction, taking 3ulPCR products carries out 1% agarose gel electrophoresis detection.Confirm pcr amplified fragment.

5th, the recovery of PCR primer

PCR primer is reclaimed with AxyPrep DNA gels QIAquick Gel Extraction Kit, and concrete operations are carried out by kit specification, is walked It is rapid as follows：

5.1 cut the Ago-Gel containing target DNA under uviol lamp is put into clean centrifuge tube, weighs weight.

5.2 add 3 Buffer DE-A of gel volume, are well mixed after 75 DEG C of heating until gel piece melts completely Change.

5.3 plus 0.5 Buffer DE-B of Buffer DE-A volumes, is well mixed；When separate DNA fragmentation is less than During 400bp, 1 isopropanol of gel volume is added.

5.4 by mixed liquor, is transferred to DNA and prepares pipe 12,000 × g centrifugations 1min.Abandon filtrate.

5.5 will prepare pipe puts back into 2ml centrifuge tubes, plus 500 μ l Buffer W1,12,000 × g centrifugation 30s, abandons filtrate.

5.6 will prepare pipe puts back into 2ml centrifuge tubes, plus 700 μ l Buffer W2,12,000 × g centrifugation 30s, abandons filtrate.With Same method is again with 700 μ l Buffer W2,12,000 × g centrifugations 1min.

5.7 will prepare during pipe puts back into 2ml centrifuge tubes, 12,000 × g centrifugations 1min.

5.8 will prepare pipe is placed in the 1.5ml centrifuge tubes of cleaning (being provided in kit), is preparing film center plus 25-30 μ L deionized waters, are stored at room temperature 1min.12,000 × g is centrifuged 1min eluted dnas.

6th, generation sequencing

Generation sequencing is carried out using the technology based on Sanger dideoxy chain terminations, sequenator used is ABI3730xl.

7th, sequence alignment, divides influenza virus type

The sequence for obtaining will be sequenced to compare in ncbi database, unknown virus are divided to corresponding classification.

Sequence table (SEQUENCE LISTING)

<110 ＞ Zhou Youliang

Hu Chunling Wang Haitaos Chen Feng

Wang Chaohui

<120 ＞ HCV gene typings chips and classifying method

<160> 8

<210> 1

<211> 20

<212> DNA

<213>Artificial sequence

<400> 1

GGTTGCTCYTTYTCTATCTT

<210> 2

<211> 19

<212> DNA

<213>Artificial sequence

<400> 2

TCATCATATCCCANGCCAT

<210> 3

<211> 26

<212> DNA

<213>Artificial sequence

<400> 3

TTTTTTTTTT-ACTAGGGACGGCAAAC

<210> 4

<211> 25

<212> DNA

<213>Artificial sequence

<400> 4

TTTTTTTTTT-AGAACAACGCCTCCC

<210> 5

<2 11> 26

<212> DNA

<213>Artificial sequence

<400> 5

TTTTTTTTTT-AGAACACCAGTAAGAG

<210> 6

<211> 25

<212> DNA

<213>Artificial sequence

<400> 6

TTTTTTTTTT-AGCTAGTCTAGGGTG

<210>7

<211> 27

<212> DNA

<213>Artificial sequence

<400> 7

TTTTTTTTTT-GTCTGGTCTAGAGCACA

<210> 8

<2 11> 24

<212> DNA

<213>Artificial sequence

<400> 8

TTTTTTTTTT-GGCTCTTACCTACG

Claims

1. a kind of method that parting is carried out using gene infected by influenza, it is characterised in that comprise the following steps：

Step S1, viral RNA are extracted；

Step S2, by viral RNA reverse transcription be cDNA；

Step S3, entered using specific primer performing PCR amplification；

Step S4, recovery pcr amplification product；

Step S5, the recovery product is sequenced；

Step S6, known array in sequencing result and public database is compared, unknown virus are divided to corresponding class Not.

2. a kind of method that parting is carried out using gene infected by influenza as claimed in claim 1, it is characterised in that the spy The nucleotide sequence of specific primer such as SEQ ID NO:1 to SEQ ID NO:Shown in 8.