CN109777891A - A kind of combination and detection method of the primer pair detecting gag gene and fluorescence probe - Google Patents
A kind of combination and detection method of the primer pair detecting gag gene and fluorescence probe Download PDFInfo
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- CN109777891A CN109777891A CN201910207471.2A CN201910207471A CN109777891A CN 109777891 A CN109777891 A CN 109777891A CN 201910207471 A CN201910207471 A CN 201910207471A CN 109777891 A CN109777891 A CN 109777891A
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- pcr amplification
- fluorescence probe
- amplification system
- concentration
- gag gene
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Abstract
The invention discloses the combinations and detection method of a kind of primer pair for detecting gag gene and fluorescence probe, it is related to slow virus detection technique field, primer pair in the combination of the primer pair and fluorescence probe includes: reverse primer shown in forward primer shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of fluorescence probe is as shown in SEQ ID NO.3.The combination can be used for the gag gene from slow virus in test sample, realize the detection to slow virus in sample by the detection of the gag gene to slow virus, use combine detection slow virus specificity with higher and sensitivity.
Description
Technical field
The present invention relates to slow virus detection technique field, in particular to a kind of primer pair for detecting gag gene and glimmering
The combination and detection method of light probe.
Background technique
Lentivirus is in Retroviridae, but its genome structure is complicated, except gag, pol and env this 3 and simple inverse
Outside the similar structural gene of Retroviral, further include 4 auxiliary genes, vif, vpr, nef, vpu and 2 adjusting gene tat and
rev。
Slow virus carrier has both a variety of advantages as a kind of strong gene delivery vehicle, such as the long duration table of gene
It reaches；Pattern of infection is wide；Division stage and nondividing phase cell can be infected and immunogenicity is relatively small etc..Although slow virus carrier can
By in the stable genome for being integrated into host cell of target gene, but they may also constitute a threat to the health of people, example
Such as integrating mediated transformation is slow virus (the replication competent with replication capacity that can infect non-target cell
lentivirus).United States Food and Drag Administration (FDA) require all cell products through retroviral vector transduction to exist
Before the product, it is necessary to carry out the detection of replication-competent virus.
Common detection Lentivirus method have the defects that it is certain, as sensitivity is low, poor specificity.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide the combinations of a kind of primer pair for detecting gag gene and fluorescence probe, which can
With the gag gene for detecting slow virus, combine detection slow virus specificity with higher and sensitivity are used.
Another object of the present invention is to provide a kind of quickly detection slow virus residual and the methods of reproducible slow virus, should
Method can slow virus residual and reproducible slow virus, specificity with higher and sensitivity effectively in test sample.
The present invention is implemented as follows:
The combination of a kind of primer pair detecting gag gene and fluorescence probe provided by the invention, the gag gene is from slow
Virus, the primer pair includes: reverse primer shown in forward primer shown in SEQ ID NO.1 and SEQ ID NO.2, described
The nucleotide sequence of fluorescence probe is as shown in SEQ ID NO.3.
Preferably, described fluorescence probe one end is marked with FAM fluorophor, and the other end is marked with TAMRA quenching group.
The combination of the combination primer pair and fluorescence probe of primer pair provided by the invention and fluorescence probe can be by real-time
The method of quantitative fluorescent PCR is realized and fast and accurately detects copying for lentiviral gene in sample by the detection to gag gene
Shellfish number, the yin and yang attribute of judgement sample have higher sensitivity and specificity.
A kind of method detecting gag gene provided by the invention comprising: it is added in PCR amplification system as described above
Primer pair and fluorescence probe combination；The PCR amplification system contains the DNA profiling for extracting from measuring samples.
Preferably, the annealing temperature of PCR amplification is 55-65 DEG C；Preferably, annealing temperature is 62 DEG C.
Preferably, dimethyl sulfoxide is also added in PCR amplification system.
Experimental result show that dimethyl sulfoxide helps to improve the specificity and sensitivity of primed probe, avoid
Non-specific amplification.
Preferably, concentration of the dimethyl sulfoxide in PCR amplification system is 1-10%；
Preferably, concentration of the dimethyl sulfoxide in PCR amplification system is 3%.
Preferably, concentration of the fluorescence probe in PCR amplification system are as follows: 10-500pmol/ml；
Preferably, concentration of the fluorescence probe in PCR amplification system is 380-420pmol/ml.
Preferably, concentration of the forward primer in PCR amplification system is 10-500pmol/ml；Preferably, it is described just
It is 240-260pmol/ml to concentration of the primer in PCR amplification system.
Preferably, concentration of the reverse primer in PCR amplification system are as follows: 10-500pmol/ml；
Preferably, concentration of the reverse primer in PCR amplification system is 240-260pmol/ml.
According to the gag gene order of slow virus, the primer pair and fluorescence probe of the specificity designed can detecte sample
In slow virus, can also specifically detect the copy number in sample containing lentiviral gene.
The present inventor is configured by the reasonably optimizing to primer and probe sequence, has its testing result more preferable
Specificity and sensitivity, realize effective detection to slow virus and its copy number in sample.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the fluorescent amplification curve of the positive criteria plasmid to various concentration in experimental example of the present invention as a result, in figure:
It is successively from left to right: 1 × 106copies/μl、1×105copies/μl、1×104copies/μl、1×103copies/μ
l、1×102copies/μl、1×101copies/μl。
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The combination of the primer pair and fluorescence probe provided in this embodiment for being used to detect slow virus gag gene, primer pair packet
It includes: reverse primer shown in forward primer shown in SEQ ID NO.1 and SEQ ID NO.2；The nucleotide of the fluorescence probe
Sequence is as shown in SEQ ID NO.3.Wherein, there is fluorophor FAM at 5 ' ends of fluorescence probe, and there is quenching group TAMRA at 3 ' ends.
The base sequence of primer pair and fluorescence probe is as shown in table 1 below.
Table 1
The method of real-time fluorescence quantitative PCR detection is carried out such as using the primer pair of the present embodiment and the combination of fluorescence probe
Under:
(1) real-time fluorescence quantitative PCR reaction system (by taking 20ul as an example) is prepared:
Fluorescence probe (concentration is 10 μM) 0.8 μ l, forward primer and reverse primer (concentration is 10 μM) each 0.5ul,
TaqpathTMQpcr Master Mix.CG reaction solution 10 μ l, 0.6 μ l of dimethyl sulfoxide (concentration in system is 3%), DNA mould
2 μ l of plate 10ng or standard plasmid solution, adds RNase-free Water to supply 20ul system and (in system, draws forward or backwards
The final concentration of 250pmol/ml of object, the final concentration of 400pmol/ml of fluorescence probe).After preparing, reaction solution is dispensed into eight companies
Guan Zhong.
(2) PCR response procedures:
Response procedures are as follows: activation UNG enzyme: 50 DEG C, 2min；Initial denaturation: 95 DEG C, 20s；(denaturation: 95 DEG C, 15s；Annealing and
Extend: 62 DEG C, 60s) 40 circulations；Fluorescence signal is collected in amplification procedure at 62 DEG C, selection fluorescence signal channel is
FAM。
(3) result judges:
If there are two multiple holes testing results in three multiple holes of sample to be negative, feminine gender herein is that multiple holes are bent without amplification
Line, another testing result Ct value are greater than the Ct value of 10copy sample, also think this sample for feminine gender.
If there are two the Ct values that multiple holes detection Ct value is less than or equal to 10copy sample for three multiple holes of sample, then it is assumed that
This sample is the positive.
If the Ct value of three multiple holes of sample is respectively less than the Ct value of 10copy sample, pattern detection result is the positive.
Experimental example 2
Sensitivity technique
(1) positive plasmid of various concentration is prepared
Contain the concentration of gag plasmid (standard plasmid) using micro UV spectrophotometer measuring, and calculate copy number, counts
It calculates formula copy number (copy/ μ L)=(6.02 × 1023Copies/mol) × (concentration g/ μ L)/(MW g/mol).Contain gag base
The plasmid MW g/mol=7040880 of cause.Plasmid is diluted to 1 × 10 using RNase-free Water10Copies/ μ L, and by
Grade is diluted to 1 × 106copies/μL、1×105copies/μL、1×104copies/μL、1×103copies/μL、1×
102copies/μL、1×101copies/μL。
(2) different using the combination of the primer pair of embodiment 1 and fluorescence probe and corresponding method detection above-mentioned steps (1)
The positive criteria plasmid of concentration.The result is shown in Figure 1.
From figure 1 it appears that the sensitivity using the combine detection of the primer pair and fluorescence probe of embodiment 1 can reach
To 1 × 101copies/μL。
(3) extremely by step (1) method dilution plasmid: 5 × 109copies/μL、5×108copies/μL、5×
107copies/μL、5×106copies/μL、5×105copies/μL、5×104copies/μL、5×103copies/μL、5
×102copies/μL、5×101copies/μL、5×100copies/μL。
(4) different using the combination of the primer pair of embodiment 1 and fluorescence probe and corresponding method detection above-mentioned steps (3)
The positive criteria plasmid of concentration.As a result 2 be see the table below.
Table 2
| Sample type | Gene Name | Fluorophor | Ct value | Copy number |
| Standard plasmid | gag | FAM | 39.114 | 1.E+01 |
| Standard plasmid | gag | FAM | 38.834 | 1.E+01 |
| Standard plasmid | gag | FAM | 37.388 | 1.E+01 |
| Standard plasmid | gag | FAM | 33.979 | 1.E+02 |
| Standard plasmid | gag | FAM | 34.096 | 1.E+02 |
| Standard plasmid | gag | FAM | 34.118 | 1.E+02 |
| Standard plasmid | gag | FAM | 30.842 | 1.E+03 |
| Standard plasmid | gag | FAM | 30.636 | 1.E+03 |
| Standard plasmid | gag | FAM | 30.772 | 1.E+03 |
| Standard plasmid | gag | FAM | 26.783 | 1.E+04 |
| Standard plasmid | gag | FAM | 26.886 | 1.E+04 |
| Standard plasmid | gag | FAM | 26.737 | 1.E+04 |
| Standard plasmid | gag | FAM | 23.069 | 1.E+05 |
| Standard plasmid | gag | FAM | 23.045 | 1.E+05 |
| Standard plasmid | gag | FAM | 23.011 | 1.E+05 |
| Standard plasmid | gag | FAM | 19.657 | 1.E+06 |
| Standard plasmid | gag | FAM | 19.625 | 1.E+06 |
| Standard plasmid | gag | FAM | 19.587 | 1.E+06 |
(5) cell sample detects
The extraction of sample DNA:
Take 1 × 106Cell sample 1ml is added in 37 DEG C of incubation 30min digest mediums of nuclease of 1 μ l into sample
DNA genome or plasmid that may be present.Then 1000rpm be centrifuged 5min, abandon supernatant, be added 1ml PBS cleaning, then from
The heart abandons supernatant, repeats twice of this process.200 μ l PBS piping and druming is added into the sample after cleaning to mix, is then added 20 μ l's
Proteinase K and RNase A are mixed, and are incubated for 2min.200ul Pure Link Genomic DNALysis/ is added after the completion of being incubated for
Binding Buffer, concussion mix, and are incubated for 10min in 55 DEG C of water-baths.
200 μ l 100%ethanol are added, concussion mixes, above-mentioned lysate is transferred in Filter column, 10000g centrifugation
1min.The liquid in collecting pipe is discarded, the genome cleaning solution 1 of 500ul is added into Filter column, 10000g is centrifuged 1min.It abandons
Fall the liquid in collecting pipe, the genome cleaning solution 2 of 500ul is added into Filter column, 10000g is centrifuged 3min.Discard collection
Pipe changes the EP pipe of 1.5ml, the eluent of 30ul is added into Filter column, is incubated for 1min, and 10000g is centrifuged 1min, will obtain
Liquid rejoin in Filter column, again be incubated for and be centrifuged, obtain sample DNA solution, and examined with micro-spectrophotometer
Survey the concentration of DNA solution.
As a result sample DNA see the table below using the primer pair of embodiment 1 and the combination of fluorescence probe and the detection of corresponding method
3.Sample 1 is detected to sample after slow-virus infection T cell, and sample 2 is slow-virus infection T cell, after cultivating a period of time
Sample detection after being cleaned.
Table 3
Remarks: sample 1 is sample detection at once after transfection, containing the plasmid largely containing gag gene in sample, therefore is examined
Surveying result is the positive.
Sample 2 is to sample after cleaning after transfection is cultivated, and the plasmid containing gag gene in sample is in the process cultivated and cleaned
Middle degradation is washed off, and does not find reproducible lentiviral gene in sample therefore testing result feminine gender.
Experimental example 2
Primed probe with different primed probes, in contrast table 1.The primed probe used in experiment such as the following table 4.
Table 4
Using the reaction condition in embodiment 1, T cell is expanded simultaneously with table 4 each different primer pair and probe combinations
DNA, the plasmid (10copies) of the gene containing gag, 293T cell DNA, VERO cell DNA, Jurkat cell DNA, C8166 cell
DNA.Testing result such as the following table 5:
Table 5
The experimental results showed that the primer pair (SEQ ID NO.1 and SEQ ID NO.2) and fluorescence probe (SEQ of gag gene 1
ID NO.3) sensitivity is higher, and specificity is more preferable.
Experimental example 3
Using the experiment condition and experiment parameter in embodiment 1, comparison is separately added into dimethyl sulfoxide and glycine betaine same
Amplification under the conditions of sample；Testing result such as the following table 6:
Table 6
Remarks: standard plasmid is the plasmid (10 copy) containing gag gene.
The experimental results showed that adding the experimental group detection sensitivity of dimethyl sulfoxide higher.
Experimental example 4
Using the primed probe in embodiment 1, the amplification situation of the standard items under different annealing temperature is verified.As a result such as table
Shown in 7.
Table 7
The results showed that the primed probe in embodiment 1 is when annealing temperature is 62 DEG C, amplification situation is preferable, multiple holes
Between repeatability it is more preferable.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Shanghai Bang Yao Biotechnology Co., Ltd
<120>a kind of combination and detection method of the primer pair for detecting gag gene and fluorescence probe
<130> 250
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>artificial sequence
<400> 1
cccatgtttt cagcattatc ag 22
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
gtcacttccc cttggttctc 20
<210> 3
<211> 22
<212> DNA
<213>artificial sequence
<400> 3
aaatcttgtg gggtggctcc tt 22
Claims (9)
1. a kind of combination of the primer pair for detecting gag gene and fluorescence probe, the gag gene come from slow virus, feature exists
In the primer pair includes: reverse primer shown in forward primer shown in SEQ ID NO.1 and SEQ ID NO.2, described glimmering
The nucleotide sequence of light probe is as shown in SEQ ID NO.3.
2. the combination of primer pair according to claim 1 and fluorescence probe, which is characterized in that fluorescence probe one end mark
Note has FAM fluorophor, and the other end is marked with TAMRA quenching group.
3. a kind of method for detecting gag gene, characterized in that it comprises: claims 1 or 2 is added in PCR amplification system
The combination of primer pair and the fluorescence probe；The PCR amplification system contains the DNA profiling for extracting from measuring samples.
4. the method for detection gag gene according to claim 3, which is characterized in that the annealing temperature of PCR amplification is 55-
65℃；Preferably, annealing temperature is 62 DEG C.
5. the method for detection gag gene according to claim 3, which is characterized in that be also added with two in PCR amplification system
Methyl sulfoxide.
6. the method for detection gag gene according to claim 5, which is characterized in that
Preferably, concentration of the dimethyl sulfoxide in PCR amplification system is 1-10%；
Preferably, concentration of the dimethyl sulfoxide in PCR amplification system is 3%.
7. according to the method described in claim 3, it is characterized in that, concentration of the fluorescence probe in PCR amplification system are as follows:
10-500pmol/ml；
Preferably, concentration of the fluorescence probe in PCR amplification system is 380-420pmol/ml.
8. according to the method described in claim 3, it is characterized in that, concentration of the forward primer in PCR amplification system is
10-500pmol/ml；Preferably, concentration of the forward primer in PCR amplification system is 240-260pmol/ml.
9. according to the method described in claim 4, it is characterized in that, concentration of the reverse primer in PCR amplification system are as follows:
10-500pmol/ml；
Preferably, concentration of the reverse primer in PCR amplification system is 240-260pmol/ml.
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Cited By (1)
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| CN111850171A (en) * | 2020-08-03 | 2020-10-30 | 浙江大学 | Method for detecting lentivirus in deep sea sediment sample |
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