CN110172504A - A kind of detection method and kit of foreign gene - Google Patents
A kind of detection method and kit of foreign gene Download PDFInfo
- Publication number
- CN110172504A CN110172504A CN201910320127.4A CN201910320127A CN110172504A CN 110172504 A CN110172504 A CN 110172504A CN 201910320127 A CN201910320127 A CN 201910320127A CN 110172504 A CN110172504 A CN 110172504A
- Authority
- CN
- China
- Prior art keywords
- sequence
- sample
- foreign gene
- tested
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention discloses a kind of detection method of foreign gene and kits, belong to field of biological detection.The detection method comprises determining that the endogenous standard gene in the foreign gene and sample to be tested for needing to detect in sample to be tested；Hybridization probe is prepared according to the sequence of the artificial sequence of foreign gene, the border sequence of foreign gene or endogenous standard gene；It extracts and purifies the genomic DNA of sample to be tested and the genomic DNA of quality-control sample and carry out fragmentation respectively；Top connection sequence is separately connected on sample to be tested DNA fragmentation and quality-control sample DNA fragmentation；The foreign gene in sample to be tested is detected using the characteristic fragment of template segments and foreign gene, determines that foreign gene whether there is in sample to be tested.False positive results caused by this method can be avoided because of microorganism infection, Aerosol Pollution, additionally it is possible to the problem of quantitative inaccuracy caused by avoiding because of factors such as PCR amplification efficiency.
Description
Technical field
The present invention relates to field of biological detection, in particular to the detection method and kit of a kind of foreign gene.
Background technique
The commercial development of genetically modified crops appears in 1996, with the development of biotechnology, transgenic plant product
Type increase year by year.According to International Agriculture biotechnology applications Servers Organization (International Service for the
Acquisition of Agri-biotech Applications, ISAAA) report, by the end of 2013, the whole world was
There are 27 kinds of genetically modified crops types and 336 kinds of genetically modified crops kinds to be developed, in this 27 years, global genetically modified crops
Cultivated area reaches 1.75 hundred million hectares.In China, by the end of 2015, there are 37 kinds of genetically modified crops to go through import, and
As processing raw material.In many countries including China because government and the public worry transgenic product food safety or
Environmental security holds mistrustful attitude to transgenic product.Many countries in order to protect the right to know of the public, including China
Control and tracking are carried out with feed to the GM food in transgenic product by legislation.It is vertical in order to adapt to transgenic product
Method require and ensure product it is legal with it is traceable, effectively and accurately transgenosis is screened, identification and quantification method is very must
It wants.
Method currently used for detecting foreign gene is substantially to be to confirm foreign gene by a transgenic event
No presence, for example, being that combined primer carries out PCR (Polymerase with the sequence in the sequence and rice on cryIAb gene
Chain Reaction, polymerase chain reaction) amplification, if there is amplified production, prove cryIAb gene and rice sequences
Connection product, thus confirmation there are foreign gene cryIAb.
In the implementation of the present invention, the inventor finds that the existing technology has at least the following problems:
It, must be according to cryIAb in new transgenosis but when cryIAb is gone in another rice varieties or corn variety
It is new detect that the genome sequence of the new rice varieties or corn variety connected in event designs new primer combination
Transgenic event, new transgenic event emerge one after another, it means that and it to design countless primer combination and be detected, this work
Amount is great.Meanwhile if new transgenic event is unknown or the sequence that connect with cryIAb is unknown, just have no idea to design
New primer combination, causes the transgenic event undetectable.Meanwhile most of transgenic element all originates from microorganism, and
May be by microorganism infection, therefore during biological growth, detecting transgenic element and not representing must be transgenic positive
Product.Existing all detection techniques are only capable of screening transgenic product according to transgenic element, it is also desirable to according to
The testing result of transgenic event is determined.Therefore, current detection technique cannot directly confirm transgenic product.Transgenosis
Similarly there is problem in event detection.Since transgenic product and transgenosis new events are all very many and diverse, existing technology is all only
It can the limited several known transgenic events of detection.If it is desired to confirm that product is non-transgenic product, it is necessary to carry out big
The detection GMOs of amount, this is not only time-consuming but also significantly increases the cost of detection, causes it in detection GMOs application
Without feasibility.
Existing transgenic detection method is all based on what PCR reaction carried out, and the aerosol that PCR product is formed may pollute
Laboratory.In order to avoid the influence of aerosol, the Laboratory Request of detection GMOs is very high, needs to form negative pressure, to experiment people
Member's operation requires also very stringent.Even so, since the false positive of detection GMOs caused by polluting also occurs often, even if
It is the laboratory for achieving qualification, also usually influences testing result because of the pollution of aerosol.Existing detection GMOs side
Method may be by the infection of microorganism, to cause false positive test results.
Summary of the invention
In order to solve problems in the prior art, the embodiment of the invention provides a kind of foreign gene detection method and reagents
Box.The technical solution is as follows:
The embodiment of the invention provides a kind of detection method of foreign gene, the detection method includes:
Determine the endogenous standard gene in the foreign gene for needing to detect in sample to be tested and the sample to be tested；
According to the artificial sequence of the foreign gene, the border sequence of the foreign gene or the endogenous standard gene
Sequence prepares hybridization probe；
It extracts and purifies the genomic DNA of the sample to be tested and the genomic DNA of quality-control sample；
The equal fragmentation of the genomic DNA of the genomic DNA of the sample to be tested and the quality-control sample is obtained to test sample
Product DNA fragmentation and quality-control sample DNA fragmentation；
It is separately connected top connection sequence on the sample to be tested DNA fragmentation and the quality-control sample DNA fragmentation, obtains band
There are the sample to be tested DNA fragmentation of the joint sequence and the quality-control sample DNA fragmentation with the joint sequence；
Using the sample to be tested DNA fragmentation for having joint sequence described in polymerase chain reaction amplification and described have
The quality-control sample DNA fragmentation of joint sequence obtains the first amplified production；
It is enriched with first amplified production using the hybridization probe, obtains enriched product；
Using enriched product described in PCR amplification, the second amplified production is obtained；
Second amplified production is purified, sequencing library is obtained；
High-flux sequence is carried out to the sequencing library, obtains high-flux sequence data；
Quality control is carried out to the high-flux sequence data, obtains qualified sequencing data；
The characteristic fragment of template segments and the foreign gene is obtained according to the sequencing data of the qualification；
The foreign gene in the sample to be tested is detected using the template segments and the characteristic fragment,
Determine that the foreign gene whether there is in the sample to be tested.
Specifically, the foreign gene for needing to detect is at least one.
Specifically, the artificial sequence is artificially to the improved sequence of the foreign gene existing for nature.
Specifically, the joint sequence includes at least one of sample bar code sequence and random barcodes sequence；
In same laboratory, when the detection method is less than or equal to one, the sample bar code is not reused.
Further, the method for obtaining the template segments includes:
Using the random barcodes having the same, have on reference genome with identical comparison start position and phase
The sequence of sequencing fragment described in one group of same comparison final position, which calculates, obtains the template segments；One group of sequencing fragment
Middle ratio is more than that the base sequence of R1 is the base sequence of the template segments；If Non-scale is more than the base sequence of R1
Column, then be calculated as any base for base sequence described in the template segments, and any base is A, T, C or G.
Specifically, the characteristic fragment includes the sequence of the foreign gene but the template being not present in nature
Segment；
The characteristic fragment includes at least one of following two sequence:
A) artificial sequence；
B) sequence of the foreign gene and the sequence except border sequence of the source gene in originating species except as noted
Catenation sequence.
Specifically, the method for the quality control includes:
Count the number TRF of the template segments of endogenous standard gene described in the sample to be tested；Statistics is described to be measured
In the template segments of the endogenous standard gene of sample, only the ratio of the template segments comprising 1 sequencing fragment, is denoted as
TRFs；
As TRF > N2, the sequencing data is determined for qualification, N2 is the threshold value for determining the sequencing data qualification.
Specifically, the method that the foreign gene in the sample to be tested is detected includes:
Calculate the number TEF of the characteristic fragment of the foreign gene in the sample to be tested；
As TEF > N3, determine that there are the foreign genes in the sample to be tested；
As TEF≤N3, determine that there is no the foreign genes in the sample to be tested.
On the other hand, the embodiment of the invention provides a kind of kit for above-mentioned detection method, the kits
It include: the hybridization probe and the joint sequence,
The hybridization probe according to the artificial sequence of the foreign gene, the border sequence of the foreign gene or it is described in
It is prepared by the sequence of source standard gene；
The joint sequence includes: at least one of sample bar code sequence and random barcodes sequence.
Characteristic fragment in the embodiment of the present invention refers to the template segments being not present in nature, makes it impossible to source
In the biological infection of foreign gene, therefore, the presence of foreign gene can be confirmed by characteristic fragment.
The embodiment of the present invention goes to fish using hybridization probe prepared by the sequence of foreign gene takes characteristic fragment, due to external source base
The sequence of cause is substantially stationary, so, the hybridization probe of preparation is suitable for all foreign genes, can detecte with identical outer
All transgenic events in all species of source gene, including unknown transgenic event.
In the embodiment of the present invention, each test sample is all made of different sample bar codes before PCR amplification and is marked
It is fixed, know whether the sample bar code of the sequencing fragment in aerosol and the sample bar code in test sample are consistent with this, according to
This identifies and excludes Aerosol Pollution in sample to be tested.For example, detecting 546 bands in the test sample of the present embodiment
There is the sequencing fragment of the foreign gene of non-test sample bar code, the Bar gene of non-test sample bar code is had including 125
Sequencing fragment, based in existing foreign gene detection method, Bar gene will be judged as positive foreign gene, produce
The false positive mistake of raw detection GMOs.But in the present embodiment, according to sample bar code, it is determined as Aerosol Pollution source
Bar gene sequencing fragment, so that being appropriately determined in test sample does not have the sequencing fragment of Bar gene.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, embodiment of the present invention will be made into one below
Step ground detailed description.The operating process or working specification for being not specified or being described in detail in the embodiment of the present invention are that common molecular is raw
Operation known to object technical staff.The reagent or biomaterial being not specified in the embodiment of the present invention are available on the market
Common agents or biomaterial are known to common molecular biology technical staff, and can buy on the market.
Embodiment
The embodiment of the invention provides a kind of detection methods of foreign gene, are suitable for detection transgenic product.Specifically,
In the mixing sample of transgenic paddy rice and transgenic corns, foreign gene 35S promoter and foreign gene cryIAb are carried out
Qualitative and quantitative detection.
The test sample of the present embodiment is transgenic paddy rice standard sample powder and transgenic corns standard sample powder, is turned
Trans-genetic hybrid rice standard sample powder and transgenic corns standard sample powder are provided by agriculture rural area portion development in science and technology center,
In, transgenic paddy rice standard sample powder and transgenic corns standard sample powder are containing 1% from tobacco mosaic disease
Poison foreign gene 35S promoter and 1% the desinsection poison protein-2 genes from bacillus thuringiensis foreign gene
CryIAb, transgenic paddy rice standard sample powder and transgenic corns standard sample powder do not contain the solution starch of antiweed
The ribalgilase foreign gene Bar of bacillus.By 1 gram of transgenic paddy rice standard sample powder and 1 gram of transgenic corns mark
Quasi- sample powder mixing, obtains mixing sample, using mixing sample as sample to be tested.In sample to be tested, foreign gene 35S is opened
The content of mover and foreign gene cryIAb are 1%.
Sample to be tested foreign gene detection method, the specific steps are as follows:
Step 1 determines endogenous standard gene in the foreign gene and sample to be tested for needing to detect in sample to be tested.
The foreign gene for needing to detect can be one or more.If it is determined that various exogenous genes, it is only necessary in step 2
Hybridization probe is prepared respectively for various exogenous genes, may be implemented in the same detection process while detecting a variety of external source bases
Because and do not increase testing cost.
In the present embodiment, determine that the foreign gene for needing to detect in sample to be tested is 35S promoter and cryIAb base
Cause determines that the endogenous standard gene in sample to be tested is ribulose-diphosphonic acid carboxylase gene RBCL, the ribulose-diphosphonic acid
Carboxylase gene is guarded in plant, so existing simultaneously among transgenic paddy rice and transgenic corns.
Step 2 is prepared according to the sequence of the border sequence of foreign gene or the artificial sequence of foreign gene and endogenous standard
Hybridization probe.
The characteristic fragment for getting two kinds of foreign genes can be fished according to hybridization probe prepared by the border sequence of foreign gene
One of, i.e. the connection sequence of exogenous gene sequence and the sequence in addition to border sequence of the foreign gene in originating species
Column.The artificial sequence of foreign gene is the sequence being artificially transformed to foreign gene existing for nature.According to foreign gene
Artificial sequence preparation hybridization probe, the another kind in the characteristic fragment for getting two kinds of foreign genes, i.e. external source base can be fished
The artificial sequence of cause.Because the characteristic fragment of foreign gene can not from microorganism infection existing for nature, keep away
The result of the false positive of the detection of foreign gene caused by microorganism infection is exempted from.
In the present embodiment, according in the sequence such as sequence table of the hybridization probe of the sequence of endogenous standard gene RBCL preparation
Shown in SEQ ID NO:1, according in the sequence such as sequence table of the hybridization probe of the border sequence of foreign gene 35S promoter preparation
It is the miscellaneous of the border sequence preparation of cryIAb according to the artificial sequence of foreign gene cryIAb, while also shown in SEQ ID NO:2
It hands over the sequence of probe as shown in SEQ ID NO:3 in sequence table, is according to the artificial sequence of foreign gene Bar, while also
The sequence of the hybridization probe of the border sequence preparation of cryIAb is as shown in SEQ ID NO:4 in sequence table.
Tobacco mosaic virus (TMV) usually infects biology, causes the microorganism infection of sample to be tested, this makes energy in sample to be tested
It enough detects the 35S promoter of tobacco mosaic virus (TMV), so as to cause the false positive results of detection foreign gene 35S promoter, is
Know that sample to be tested, can be except 35S promoter according to tmv cdna group whether by tobacco mosaic virus infection
Region prepare hybridization probe, for show sample to be tested whether by tobacco mosaic virus infection, the hybridization probe prepared with this
Sequence is as shown in SEQ ID NO:5 in sequence table.
Step 3 is extracted and purifies sample to be tested genomic DNA and quality-control sample genomic DNA.
By Tiangeng biochemical technology Co., Ltd production plant genes group extracts kit operating instruction extract to
The genomic DNA of sample and the genomic DNA of quality-control sample, and be dissolved in 20 μ L pure water.
Step 4, by the genomic DNA fragment of the genomic DNA of sample to be tested and quality-control sample, obtain sample to be tested
DNA fragmentation and quality-control sample DNA fragmentation.
In sample to be tested sample making course, pure water is become exposed in sample preparation environment from sample to be tested sample preparation, to test sample
After product sample preparation, salmon sperm dna is added into the pure water being exposed in sample preparation environment, the total amount of salmon sperm dna is made to be greater than DNA
The lowest threshold of extraction limits.Pure water after salmon sperm dna is added is quality-control sample.In the present embodiment, the minimum threshold of DNA extraction
Value limit is set as 50ng, has been actually added into 100ng salmon sperm dna.The pure water being exposed in sample preparation environment can be used for knowing to be measured
Whether sample occurs cross contamination during the preparation process.Specifically, because not containing any nucleic acid composition in pure water, if
Any nucleic acid composition in addition to salmon sperm dna detected in quality-control sample should all be from cross contamination, that is, when detecting
Cross contamination has occurred.
Using ultrasonic wave or sonar by the genome of the genomic DNA of the sample to be tested of 100ng and the quality-control sample of 100ng
DNA fragmentation in the present embodiment, carries out fragmentation processing using ultrasonic wave, two kinds of genomic DNAs after fragmentation pass through respectively
1.5% agarose gel electrophoresis detection, and by the operation of Tiangeng biochemical technology Co., Ltd production PCR product QIAquick Gel Extraction Kit
Illustrate to recycle DNA fragmentation, the DNA fragmentation of acquisition is between 300bp to 800bp.
Sample to be tested DNA fragmentation and quality-control sample DNA fragmentation are separately connected top connection sequence by step 5, and acquisition has and connects
The sample to be tested DNA fragmentation of header sequence and quality-control sample DNA fragmentation with joint sequence.
Joint sequence includes at least one of sample bar code sequence and random barcodes sequence.Existing high pass measures
Sample bar code is added in the method for sequence by way of PCR amplification, can lead to a sample by way of " bar code jump "
Sequencing data be mixed into another sample, if without containing foreign gene sample to be tested in be mixed with containing foreign gene
The sequence of sample to be tested will lead to the false positive of foreign gene detection.In the good situation of laboratory ventilation, one day (24
Hour) Laboratory air may be implemented sufficiently exchanged with extraneous, it is therefore, (small no more than 24 in one day in same laboratory
When), it is not possible to use identical sample bar code.If detection time is greater than or equal to one day (24 hours) in same laboratory
When, then sample bar code may be reused, and can sentence in this way to avoid the false positive because of foreign gene caused by Aerosol Pollution
It is fixed.If laboratory ventilation is bad or in order to which maximum possible prevents Aerosol Pollution, sample bar code can be extended and do not repeat to make
Period.In the present embodiment, it is not reused using sample bar code in 30 days.
In the present embodiment, sample bar code is added before PCR amplification.In sequencing data, the sequence of random barcodes, base
Because the start position and final position sequencing fragment all the same compared in group is from identical template segments, so as to sentence
The number for determining the template segments in sequencing data avoids influence of the PCR amplification efficiency to detection foreign gene accuracy.
Two double-stranded DNAs of joint sequence are annealed in PCR instrument, annealing can be such that two double-stranded DNAs are respectively formed
The joint sequence of double-stranded form, cycle of annealing are as follows: 94 DEG C 5 minutes and 37 DEG C 10 minutes.In the present embodiment, it is sequenced two in sample
The sequence of double-stranded DNA is respectively as shown in SEQ ID NO:7 in SEQ ID NO:6 in sequence table and sequence table, in sequence table
In SEQ ID NO:6 and sequence table SEQ ID NO:7, acacgatgactc and gagtcatcgtgt are the sample of sample to be tested
The sequence of the sequence of product bar code, the sample bar code of the sample bar code and quality-control sample of sample to be tested is all different, Quality Control
The sample bar code of sample is ctcgcatactac and its complementary series.N is random barcodes in sequence table SEQ ID NO:7
Sequence, n represent any one base in tetra- kinds of bases of A, T, C and G.
It (is purchased in Shijiazhuang Bo Ruidi biotechnology by GenoBaits DNA-seq Kit for illumina kit
Co., Ltd, article No.: K00 002) operating instruction, the DNA fragmentation of the DNA fragmentation of sample to be tested and quality-control sample is connected respectively
Connect the above-mentioned joint sequence formed by annealing.
The sample to be tested for connecting top connection sequence and quality-control sample are blended together as test sample.Due to test specimens
Sample to be tested and quality-control sample in product have different sample bar codes, therefore, can distinguish from final sequencing data
Out.
Step 6, the sample to be tested DNA fragmentation using PCR amplification with joint sequence and the Quality Control sample with joint sequence
Product DNA fragmentation obtains the first amplified production.
It (is purchased in Shijiazhuang Bo Ruidi biotechnology by GenoBaits DNA-seq Kit for illumina kit
Co., Ltd, article No.: K00 002) operating instruction amplification assay sample in, the sample to be tested DNA fragmentation with joint sequence
With the quality-control sample DNA fragmentation for having joint sequence, amplified production is obtained, after purifying amplified production, obtains the first amplified production.
To guarantee that the first amplified production increased within the linear amplification phase, to increase the stability of detection, PCR cycle number is no more than 20
It is a.In the present embodiment, PCR cycle number is 20, and the sequence of the primer of PCR amplification is respectively such as SEQ ID NO:8 in sequence table
With in sequence table shown in SEQ ID NO:9.
Step 7 is enriched with the first amplified production using hybridization probe, obtains enriched product；
Using PCR amplification enriched product, the second amplified production is obtained；
The second amplified production is purified, sequencing library is obtained.
It (is purchased in Shijiazhuang Bo Ruidi biotechnology by GenoBaits DNA-seq Kit for illumina kit
Co., Ltd, article No.: K00 002) operating instruction, capture and be enriched with the first amplified production using hybridization probe, capture first
It is purified after amplified production, obtains enriched product.Used hybridization probe is all hybridization probes prepared in step 2
Mixed probe.
It (is purchased in Shijiazhuang Bo Ruidisheng also with GenoBaits DNA-seq Kit for illumina kit
Object Technology Co., Ltd., article No.: K00 002) operating instruction use PCR amplification enriched product, PCR amplification cycles number be 25
It is a, the second amplified production is obtained, the second amplified production is purified, obtains sequencing library.The primer sequence of PCR amplification is respectively such as sequence
In list in SEQ ID NO:10 and sequence table shown in SEQ ID NO:11.
Step 8 carries out high-flux sequence to sequencing library, obtains high-flux sequence data.
By the HiSeq Xten sequenator of Illumina company and the operating instruction of 2 × 150bp both-end sequencing kit, adopt
High-flux sequence detection is carried out to the library of building with the both-end of 2 × 150bp sequencing mode, the base data volume of sequencing is set as
Greater than 2G.
In the same channel of test sample, also sequencing has detected the DNA sequence dna of people, the DNA sequence dna of people have with to
The sample bar code that sample and quality-control sample are all different.Therefore, if the people that the sequencing fragment of test sample can only compare
With reference on genome, illustrating in the presence of " jump of bar code ".But in the present embodiment, do not detect that bar code jump is existing
As.
Step 9 carries out quality control to high-flux sequence data, obtains qualified sequencing data；According to qualified sequencing
The characteristic fragment of data acquisition template segments and foreign gene.
High-flux sequence data carry out the ratio that the main method of quality control reaches Q30 standard for mass value
70%, then it is assumed that up-to-standard.In the present embodiment, which reaches 85%, and therefore, sequencing data is qualified.
The method for obtaining template segments includes: using random barcodes having the same, and having on reference genome has
The identical sequence calculating acquisition template segments for comparing start position and identical one group of sequencing fragment for comparing final position.At this
It include all foreign genes and endogenous standard gene with reference to genome in embodiment.Ratio is more than R1's in this group of sequencing fragment
Base sequence is the base sequence of template segments, and the value of R1 should be greater than or be equal to 50%.If Non-scale is more than the base sequence of R1,
The base sequence in template segments is then calculated as any base；Any base is one of tetra- kinds of bases of A, T, C or G.
Base in random barcodes is any one in tetra- kinds of bases of A, T, C or G, and therefore, random barcodes have
4mKind, (m >=4, and be positive integer), wherein m is the length of the base sequence of random barcodes.In the present embodiment, m=8,
Therefore, the type of random barcodes can achieve 48, i.e., 65536 kinds, along on reference gene comparison start position with than
To final position, a combination thereof number be it is very huge, can guarantee that different template segments connect different random bar shapeds in this way
Code.All exogenous gene sequences and endogenous standard gene sequence are classified as with reference to genome sequence.
In the present embodiment, sequencing is obtained by BWA software (version number: 0.7.9a-r786) and its default parameters
Reads is compared onto reference genome, and according to the above method, i.e., random barcodes having the same have tool on reference genome
There are identical comparison start position and the identical principle for comparing final position, determines one group of sequencing for belonging to each template segments
Segment.The R1=60% of the present embodiment setting, determines each base in template segments according to the value of R1, to obtain all moulds
Plate segment.
Using sample bar code, the template segments in test sample are divided into the template segments and Quality Control sample of sample to be tested
The template segments of product.
By way of simply adding up, the number of the template segments of endogenous standard gene in sample to be tested, the number are counted
Mesh TRF, in the present embodiment, TRF=312.12K, wherein 1K=1000；Count the template of the endogenous standard gene of sample to be tested
In segment, the only ratio TRFs of the template segments comprising 1 sequencing fragment, in the present embodiment, TRFs=2.11%.According to TRF
With the value of TRFs, determine whether sequencing data is qualified, if unqualified, determines underproof reason, and provide subsequent operation mode,
Specific judgment principle is as follows:
As TRF > N2, determines that sequencing data is qualified, then can carry out foreign gene and be detected；
As N1<TRF≤N2 and TRFs>=R, determines that the amount of sequencing data is insufficient, need to re-start high-flux sequence；
N1<N2。
As N1 < TRF≤N2 and TRFs < R or TRF≤N1, determines that sequencing library building is unsuccessful, need structure again
Build sequencing library.
Specifically, N2 is the threshold value for determining sequencing data qualification.The value of N2 depends on the inspection for the foreign gene for wishing to reach
Survey lower limit.For example, be 0.1% it is generally desirable to the Monitoring lower-cut of the foreign gene reached in detection GMOs, then, work as N2
When=10000,0.1% foreign gene can averagely be detected 10 template segments.It is calculated according to bi-distribution, detects 2
The probability of the template segments of above foreign gene is greater than 99.95%.Therefore, in the present embodiment, N2=10000 is taken.Accordingly
Ground, the TRF > N2 of the present embodiment determine that sequencing data is qualified, can carry out the detection of foreign gene.
As TRF≤N2, the template number of endogenous standard gene is insufficient for the requirement of foreign gene Monitoring lower-cut, needs
Again it to test.At this point, being divided into two kinds of situations again: the first situation, as N1<TRF≤N2 and TRFs>=R, (wherein, N1 is
Construct the unsuccessful threshold value of sequencing library, N1≤1000.R be sequencing saturation degree threshold value, and R > 50%), illustrate foreign gene
Although the value of N2 is not achieved in template number, but have been over certain quantity, that is, has been more than the value of N1, simultaneously as TRFs
>=R illustrates that there are also the template segments of an a certain amount of only sequencing fragment in sequencing data, meanwhile, also there are also certain for explanation
The template segments of 0 sequencing fragment of amount, i.e., there are also a certain amount of template segments being not detected.Therefore, it can be surveyed by increasing
The method of sequence amount obtains the template segments of enough numbers, to meet the requirement of foreign gene Monitoring lower-cut.We are to more than 200 times
Sequencing data amount is insufficient but the actually detected experiment for the sequencing fragment that sequencing library can supply endogenous standard gene is surveyed by mending
Statistics, when N1, which is set as 1000, R, is set as 70%, 95% experiment, which increases one times of sequencing amount, can mend enough endogenous standard base
The sequencing fragment of cause.Therefore, in the present embodiment, N1 is set as 1000, R and is set as 70%.
As TRF≤N1, endogenous standard gene sequencing fragment is very little, and maximum probability needs the reason is that library construction is unsuccessful
Rebuild library.As N1 < TRF≤N2 and TRFs < R, it is clear that have a sequencing fragment of certain data, but due to TRFs value compared with
It is small, illustrate undetected template segments limited amount, therefore, it is also desirable to rebuild library.
The embodiment of the present invention can be monitored by multiple key nodes of the final detection data to detection process.Example
Such as, foreign gene failure is detected by PCR mode, it may be possible to which template quantity is insufficient, it is also possible to the failure of PCR primer.Reason is not
Together, subsequent processing mode is different, is possible to due to two kinds, subsequent how to handle is indefinite.
Step 10 detects the foreign gene in sample to be tested using template segments and characteristic fragment, determines external source
Gene whether there is in sample to be tested.
Obtain the template piece that the characteristic fragment of foreign gene refers to the sequence including foreign gene but is not present in nature
Section；The characteristic fragment of foreign gene includes at least one of following two sequence: (a) artificial sequence；(b) foreign gene
The catenation sequence of sequence and the sequence in addition to border sequence of the foreign gene in originating species.
The template segments obtained in step 9 are compared by BWA software (version number: 0.7.9a-r786) and its default parameters
Onto reference genome.Due to the sequence of the natural anti insect gene in the sequence and bacillus thuringiensis of foreign gene cryIAb
Homology is 85% or more, therefore, the impossible Su Yunjin in nature of the full sequence of foreign gene cryIAb
The infection of bacillus, therefore, the template segments than right foreign gene cryIAb sequence think to contain artificial sequence,
Characteristic fragment as foreign gene.The sequence of the tobacco mosaic virus (TMV) of sequence and nature in foreign gene 35S promoter
Unanimously, therefore, when reservation has characteristic fragment of the following template segments for comparing feature as foreign gene 35S: only part mould
The sequence of plate sequence and the success of foreign gene 35S promoter sequence alignment, another part do not compare successful template sequence with
Tobacco mosaic virus (TMV) is also unsuccessful with reference to genome alignment.
In the present embodiment, by the above process, the number of the characteristic fragment of foreign gene in sample to be tested is calculated, TEF is denoted as；
Wherein, the number of the characteristic fragment of foreign gene 35S promoter, i.e. TEF (35S)=1.58 K (1K=1000), foreign gene
The number of the characteristic fragment of gene cryIAb, i.e. TEF (cryIAb)=3.21K (1K=1000).The feature of foreign gene Bar
The number of segment, i.e. TEF (Bar)=0.
As TEF > N3, determine that there are foreign genes in sample to be tested；As TEF≤N3, determine not deposit in sample to be tested
In foreign gene.Since characteristic fragment can not be from biological infection, and sample bar code in turn avoids the pollution of aerosol
The problem of with bar code jump, therefore, as long as there is a characteristic fragment, it can determine that there are foreign genes in sample to be tested.
But for the sake of security, in the present embodiment, take N3=5.Due to TEF (35S)=1.58K > 5 and TEF (cryIAb)=3.21K
Therefore (1K=1000) > 5 determines that there are foreign gene 35S promoters and foreign gene gene cryIAb in sample to be tested.By
In TEF (Bar)=0≤5, therefore, determine that foreign gene Bar is not present in sample to be tested.
The embodiment of the invention also provides a kind of kit for above-mentioned detection method, which includes: that hybridization is visited
Needle and joint sequence,
Hybridization probe is according to the sequence of the artificial sequence of foreign gene, the border sequence of foreign gene or endogenous standard gene
Preparation；
Joint sequence includes: at least one of sample bar code sequence and random barcodes sequence.
Since existing foreign gene detection method has no idea absolutely to exclude the biological infection containing foreign gene, because
This, when detecting the foreign gene of biological source in sample to be tested, can not determine the type of foreign gene.For example, utilizing
Existing method detects the presence of 35S promoter, may be the 35S promoter being transferred to, i.e. foreign gene, it is also possible to
Sample to be tested is by the 35S promoter after tobacco mosaic virus infection, therefore, has no idea to confirm that there are external source bases in sample to be tested
Because of 35S promoter.
Characteristic fragment in the embodiment of the present invention refers to the template segments being not present in nature, makes it impossible to source
In the biological infection of foreign gene, therefore, the presence of foreign gene can be confirmed by characteristic fragment.For example, in the present embodiment
It confirmed that 35S promoter derives from foreign gene, rather than derive from tobacco mosaic virus (TMV), it is thus identified that sample to be tested is to turn
Gene prod.In step 2, the region in tmv cdna group except 35S promoter devises hybridization probe sequence
Column, for showing test sample whether by the infection of tobacco mosaic virus (TMV).In fact, detecting the corresponding tobacco of hybridization probe
Mosaic virus sequencing fragment totally 321, this suffices to show that the test sample, and there are the infection of tobacco mosaic disease.It detects altogether in laboratory
Nearly 10000 test samples, it was demonstrated that all there is tobacco mosaic disease in most of biological sample, this be why traditional method
35S promoter etc. can only be derived to the foreign gene of nature as screening, without can confirm that whether product is that transgenosis produces
The reason of product.
The embodiment of the present invention goes to fish using hybridization probe prepared by the sequence of foreign gene takes characteristic fragment, due to external source base
The sequence of cause is substantially stationary, so, the hybridization probe of preparation is suitable for all foreign genes, can detecte with identical outer
All transgenic events in all species of source gene, including unknown transgenic event.Such as 35S promoter and resistance marker
Etc. the substantially stationary foreign gene of sequences, almost all used in all transgenic product, therefore, the embodiment of the present invention passes through
The hybridization probe of 35S promoter preparation can detecte the most transgenic product of confirmation, no matter the transgenosis thing in the product
Whether part is it is known that this is the technical effect that traditional method is not accomplished.
In the embodiment of the present invention, each test sample is all made of different sample bar codes before PCR amplification and is marked
It is fixed, know whether the sample bar code of the sequencing fragment in aerosol and the sample bar code in test sample are consistent with this, according to
This identifies and excludes Aerosol Pollution in sample to be tested.For example, detecting 546 bands in the test sample of the present embodiment
There is the sequencing fragment of the foreign gene of non-test sample bar code, the Bar gene of non-test sample bar code is had including 125
Sequencing fragment, based in existing foreign gene detection method, Bar gene will be judged as positive foreign gene, produce
The false positive mistake of raw detection GMOs.But in the present embodiment, according to sample bar code, it is determined as Aerosol Pollution source
Bar gene sequencing fragment, so that being appropriately determined in test sample does not have the sequencing fragment of Bar gene.
The present invention can detect simultaneously and confirm various exogenous genes, only when every kind of foreign gene detects failure,
It will lead to and transgenic product is mistaken for non-transgenic product, false negative rate also greatly reduces.For example, in the present embodiment,
Simultaneously confirmed that foreign gene cryIAb and 35S promoter are present in sample to be tested, significantly reduce in existing method, only according to
Rely and determines the risk of the false positive conclusion of transgenic positive in the transgenic event of foreign gene cryIAb.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
<110>Biotechnology Co., Ltd is illustrated in Wuhan
<120>detection method and kit of a kind of foreign gene
<160>11
<170> SIPOSequenceListing 1.0
<210> 1
<211>110
<212>DNA
<213> Oryza.sativa
<400> 1
ttgtgagaat tcttaattca tgagttgtag ggagggacgt atgtcaccac aaacagaaac 60
taaagcaagt gttggattta aagctggtgt taaggattat aaattgactt 110
<210> 2
<211>120
<212> DNA
<213> Oryza.sativa
<400> 2
ggattgatgt gacatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca 60
agacccttcc tctatataag gaagttcatt tcatttggag aggacacgct gaaatcacca 120
<210> 3
<211>120
<212> DNA
<213> Tobacco mosaic virus
<400> 3
acgaatgcat tccatacaac tgcttgagta acccagaagt tgaagtactt ggtggagaac 60
gcattgaaac cggttacact cccatcgaca tctccttgtc cttgacacag tttctgctca 120
<210> 4
<211>120
<212> DNA
<213> Streptomyces hygroscopicus
<400> 4
tggggatcta ccatgagccc agaacgacgc ccggccgaca tccgccgtgc caccgaggcg 60
gacatgccgg cggtctgcac catcgtcaac cactacatcg agacaagcac ggtcaacttc 120
<210> 5
<211>120
<212> DNA
<213>bacillus thuringiensis (Bacillus thuringiensis)
<400> 5
attacatccc gaagctagct caaatcagaa agcctctgca agccaagctt aaagaaaacg 60
ttccatggag atggacaaaa gaggataccc tctacatgca aaaggtgaag aaaaatctgc 120
<210> 6
<211>42
<212> DNA
<213> Tobacco mosaic virus
<400> 6
tctttcccta cacgacgctc ttccgatcta cacgatgact ct 42
<210> 7
<211>45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gagtcatcgt gtnnnnnnnn atggaattct cgggtgccaa ggaac 45
<210> 8
<211>71
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
aatgatacgg cgaccaccga gatctacaca tgcaatgnac actctttccc tacacgacgc 60
tcttccgatc t 71
<210>9
<211>66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400>9
caagcagaag acggcatacg agatgcattg gcgtgactgg agttccttgg cacccgagaa 60
ttccat 66
<210>10
<211>29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400>10
aatgatacgg cgaccaccga gatctacac 29
<210>11
<211>24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400>11
caagcagaag acggcatacg agat 24
Claims (9)
1. a kind of detection method of foreign gene, which is characterized in that the detection method includes:
Determine the endogenous standard gene in the foreign gene for needing to detect in sample to be tested and the sample to be tested；
According to the sequence of the artificial sequence of the foreign gene, the border sequence of the foreign gene or the endogenous standard gene
Prepare hybridization probe；
It extracts and purifies the genomic DNA of the sample to be tested and the genomic DNA of quality-control sample；
By the equal fragmentation of the genomic DNA of the genomic DNA of the sample to be tested and the quality-control sample, sample to be tested is obtained
DNA fragmentation and quality-control sample DNA fragmentation；
It is separately connected top connection sequence on the sample to be tested DNA fragmentation and the quality-control sample DNA fragmentation, has obtained band
State the sample to be tested DNA fragmentation of joint sequence and the quality-control sample DNA fragmentation with the joint sequence；
Using described in polymerase chain reaction amplification have joint sequence the sample to be tested DNA fragmentation and it is described have connector
The quality-control sample DNA fragmentation of sequence obtains the first amplified production；
It is enriched with first amplified production using the hybridization probe, obtains enriched product；
Using enriched product described in PCR amplification, the second amplified production is obtained；
Second amplified production is purified, sequencing library is obtained；
High-flux sequence is carried out to the sequencing library, obtains high-flux sequence data；
Quality control is carried out to the high-flux sequence data, obtains qualified sequencing data；
The characteristic fragment of template segments and the foreign gene is obtained according to the sequencing data of the qualification；
The foreign gene in the sample to be tested is detected using the template segments and the characteristic fragment, is determined
The foreign gene whether there is in the sample to be tested.
2. detection method according to claim 1, which is characterized in that the foreign gene that the needs detect is at least one
Kind.
3. detection method according to claim 1, which is characterized in that the artificial sequence is artificially to existing for nature
The improved sequence of foreign gene.
4. detection method according to claim 1, which is characterized in that the joint sequence include sample bar code sequence and
At least one of random barcodes sequence；
In same laboratory, when the detection method is less than or equal to one, the sample bar code is not reused.
5. detection method according to claim 4, which is characterized in that the method for obtaining the template segments includes:
Using the random barcodes having the same, have on reference genome with identical comparison start position and identical
The sequence for comparing sequencing fragment described in one group of final position, which calculates, obtains the template segments；Compare in one group of sequencing fragment
Example is more than that the base sequence of R1 is the base sequence of the template segments；If Non-scale is more than the base sequence of R1,
Base sequence described in the template segments is then calculated as any base, any base is A, T, C or G.
6. detection method according to claim 1, which is characterized in that the characteristic fragment includes the sequence of the foreign gene
Column but the template segments being not present in nature；
The characteristic fragment includes at least one of following two sequence:
A) artificial sequence；
B) company of the sequence of the foreign gene and the sequence except border sequence of the source gene in originating species except as noted
Connect sequence.
7. detection method according to claim 1, which is characterized in that the method for quality control includes:
Count the number TRF of the template segments of endogenous standard gene described in the sample to be tested；Statistics is in the sample to be tested
Endogenous standard gene template segments in, only comprising 1 sequencing fragment template segments ratio, be denoted as TRFs；
As TRF > N2, the sequencing data is determined for qualification, N2 is the threshold value for determining the sequencing data qualification.
8. detection method according to claim 1, which is characterized in that the foreign gene in the sample to be tested is detected
Method include:
Calculate the number TEF of the characteristic fragment of the foreign gene in the sample to be tested；
As TEF > N3, determine that there are the foreign genes in the sample to be tested；
As TEF≤N3, determine that there is no the foreign genes in the sample to be tested.
9. a kind of kit for detection method as described in any one of claims 1 to 8, which is characterized in that the reagent
Box includes: the hybridization probe and the joint sequence,
The hybridization probe is according to the artificial sequence of the foreign gene, the border sequence of the foreign gene or the endogenous mark
It is prepared by the sequence of quasi- gene；
The joint sequence includes: at least one of sample bar code sequence and random barcodes sequence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910320127.4A CN110172504A (en) | 2019-04-19 | 2019-04-19 | A kind of detection method and kit of foreign gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910320127.4A CN110172504A (en) | 2019-04-19 | 2019-04-19 | A kind of detection method and kit of foreign gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110172504A true CN110172504A (en) | 2019-08-27 |
Family
ID=67689799
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910320127.4A Pending CN110172504A (en) | 2019-04-19 | 2019-04-19 | A kind of detection method and kit of foreign gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110172504A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112921076A (en) * | 2021-04-02 | 2021-06-08 | 中国农业大学 | Pig 50K liquid phase chip based on target capture sequencing and application thereof |
| CN113957130A (en) * | 2021-09-27 | 2022-01-21 | 江汉大学 | Method for identifying transgenic event based on high-throughput sequencing and probe enrichment |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705304A (en) * | 2009-12-08 | 2010-05-12 | 上海市农业科学院 | Internal standard gene suitable for detecting transgene carnation exogenous gene, preparation method and application thereof |
| CN102392040A (en) * | 2011-11-10 | 2012-03-28 | 南方医科大学 | Recombinant plasmid for detecting components in transgenic plant and application thereof |
| CN104694635A (en) * | 2015-02-12 | 2015-06-10 | 北京百迈客生物科技有限公司 | Method for constructing high-flux simplified genome sequencing library |
| CN105567832A (en) * | 2016-01-29 | 2016-05-11 | 江汉大学 | Microorganism drug resistance gene detection method |
| CN105586411A (en) * | 2016-01-29 | 2016-05-18 | 江汉大学 | Method for detecting paddy rice transgenic ingredients |
| WO2016160844A2 (en) * | 2015-03-30 | 2016-10-06 | Cellular Research, Inc. | Methods and compositions for combinatorial barcoding |
| CN108179174A (en) * | 2018-01-15 | 2018-06-19 | 武汉爱基百客生物科技有限公司 | A kind of high-throughput construction method for simplifying gene order-checking library |
-
2019
- 2019-04-19 CN CN201910320127.4A patent/CN110172504A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705304A (en) * | 2009-12-08 | 2010-05-12 | 上海市农业科学院 | Internal standard gene suitable for detecting transgene carnation exogenous gene, preparation method and application thereof |
| CN102392040A (en) * | 2011-11-10 | 2012-03-28 | 南方医科大学 | Recombinant plasmid for detecting components in transgenic plant and application thereof |
| CN104694635A (en) * | 2015-02-12 | 2015-06-10 | 北京百迈客生物科技有限公司 | Method for constructing high-flux simplified genome sequencing library |
| WO2016160844A2 (en) * | 2015-03-30 | 2016-10-06 | Cellular Research, Inc. | Methods and compositions for combinatorial barcoding |
| CN105567832A (en) * | 2016-01-29 | 2016-05-11 | 江汉大学 | Microorganism drug resistance gene detection method |
| CN105586411A (en) * | 2016-01-29 | 2016-05-18 | 江汉大学 | Method for detecting paddy rice transgenic ingredients |
| CN108179174A (en) * | 2018-01-15 | 2018-06-19 | 武汉爱基百客生物科技有限公司 | A kind of high-throughput construction method for simplifying gene order-checking library |
Non-Patent Citations (1)
| Title |
|---|
| 张欢欢 等: "仙台病毒核酸测序检测方法的建立", 《实验动物与比较医学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112921076A (en) * | 2021-04-02 | 2021-06-08 | 中国农业大学 | Pig 50K liquid phase chip based on target capture sequencing and application thereof |
| CN113957130A (en) * | 2021-09-27 | 2022-01-21 | 江汉大学 | Method for identifying transgenic event based on high-throughput sequencing and probe enrichment |
| CN113957130B (en) * | 2021-09-27 | 2023-12-22 | 江汉大学 | Method for identifying transgenic event based on high-throughput sequencing and probe enrichment |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106566876B (en) | Oligonucleotide probe and obtaining method thereof | |
| CN110172504A (en) | A kind of detection method and kit of foreign gene | |
| CN111808975A (en) | Deer species source identification primer pair of pilose antler product and identification method thereof | |
| CN106521018A (en) | Primer and method for high-flux detection of transgenic maize containing NOS terminator | |
| CN106520958B (en) | Method for developing microsatellite marker locus and method for detecting length of microsatellite marker in microsatellite marker locus | |
| CN108103220A (en) | A kind of method of the transgenic element selective mechanisms of high throughput | |
| CN111793707A (en) | Gene editing transgenic crop editing site specificity PCR method and application thereof | |
| CN114231603B (en) | Primer, reagent, identification method and kit for identifying paeonia boita | |
| CN111996274B (en) | Large-scale quantitative detection method for plant pathogenic fungi by high-throughput sequencing | |
| CN113564266B (en) | SNP typing genetic marker combination, detection kit and application | |
| CN112280905B (en) | Method for detecting southern bean mosaic virus and tobacco ringspot virus by using multiple DPO-RT-PCR | |
| CN112779274B (en) | Ribosomal RNA gene of mulberry plaster disease pathogenic bacteria and application thereof | |
| CN109097488A (en) | For synchronizing nucleic acid, method and the kit of five kinds of dog diarrhea virus of detection and identification | |
| CN110923304B (en) | Molecular marker, primer pair and method for identifying sex of ginkgo biloba | |
| CN114164292A (en) | Development and application of KASP marker of rice blast resistance gene Pid4 | |
| CN108707663B (en) | Reagent for cancer sample miRNA sequencing quantitative result evaluation, preparation method and application | |
| CN111218520A (en) | Transgenic soybean GTS-40-3-2 strain EFIRM detection probe and application thereof | |
| CN106520959B (en) | Development method of orchid microsatellite marker locus and method for detecting length of microsatellite marker in microsatellite marker locus | |
| CN106520961B (en) | Corn microsatellite marker locus development method and length detection method of microsatellite markers in microsatellite marker locus | |
| CN115820873B (en) | DNA bar code of pear stem bees and application thereof | |
| CN117757979B (en) | Primer group, kit and identification method for identifying soybean varieties | |
| CN110819721B (en) | Molecular marker for identifying high-yield Yili geese and application thereof | |
| CN112322794B (en) | Kit for detecting peanut dwarf virus and tomato ringspot virus by using multiple DPO-RT-PCR | |
| CN114657275B (en) | Primer pair combination, kit and detection method for detecting transgenic alfalfa | |
| CN114369676B (en) | Primer combination, kit, detection method and application for detecting transgenic components of tobacco |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190827 |
|
| WD01 | Invention patent application deemed withdrawn after publication |