CN109988858B - Multiple fluorescence PCR gene locus, primer and detection method of transgenic tobacco - Google Patents
Multiple fluorescence PCR gene locus, primer and detection method of transgenic tobacco Download PDFInfo
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Abstract
The application discloses a transgenic tobacco multiplex fluorescence PCR gene locus, a primer and a detection method thereof, which comprise loci, primers and probes of Nr, btc, cpTI, EPSPS, CMV-cp, PVY-cp, TMV-cp, CMV-sat, PVY-nib and TMV-54D genes. The application simultaneously uses endogenous genes and specific exogenous genes of the tobacco transgenic strain to design the specific primer probe set, and each primer probe can not cause mutual interference, can only amplify specific target sequences and excite fluorescent signals, has no amplification and fluorescent signals to non-target sequences, and has good specificity and high detection sensitivity.
Description
Technical Field
The application relates to the field of gene detection, in particular to a multiple fluorescence PCR gene locus, a primer and a detection method of transgenic tobacco.
Background
In 1983, the first transgenic crop in the world, namely transgenic tobacco, was developed successfully in the united states; in 1994, the extended fresh-keeping transgenic tomatoes of Monsanto company in the United states were approved for sale in the United states; transgenic crops have been widely used worldwide for decades, and have also led to numerous disputes.
The related specifications of the transgenic tobacco released in China are national tobacco law 1998 168, tobacco genetic engineering research and application management method, national tobacco science 2003 387, notification of enhanced monitoring of transgenic tobacco by national tobacco specialty agency, and several documents for enhanced export of transgenic tobacco detection are successively conveyed in 2005 to 2006. Tobacco has been found in spot inspection in some detection institutions because of its earliest research and its broader scope as a transgenic mode crop. Currently, PCR technology is the mainstream method for detecting and identifying transgenic products, and by detecting a foreign gene in the DNA of a sample, whether the sample contains the transgenic component is determined.
The extraction of DNA in the sample can be classified into automatic extraction from the operation mode, such as a self-checking workstation which is not yet popularized; semi-automated extraction, such as nucleic acid extractors that have become popular; manual extraction, such as the current common commercial kit, self-made reagent and the like.
The DNA loss of seeds and fresh tobacco leaves is small and complete, the three methods are applicable, the DNA purity OD260/OD280 is usually 1.6-1.8, the concentration is in ng/mu L level, and the DNA can be slightly diluted or directly used for PCR detection.
The tobacco leaves and cigarettes after baking have large DNA loss and are fragmented, and the yield of an automatic and semi-automatic method is lower; manual extraction is generally used. Because the types and research modes of the samples are very different, the manual extraction method is also widely varied and has obvious difference, and the effects are uneven.
The first generation PCR technology, commonly called ordinary PCR technology, designs the primer of the species specific gene, mixes the sample DNA with the primer and the amplifying reagent, then carries out repeated amplification to the target gene by a PCR instrument, and then identifies the amplified product by electrophoresis, thus judging whether the species source component is contained.
The second generation PCR technique is real-time quantitative fluorescent PCR, which is commonly abbreviated as fluorescent PCR. In addition to designing the primers for the species-specific genes, a fluorescent probe is designed. The sample DNA, the primer, the probe and the amplification reagent are uniformly mixed, and then the target gene is repeatedly amplified by a real-time quantitative fluorescent PCR instrument (usually abbreviated as fluorescent PCR instrument), the fluorescent signal accumulation of the probe is synchronous with the gene amplification, and compared with the common PCR, the specificity during the amplification is improved, and the detection result is obtained immediately after the amplification is finished.
Multiple real-time quantitative fluorescent PCR (hereinafter referred to as multiple fluorescent PCR) belongs to one kind of fluorescent PCR, and for a plurality of species-specific genes, primers and probes are designed one by one, and fluorescent groups with different wavelengths are respectively marked on different probes. And (3) uniformly mixing sample DNA, primers, probes and an amplification reagent, repeatedly amplifying a plurality of target genes by a multichannel fluorescent PCR instrument, and monitoring a plurality of fluorescent signals to meet the requirement of simultaneously detecting a plurality of target genes. Compared with the common fluorescent PCR, the detection efficiency can be doubled and the cost of other reagents can be reduced by one time by adding one set of primers and probes.
The patent application part of the PCR technology related to the identification of transgenic components in the Chinese patent database has hundreds of parts, but most of the parts are a common PCR method and a fluorescent PCR method, and the research on tobacco transgenes by utilizing the multiple fluorescent PCR technology is still blank in China, so the method for detecting the tobacco transgenes by providing the multiple fluorescent PCR is the current problem to be solved urgently.
Disclosure of Invention
The application provides a multiple fluorescence PCR gene locus, a primer and a detection method thereof for transgenic tobacco, and aims to provide a detection method for the transgenic tobacco, which has high efficiency, low cost and accurate detection result.
The specific technical scheme is as follows:
specific endogenous genes and exogenous genes selected in the present application include:
(1) Endogenous genes: nr, english name: nicotiana tabacum nitrate reductase; chinese name: tobacco nitroreductase.
(2) Exogenous genes/universal elements: caMV35S, english name: 35promoter form cauliflomer mosaic virus; chinese name: the cauliflower mosaic virus 35S promoter.
(3) Exogenous genes/universal elements: NOS, english name: nopaline synthase terminator; chinese name: nopaline synthase 3' transcription terminator.
(4) Exogenous genes/universal elements: NPT II, english name: neomycin phosphotransferase II; chinese name: neomycin-3' -phosphotransferase.
(5) Exogenous gene/strain gene: btc, english name: bacillus thuringiensis CryIA (b)/CryIA (c) fusion gene; chinese name: genes encoding the insecticidal crystal proteins of bacillus thuringiensis CryIA (b) and CryIA (c) are artificially spliced.
(6) Exogenous gene/strain gene: cpTI, english name: cowpea trypsin inhibitor; chinese name: cowpea trypsin inhibitor.
(7) Exogenous gene/strain gene: EPSPS, english name: 5-enolpyruvylshikimate-3-phosphate synthase; chinese name: 5-shikimate-3-phosphate synthase.
(8) Exogenous gene/strain gene: CMV-cp, english name: cucumber mosaic virus coat protein; chinese name: tobacco flower leaf coat protein.
(9) Exogenous gene/strain gene: PVY-cp, english name: potato Y virus coat protein; chinese name: potato virus Y coat protein.
(10) Exogenous gene/strain gene: TMV-cp, english name: tobacco mosaic virus coat protein; chinese name: tobacco mosaic virus coat protein.
(11) Exogenous gene/strain gene: CMV-sat, english name: cucumber mosaic virus microsatellite RNA; chinese name: cucumber mosaic virus microsatellite RNA.
(12) Exogenous gene/strain gene: PVY-Nib, english name: potato Y virus nuclear inclusion b; chinese name: potato virus Y replicase.
(13) Exogenous gene/strain gene: TMV-54kD, english name: tobacco mosaic virus 54kD protein; chinese name: tobacco mosaic virus replicase.
The application provides a multiplex fluorescence PCR gene locus for detecting transgenic tobacco, which comprises the following steps:
nr gene: the detection site is 501 to 630bp of JN384020.1 in Genbank with NCBI.
Btc gene: the detection site is 540 to 657bp of EU816953.1 of EU816953.1 in Genbank of NCBI.
CpTI gene: the detection site is 599 to 720bp of AJ271752.1 of AJ271752.1 in Genbank of NCBI.
EPSPS gene: the detection site is 243 to 614bp of AY592954.1 of AY592954.1 in Genbank of NCBI.
CMV-cp gene: the detection site is 312 to 436bp of KJ513406.1 of KJ513406.1 in Genbank of NCBI.
PVY-cp gene: the detection site is 317 to 407bp of KX376949.1 of KX376949.1 in Genbank of NCBI.
TMV-cp Gene: the detection site is 285 to 424bp of KJ508033.1 of KJ508033.1 in Genbank of NCBI.
CMV-sat Gene: the detection site is 224 to 358bp of EF363687.1 of EF363687.1 in Genbank of NCBI.
PVY-nib Gene: the detection site is 1305 to 1453bp of EF470240.1 of EF470240.1 in Genbank of NCBI.
TMV-54D Gene: the detection site is 559 to 651bp of X66047.1 of X66047.1 in Genbank of NCBI.
The multiplex fluorescence PCR gene locus also comprises CaMV35S, NPT II and NOS gene loci which are universal gene loci.
When the primers and probes of endogenous genes and strain genes are designed, tens of related sequences are searched in Genbank of NCBI, a conserved region is compared by using MegAlign of Lasergene v7.1.0, the primers and probes are designed by using Oligo v7.0.2, and the primers-Blast of NCBI are firstly used for checking, and finally the actual verification is carried out.
The application also provides a multiplex fluorescence PCR primer for detecting transgenic tobacco aiming at the multiplex fluorescence PCR gene locus, which comprises the following components:
a single primer of Nr gene, a triple primer of Btc, cpTI, EPSPS gene, a triple primer of CMV-cp, PVY-cp and TMV-cp genes, and a triple primer of CMV-sat, PVY-Nib and TMV-54kD genes aiming at the multiple fluorescence PCR gene loci;
the primers of the Nr gene are as follows: SEQ ID NO.1 and SEQ ID NO.2;
the primer of the Btc gene is as follows: SEQ ID NO.9 and SEQ ID NO.10;
the primers of the CpTI gene are as follows: SEQ ID NO.11 and SEQ ID NO.12;
the primers of the EPSPS gene are as follows: SEQ ID NO.13 and SEQ ID NO.14;
the CMV-cp gene primers are: SEQ ID NO.15 and SEQ ID NO.16;
the PVY-cp gene primers are as follows: SEQ ID NO.17 and SEQ ID NO.18;
the primers of the TMV-cp gene are as follows: SEQ ID NO.19 and SEQ ID NO.20;
the CMV-sat gene primers are as follows: SEQ ID NO.21 and SEQ ID NO.22;
the PVY-Nib gene primers are as follows: SEQ ID No.23 and SEQ ID No.24;
the primers of the TMV-54kD gene are as follows: SEQ ID No.25 and SEQ ID No.26.
The multiplex fluorescence PCR primer also comprises triple primers of CaMV35S, NPT II and NOS genes,
the primer of CaMV35S gene is: SEQ ID NO.3 and SEQ ID NO.4;
the primers for the NPT II gene are: SEQ ID NO.5 and SEQ ID NO.6;
the primers for the NOS gene are: SEQ ID NO.7 and SEQ ID NO.8;
the application also provides a multiplex fluorescence PCR probe for detecting transgenic tobacco, which is characterized by comprising a single probe of Nr gene, a triple probe of Btc, cpTI, EPSPS gene, a triple probe of CMV-cp, PVY-cp and TMV-cp gene, and a triple probe of CMV-sat, PVY-Nib and TMV-54kD gene;
the probe of the Nr gene is SEQ ID NO.27, and the fluorescent probe is modified into: 5'-FAM,3' -MGB;
the probe of the Btc gene is SEQ ID NO.31, and the fluorescent probe is modified into: 5'-FAM,3' -MGB;
the probe of the CpTI gene is SEQ ID NO.32, and the fluorescent probe is modified into: 5'-HEX,3' -MGB;
the probe of the EPSPS gene is SEQ ID NO.33, and the fluorescent probe is modified into: 5'-TAMRA,3' -MGB;
the probe of the CMV-cp gene is SEQ ID NO.34, and the fluorescent probe is modified into: 5'-FAM,3' -MGB;
the PVY-cp gene probe is SEQ ID NO.35, and the fluorescent probe is modified into: 5'-HEX,3' -MGB;
the probe of the TMV-cp gene is SEQ ID NO.36, and the fluorescent probe is modified into: 5'-TAMRA,3' -MGB;
the probe of the CMV-sat gene is SEQ ID NO.37, and the fluorescent probe is modified into: 5'-FAM,3' -MGB;
the PVY-Nib gene probe is SEQ ID NO.38, and the fluorescent probe is modified into: 5'-HEX,3' -MGB;
the probe of the TMV-54kD gene is SEQ ID NO.39, and the fluorescent probe is modified as follows: 5'-TAMRA,3' -MGB.
The multiplex fluorescence PCR probe also comprises triple probes of CaMV35S, NPT II and NOS genes, the probes of the CaMV35S genes are SEQ ID NO.28, and the fluorescent probes are modified into: 5'-FAM,3' -MGB;
the probe of NPT II gene is SEQ ID NO.29, and the fluorescent probe is modified into: 5'-HEX,3' -MGB;
the probe of NOS gene is SEQ ID NO.30, and the fluorescent probe is modified into: 5'-TAMRA,3' -MGB;
the application also provides a method for detecting multiple fluorescence PCR of the transgenic tobacco, wherein the transgenic tobacco is selected from seeds, fresh tobacco leaves, cigarettes after baking or cigarettes.
Preferably, the method comprises the steps of:
step 1: sample treatment is carried out, and sample DNA is extracted;
step 2: establishing a multiplex fluorescence PCR reaction system comprising the primer probe of claim 2 under the following reaction conditions: 20-120s or 10-15min at 95 ℃; collecting fluorescence signals after 5-60s at 95 ℃ and 20-120s at 60 ℃ for 40 cycles;
step 3: and judging the detection result of the transgenic components of the tobacco.
Preferably, the step 1 specifically includes:
when the samples are seeds and fresh tobacco leaves, extracting sample DNA by using a self-checking workstation, a nucleic acid extractor, a kit, a CTAB method and an SDS method;
when the samples are cured tobacco leaves and cigarettes, the sample DNA is extracted by adopting the following steps:
crushing a sample, adding n-hexane and CTAB extract, uniformly mixing, standing until the water phase and the organic phase are obviously layered, centrifuging, obviously layering the water phase and the organic phase, taking the water phase, centrifuging the water phase, taking a supernatant water phase, extracting DNA, and concentrating the DNA sample for subsequent detection.
Preferably, the mass of the crushed sample is not less than 10g;
the dosage of the n-hexane is the same as the volume of the sample;
the consumption of the CTAB extract is 20mL;
the rotational speed of the water phase and the organic phase is not less than 3k rpm, and the centrifugation time is not less than 10min;
the rotational speed of the water phase is not less than 6k rpm, and the centrifugation time is not less than 10min;
the DNA sample was concentrated to a volume of 10-100. Mu.L.
The method for amplification and detection on the multiplex fluorescence PCR instrument comprises the steps of preparing a reaction system by selecting 25 mu L or 50 mu L of a system, respectively preparing a single reaction system A aiming at Nr genes, a triple reaction system B aiming at CaMV35S, NPT II and NOS genes, a triple reaction system C aiming at Btc, cpTI, EPSPS genes, a triple reaction system D aiming at CMV-cp, PVY-cp and TMV-cp genes, setting reaction conditions and judging results according to judging conditions. To facilitate understanding of the judgment conditions, the relevant definitions will be briefly described.
Fluorescent signal detection gene: FAM fluorescent signal in system A detects Nr gene, FAM, HEX, TAMRA fluorescent signal in system B detects CaMV35S, NPT II and NOS genes respectively, FAM, HEX, TAMRA fluorescent signal in system C detects Btc, cpTI, EPSPS genes respectively, FAM, HEX, TAMRA fluorescent signal in system D detects CMV-cp, PVY-cp and TMV-cp genes respectively, FAM, HEX, TAMRA fluorescent signal in system E detects CMV-sat, PVY-Nib and TMV-54kD genes respectively.
Positive fluorescent signal detection: FAM, HEX or TAMRA has fluorescence logarithmic growth and Ct value less than or equal to 30.0; or 30.0< Ct <40.0 and the post-recheck Ct <40.0, both of which are positive for the fluorescent signal detection.
Fluorescent signal detection negative: FAM, HEX or TAMRA has fluorescence logarithmic growth and Ct value is more than or equal to 40.0; or 30.0< Ct value <40.0 and Ct value after recheck is more than or equal to 40.0, both of which are negative for the fluorescent signal detection.
Preferably, the judging method in the step 3 specifically includes:
(1) Quality control standard: the positive control Nr, caMV35S, NPT II, NOS, btc, cpTI, EPSPS, CMV-cp, PVY-cp, TMV-cp, CMV-sat, PVY-Nib and TMV-54kD all have fluorescence logarithmic growth, the Ct value is less than or equal to 30.0, the negative control and the blank control have no fluorescence signal and fluorescence logarithmic growth, the Ct value is more than or equal to 40.0, and the tobacco component detection in the step (2) is carried out;
(2) And (3) detecting tobacco components: sample Nr is negative in detection, which shows that the sample does not contain tobacco DNA, or the tobacco DNA content is insufficient for transgene component detection and judgment; detecting positive sample Nr, wherein the sample contains tobacco DNA, and judging the transgenic component of the step (3);
(3) Determination of transgenic Components: the CaMV35S or/and NPT II or/and NOS detection is negative, which indicates that the sample does not detect the transgenic components of the CaMV35S or/and NPT II or/and NOS; the CaMV35S or/and NPT II or/and NOS is detected positively, which shows that the sample detects transgenic components of the CaMV35S or/and NPT II or/and NOS, and the strain identification of the step (4) is carried out;
(4) And (3) strain identification: btc or/and CpTI or/and EPSPS or/and CMV-cp or/and PVY-cp or/and TMV-cp or/and CMV-sat or/and PVY-Nib or/and TMV-54kD detection is negative, which shows that the sample contains Btc or/and CpTI or/and EPSPS or/and CMV-cp or/and PVY-cp or/and TMV-cp or/and CMV-sat or/and PVY-Nib or/and TMV-54kD strain genes; btc or/and CpTI or/and EPSPS or/and CMV-cp or/and PVY-cp or/and TMV-cp or/and CMV-sat or/and PVY-Nib or/and TMV-54kD, indicating that the sample contains Btc or/and CpTI or/and EPSPS or/and CMV-cp or/and PVY-cp or/and TMV-cp or/and CMV-sat or/and PVY-Nib or/and TMV-54kD strain genes.
The reaction system A, B, C, D, E can be prepared together and then detected simultaneously according to the reaction conditions, so that the detection efficiency is improved; the reaction system A can be prepared for detection, the reaction system B can be prepared for detection if the result is positive, and the reaction system C, D, E can be prepared for detection if the result is positive, so that the operation and the reagent are saved.
The inventor screens Nr genes as tobacco endogenous genes and CaMV35S, NPT II and NOS as general elements of transgenic tobacco according to current literature reports, standard revision conditions and related research conditions, and the detection range can cover most of current commercial transgenic tobacco and products. In line identification, common transgenic tobacco traits are expressed through one or more exogenous functional genes such as Btc, cpTI, EPSPS, CMV-cp, PVY-cp, TMV-cp, CMV-sat, PVY-Nib, TMV-54kD and the like, and the line identification is carried out through corresponding gene detection. The application is favorable for fully detecting the transgenic components of different tobacco strains by combining the endogenous genes, the universal elements and the functional genes because the transgenic technology is developed rapidly and the transgenic tobacco strains in experimental study are numerous.
Furthermore, it should be noted that: the negative control in the method is marked as non-transgenic and non-tobacco source DNA, such as non-transgenic animal DNA, non-transgenic plant DNA except tobacco, and the like, and has wide sources and convenient preparation. But care should be taken: genes such as CaMV35S, NPT II, NOS, btc, cpTI, EPSPS, CMV-cp, PVY-cp, TMV-cp, CMV-sat, PVY-Nib and TMV-54kD are also obtained from corresponding species in nature and are applied to genetic engineering, and DNA of the corresponding species is not deliberately used as a negative control, so that experiments fail.
The application also provides a kit for detecting the multiplex fluorescence PCR of the transgenic tobacco, which comprises the PCR primer provided by the multiplex fluorescence PCR primer for detecting the transgenic tobacco, the PCR probe provided by the multiplex fluorescence PCR probe for detecting the transgenic tobacco, a fluorescence PCR reagent, a positive control, a negative control and a blank control.
Preferably, the positive control is a DNA fragment which can be amplified by the multiplex fluorescent PCR primer and can be detected by a PCR probe, and the concentration is 10 5 A copes/. Mu.L stage;
the PCR probes of claim 2, wherein the concentration of each probe is 10. Mu. Mol/L;
the PCR primers of claim 1 are all 10. Mu. Mol/L;
the negative control is non-transgenic and non-tobacco-derived DNA;
the blank was 3d H 2 O。
From the above technical scheme, the application has the following advantages:
(1) The application simultaneously uses endogenous genes and specific exogenous genes of the tobacco transgenic strain to design the specific primer probe set, and each primer probe can not cause mutual interference, can only amplify specific target sequences and excite fluorescent signals, has no amplification and fluorescent signals to non-target sequences, and has good specificity and high detection sensitivity.
(2) Compared with the common PCR method of the conventional related standard SN/T1200-2003 'qualitative PCR detection method of the transgene component in tobacco', the fluorescent PCR method of GB/T24310-2009 'transgene detection method of tobacco and tobacco products', the common PCR method, the nest PCR method and the semi-nest PCR method are mixed, the multiplex fluorescent PCR method is unified for detecting a plurality of target genes simultaneously, the detection efficiency is improved, and the practical application of detection personnel is facilitated.
(3) According to the application, the primer probe is designed according to the fusion gene Cry1A (b)/(c), namely Btc gene of Cry1Ab and Cry1Ac, so that the coverage of Cry1Ac in GB/T24310-2009 is wider. In addition, the length of the probe is in the normal range of 15-25bp, and the specificity is improved to a certain extent compared with 40bp of the Nr gene probe in GB/T24310-2009. Finally, non-fluorescent NFQ-MGB is selected as a quenching group, and the specificity of the fluorescent TAMRA is stronger.
(4) The detection method provided by the application is favorable for the full detection of the transgenic components of different strains of tobacco, and has smaller standard error (the initial concentration of the standard 3kbp plasmid is 10) compared with the multiplex PCR method 5 At the stage, the standard deviation of quantification is 10 4 The level), the detection time is shorter (the detection result is obtained after about 1h of the machine is started), and the generated toxic and harmful substances are fewer (the using amount of fluorescent dye is ng/mu L level); the relative fluorescence PCR method has the advantages of simultaneously detecting a plurality of target genes and saving reagent cost; compared with the common PCR method, the method has the two advantages.
(5) When the kit selects the outsourcing reagent suit suitable for the multiplex fluorescence PCR primer system, the model with the thermosensitive Taq antibody is selected, the nonspecific amplification caused by nonspecific annealing of the primer or primer dimer under the low temperature condition is inhibited, the storage time of the kit is prolonged, and more freeze thawing times are allowed; the fluorescent PCR reagent comprises ROX dye, and can improve detection accuracy for a multiplex fluorescent PCR instrument requiring the ROX dye.
(6) The primer, the probe, the kit and the method provided by the application are applied to the related scientific research of the unit (the detection limit is 0.01% by mass fraction), and the detection unit in the other same industry is verified to pass, so that hundreds of batches of samples have no detection omission and false detection conditions and are the same as the detection result of the standard method; practices show that compared with the standard method, the method disclosed by the application has the advantages of saving the operation time by more than 70%, saving the reagent cost by more than 70%, and having a good practical value.
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In order to more clearly illustrate the embodiments of the application or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the application, and that other drawings can be obtained from these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a diagram of the detection results of multiple fluorescent PCR gene loci, primers and detection methods of transgenic tobacco in an embodiment of the application.
Detailed Description
The technical solutions of the present application will be clearly and completely described in conjunction with the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments. Based on the embodiments of the present application, other embodiments that may be obtained by those of ordinary skill in the art without making any inventive effort are within the scope of the present application.
The multiple fluorescence PCR gene locus, primer and detection method of the transgenic tobacco provided by the application are further described below.
The application provides a multiplex fluorescence PCR primer probe set for detecting tobacco transgenic components, which is specifically shown in table 1: the kit comprises a single primer probe set aiming at Nr genes, a triple primer probe set aiming at CaMV35S, NPT II and NOS genes, a triple primer probe set aiming at Btc, cpTI, EPSPS genes, a triple primer probe set aiming at CMV-cp, PVY-cp and TMV-cp genes, and a triple primer probe set aiming at CMV-sat, PVY-Nib and TMV-54kD genes.
TABLE 1 primer probe mapping table
Embodiment one construction and verification of multiple fluorescent kit for detecting transgenic components of tobacco
(1) Primer probe group: as shown in Table 2, the primer and probe were synthesized by a company having the ability to synthesize the primer and probe, and in this example, the dried primer and probe powder was diluted to 100. Mu. Mol/L to prepare a stock solution, and 10. Mu. Mol/L was prepared as a use solution according to Table 1.
TABLE 2 preparation of 100. Mu. Mol/L stock solution to 10. Mu. Mol/L use solution
* : packaging with 1.5mL transparent centrifuge tubes; #: the tubes were individually packed in 1.5mL black centrifuge tubes.
(2) Fluorescent PCR reagents: common commercial fluorescent PCR reagents, selected in this examplePath-ID TM qPCR Master Mix。
(3) Positive control: all primer stock solutions were taken and diluted to 10. Mu. Mol/L, respectively. Extracting transgenic tobacco strain DNA containing Nr, caMV35S, NPT II, NOS, btc, cpTI, EPSPS, CMV-cp, PVY-cp, TMV-cp, CMV-sat, PVY-Nib and TMV-54kD, amplifying on ordinary PCR instrument with corresponding primer and ordinary PCR reagent, cutting target band, recovering, and transferring to TaKaRa T-Vector pMD TM 20 vector, replicated with competent E.coli cell JM109, extracted pMT-20 plasmid, diluted to 10 respectively 7 The concentration of the cobies/. Mu.L was 1. Mu.L each, and the mixture was mixed with 3. 3d H 2 O is fixed to 100 mu L, and the final concentration is 10 5 The copies/. Mu.L.
(4) Negative control: non-transgenic and non-tobacco-derived DNA was selected.
(5) Blank control: 3d H 2 O。
(6) The multichannel fluorescence PCR instrument was selected from ABI quantsudio 5, and the sample characteristics were confirmed as shown in Table 3, the reaction solution was prepared as shown in Table 4, and the reaction was performed as shown in Table 5.
Table 3 sample characteristics
TABLE 4 25. Mu.L reaction system (unit: mu.L)
TABLE 5 reaction conditions
(7) The reaction results are shown in fig. 1 and table 6:
in FIG. 1, curves 1 to 13, 14 to 26, 27 to 39, and 40 to 52 are fluorescence signals of positive control, negative control, addition control, nr (FAM) of blank control, caMV35S (FAM), NPT II (HEX), NOS (TAMRA), btc (FAM), cpTI (HEX), EPSPS (TAMRA), CMV-cp (FAM), PVY-cp (HEX), TMV-cp (TAMRA), CMV-sat (FAM), PVY-Nib (HEX), TMV-54kD (TAMRA), respectively.
Table 6 sample detection results: actual Ct value and S-shaped curve
(8) Verification result: the method is consistent with the result of detecting the transgenic tobacco components of the sample according to GB/T24310-2009 'method for detecting the transgenic tobacco and tobacco products'. The positive control, the standard adding control, the negative control and the blank control are arranged, so that the kit has the advantages of effective components, good specificity, small standard error, short detection time and reagent cost saving.
Example two detection of samples of three transgenic tobacco lines Btc/CpTI/EPSPS, CMV-cp/PVY-cp/TMV-cp, CMV-sat/PVY-Nib/TMV-54kD
Three transgenic tobacco line seeds and fresh tobacco leaves obtained from the agricultural sciences of Guizhou province, namely Btc/CpTI/EPSPS, CMV-cp/PVY-cp/TMV-cp, CMV-sat/PVY-Nib/TMV-54kD, are sequentially coded into #1, #2 and #3 samples. After ball milling and crushing the sample, extracting sample DNA on a small magnetic rack (Qiagen MagAttract Magnetic Rack) by using an outsourcing DNA extraction kit (QiagenMagAttract Hmw DNA kit); the DNA purities were OD260/OD 280=1.79, and the concentrations were 182.7 ng/. Mu.L, 150.3 ng/. Mu.L, 194.6 ng/. Mu.L, respectively, and were stored at-20 ℃. Then respectively diluting the DNA to 50 ng/. Mu.L for detection, wherein the primer needle group, the positive control, the negative control, the blank control and the like are all prepared according to the kit components in the first embodiment, and the outsourced fluorescent PCR reagent isMultiplex PCR Kit, on-machine detection (ABI fluorescence PCR instrument model number is Step one plus).
The test results are shown in Table 7 and Table 8:
(1) Quality control standard: the positive control Nr, caMV35S, NPT II, NOS, btc, cpTI, EPSPS, CMV-cp, PVY-cp, TMV-cp, CMV-sat, PVY-Nib and TMV-54kD all have fluorescence logarithmic growth, the Ct value is less than or equal to 30.0, the negative control and the blank control have no fluorescence signal and fluorescence logarithmic growth, the Ct value is more than or equal to 40.0, and the detection of sample components can be performed.
(2) All samples Nr tested positive, indicating that all samples contained tobacco DNA, and transgene component determination could be performed.
(3) All samples were positive for CaMV35S and NPT II and NOS, indicating that all samples detected transgenic components for CaMV35S and NPT II and NOS, and strain identification was performed.
(4) The# 1 tobacco samples Btc (FAM), cpTI (HEX), EPSPS (TAMRA) tested positive, indicating that the samples contained Btc, cpTI, EPSPS strain genes.
(5) The# 2 tobacco samples CMV-cp (FAM), PVY-cp (HEX) and TMV-cp (TAMRA) were tested positive, indicating that the samples contain CMV-cp, PVY-cp and TMV-cp strain genes.
(6) The# 3 tobacco samples CMV-sat (FAM), PVY-Nib (HEX) and TMV-54kD (TAMRA) were tested positive, indicating that the samples contain CMV-sat, PVY-Nib and TMV-54kD strain genes.
(7) Comparison result: the method is consistent with the result of detecting the transgenic tobacco components of the sample according to GB/T24310-2009 'method for detecting the transgenic tobacco and tobacco products'.
Table 7 control test results: actual Ct value and S-shaped curve
Table 8 sample detection results: actual Ct value and S-shaped curve
Example three detection of non-transgenic tobacco line samples
Non-transgenic tobacco samples (cured tobacco leaf powder) were obtained from the Guizhou quality control institute. (1) crushing a sample by a ball mill, wherein the mass of the crushed sample is 10.0g after treatment; (2) Adding 10mL of normal hexane and 20mL of CTAB extract, homogenizing for 6h at 120rpm in an air shaking table, and mixing evenly with each other or reversely; (3) Centrifuging at 10k rpm for 10min to separate the water phase and the organic phase, removing the organic phase and removing the water phase; (4) Centrifuging the obtained water phase at 10k rpm for 10min, and removing the supernatant water phase; (5) DNA extraction with a large-volume DNA extraction kit (Qiagen DNeasy Plant Maxi Kit); (6) The DNA sample was concentrated to 10. Mu.L by a column membrane concentration tube (Millipore Microcon DNA fast flow (PCR grade)) and stored at-20℃with a DNA purity of OD260/OD 280=1.71 and a DNA concentration of 8.60 ng/. Mu.L.
The primer group, the probe group, the positive control, the negative control and the blank control are all the kit components in the first embodiment, and the outsourced fluorescent PCR reagent is ABI TaqMan TM Environmental Master Mix2.0, on-machine detection (ABI fluorescence PCR instrument)Model 7500 Fast).
The detection results are shown in Table 9:
(1) Quality control standard: the positive control Nr, caMV35S, NPT II, NOS, btc, cpTI, EPSPS, CMV-cp, PVY-cp, TMV-cp, CMV-sat, PVY-Nib and TMV-54kD all have fluorescence logarithmic growth, the Ct value is less than or equal to 30.0, the negative control and the blank control have no fluorescence signal and fluorescence logarithmic growth, the Ct value is more than or equal to 40.0, and the detection of sample components can be performed.
(2) The sample Nr is detected positively, which shows that the sample contains tobacco DNA and can be used for judging transgenic components.
(3) Samples CaMV35S and NPT II and NOS are negative, which indicates that all samples do not detect transgenic components of CaMV35S and NPT II and NOS, and are non-transgenic tobacco, and strain identification is not needed.
Table 9 sample test results: actual Ct value and S-shaped curve
In conclusion, the multiplex fluorescence PCR primer, the probe, the method and the kit provided by the application can be used for carrying out specific amplification on the target genes of tobacco and transgenic tobacco, generating fluorescence signals in the amplification, effectively being applicable to the detection of transgenic components of tobacco related products, conforming to the result of a related standard detection method, saving the detection time, reducing the detection cost and having better practical value.
The above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application.
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Claims (5)
1. A method for detecting multiplex fluorescence PCR of transgenic tobacco, comprising the steps of:
step 1: sample treatment is carried out, and sample DNA is extracted;
step 2: establishing a multiplex fluorescence PCR reaction system of a multiplex fluorescence PCR primer probe of the transgenic tobacco, wherein the reaction conditions are as follows: 20-120s or 10-15min at 95 ℃; collecting fluorescence signals after 5-60s at 95 ℃ and 20-120s at 60 ℃ for 40 cycles;
step 3: the judgment of the detection result of the transgenic components of the tobacco comprises the following steps: (1) quality control standard: the positive control Nr, caMV35S, NPT II, NOS, btc, cpTI, EPSPS, CMV-cp, PVY-cp, TMV-cp, CMV-sat, PVY-Nib and TMV-54kD all have fluorescence logarithmic growth, the Ct value is less than or equal to 30.0, the negative control and the blank control have no fluorescence signal and fluorescence logarithmic growth, the Ct value is more than or equal to 40.0, and the tobacco component detection in the step (2) is carried out;
(2) And (3) detecting tobacco components: sample Nr is negative in detection, which shows that the sample does not contain tobacco DNA, or the tobacco DNA content is insufficient for transgene component detection and judgment; detecting positive sample Nr, wherein the sample contains tobacco DNA, and judging the transgenic component of the step (3);
(3) Determination of transgenic Components: the CaMV35S or/and NPT II or/and NOS detection is negative, which indicates that the sample does not detect the transgenic components of the CaMV35S or/and NPT II or/and NOS; the CaMV35S or/and NPT II or/and NOS is detected positively, which shows that the sample detects transgenic components of the CaMV35S or/and NPT II or/and NOS, and the strain identification of the step (4) is carried out;
(4) And (3) strain identification: btc or/and CpTI or/and EPSPS or/and CMV-cp or/and PVY-cp or/and TMV-cp or/and CMV-sat or/and PVY-Nib or/and TMV-54kD detection is negative, which shows that the sample does not contain Btc or/and CpTI or/and EPSPS or/and CMV-cp or/and PVY-cp or/and TMV-cp or/and CMV-sat or/and PVY-Nib or/and TMV-54kD strain genes; btc or/and CpTI or/and EPSPS or/and CMV-cp or/and PVY-cp or/and TMV-cp or/and CMV-sat or/and PVY-Nib or/and TMV-54kD, indicating that the sample contains Btc or/and CpTI or/and EPSPS or/and CMV-cp or/and PVY-cp or/and TMV-cp or/and CMV-sat or/and PVY-Nib or/and TMV-54kD strain genes;
the transgenic tobacco is selected from seeds, fresh tobacco leaves, cigarettes or cigarettes after baking;
the multiplex fluorescence PCR primer of the transgenic tobacco comprises:
a single primer of Nr gene aiming at multiple fluorescence PCR gene loci of transgenic tobacco, a triple primer of CaMV35S, NPT II and NOS genes, a triple primer of Btc, cpTI, EPSPS genes, a triple primer of CMV-cp, PVY-cp and TMV-cp genes, and a triple primer of CMV-sat, PVY-Nib and TMV-54kD genes;
the primers of the Nr gene are as follows: SEQ ID NO.1 and SEQ ID NO.2;
the primer of the CaMV35S gene is as follows: SEQ ID NO.3 and SEQ ID NO.4;
the primers of the NPT II gene are as follows: SEQ ID NO.5 and SEQ ID NO.6;
the primers of the NOS gene are as follows: SEQ ID NO.7 and SEQ ID NO.8;
the primer of the Btc gene is as follows: SEQ ID NO.9 and SEQ ID NO.10;
the primers of the CpTI gene are as follows: SEQ ID NO.11 and SEQ ID NO.12;
the primers of the EPSPS gene are as follows: SEQ ID NO.13 and SEQ ID NO.14;
the CMV-cp gene primers are: SEQ ID NO.15 and SEQ ID NO.16;
the PVY-cp gene primers are as follows: SEQ ID NO.17 and SEQ ID NO.18;
the primers of the TMV-cp gene are as follows: SEQ ID NO.19 and SEQ ID NO.20;
the CMV-sat gene primers are as follows: SEQ ID NO.21 and SEQ ID NO.22;
the PVY-Nib gene primers are as follows: SEQ ID No.23 and SEQ ID No.24;
the primers of the TMV-54kD gene are as follows: SEQ ID No.25 and SEQ ID No.26;
a multiplex fluorescent PCR gene locus for transgenic tobacco comprising:
nr gene locus JN384020.1;
btc locus EU816953.1;
CpTI locus AJ271752.1;
EPSPS gene locus AY592954.1;
CMV-cp locus KJ513406.1;
PVY-cp gene locus KX376949.1;
TMV-cp gene locus KJ508033.1;
the CMV-sat gene locus is EF363687.1;
the PVY-nib gene locus is EF470240.1;
TMV-54D gene locus X66047.1;
the multiplex fluorescence PCR probes of the transgenic tobacco comprise a single probe of Nr gene, triple probes of CaMV35S, NPT II and NOS genes, a triple probe of Btc, cpTI, EPSPS genes, triple probes of CMV-cp, PVY-cp and TMV-cp genes, and triple probes of CMV-sat, PVY-Nib and TMV-54kD genes;
the probe of the Nr gene is SEQ ID NO.27, and the fluorescent probe is modified into: 5'-FAM,3' -MGB;
the probe of the CaMV35S gene is SEQ ID NO.28, and the fluorescent probe is modified as follows: 5'-FAM,3' -MGB;
the probe of the NPT II gene is SEQ ID NO.29, and the fluorescent probe is modified into: 5'-HEX,3' -MGB;
the probe of the NOS gene is SEQ ID NO.30, and the fluorescent probe is modified into: 5'-TAMRA,3' -MGB;
the probe of the Btc gene is SEQ ID NO.31, and the fluorescent probe is modified into: 5'-FAM,3' -MGB;
the probe of the CpTI gene is SEQ ID NO.32, and the fluorescent probe is modified into: 5'-HEX,3' -MGB;
the probe of the EPSPS gene is SEQ ID NO.33, and the fluorescent probe is modified into: 5'-TAMRA,3' -MGB;
the probe of the CMV-cp gene is SEQ ID NO.34, and the fluorescent probe is modified into: 5'-FAM,3' -MGB;
the PVY-cp gene probe is SEQ ID NO.35, and the fluorescent probe is modified into: 5'-HEX,3' -MGB;
the probe of the TMV-cp gene is SEQ ID NO.36, and the fluorescent probe is modified into: 5'-TAMRA,3' -MGB;
the probe of the CMV-sat gene is SEQ ID NO.37, and the fluorescent probe is modified into: 5'-FAM,3' -MGB;
the PVY-Nib gene probe is SEQ ID NO.38, and the fluorescent probe is modified into: 5'-HEX,3' -MGB;
the probe of the TMV-54kD gene is SEQ ID NO.39, and the fluorescent probe is modified as follows: 5'-TAMRA,3' -MGB.
2. The method according to claim 1, wherein the step 1 is specifically:
when the samples are seeds and fresh tobacco leaves, extracting sample DNA by using a self-checking workstation, a nucleic acid extractor, a kit, a CTAB method and an SDS method;
when the samples are cured tobacco leaves and cigarettes, the sample DNA is extracted by adopting the following steps:
crushing a sample, adding n-hexane and CTAB extract, uniformly mixing, standing until the water phase and the organic phase are obviously layered, centrifuging, obviously layering the water phase and the organic phase, taking the water phase, centrifuging the water phase, taking a supernatant water phase, extracting DNA, and concentrating the DNA sample for subsequent detection.
3. The method according to claim 2, wherein the mass of the crushed sample is not less than 10g;
the dosage of the n-hexane is the same as the volume of the sample;
the consumption of the CTAB extract is 20mL;
the rotational speed of the water phase and the organic phase is not less than 3k rpm, and the centrifugation time is not less than 10min;
the rotational speed of the water phase is not less than 6k rpm, and the centrifugation time is not less than 10min;
the DNA sample was concentrated to a volume of 10-100. Mu.L.
4. A kit for detecting multiplex fluorescence PCR of transgenic tobacco, which is characterized by comprising multiplex fluorescence PCR gene loci, multiplex fluorescence PCR primers, multiplex fluorescence PCR probes, fluorescence PCR reagents, positive control, negative control and blank control in the method of claim 1.
5. The kit according to claim 4, wherein the positive control is a DNA fragment which can be amplified by the multiplex fluorescent PCR primer according to claim 1 and can be detected by the PCR probe, and the concentration is 10 5 A copes/. Mu.L stage;
the concentration of the PCR probes is 10 mu mol/L;
the concentration of the PCR primers is 10 mu mol/L;
the negative control is non-transgenic and non-tobacco-derived DNA;
the blank was 3d H 2 O。
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