CN111621585B - Rapid detection kit for simultaneously detecting multiple transgenic rape lines and application thereof - Google Patents
Rapid detection kit for simultaneously detecting multiple transgenic rape lines and application thereof Download PDFInfo
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Abstract
The invention relates to a PCR micro-fluidic chip specially used for identifying transgenic rape strains. The invention designs and screens a series of PCR amplification primers of common transgenic rape elements with high specificity and high accuracy, which are suitable for a microfluidic chip, and the primers are combined to the PCR microfluidic chip by freeze-drying, thereby realizing high-throughput screening detection of endogenous genes and a plurality of exogenous genes in one PCR amplification reaction. The PCR microfluidic chip of the pre-freezing primer has good stability under the conditions of normal temperature and repeated freezing and thawing, and has good commercial application value.
Description
Technical Field
The invention relates to the technical field of agricultural transgenic detection, in particular to a microfluidic fluorescence PCR chip kit containing a primer group capable of screening 10 transgenic rape lines simultaneously and application thereof.
Background
Transgenic crops are grown commercially on a large scale in 1996, and the global planting area in 2017 reaches 1.898 hundred million hectares. Since the first commercialization of transgenic crops, transgenic crops have been accompanied by different voices. On one hand, the transgenic crops really greatly increase the yield of the crops, reduce the use of pesticides and protect the environment; on the other hand, however, the potential uncertainty of transgenic crops with respect to environment, society and economy is under question in many ways. Therefore, it has become an international practice to strengthen the safety supervision of the transgenosis and establish the identification system of the transgenosis products, and countries and regions such as the united states, european union, japan, korea and the like implement quantitative identification, while China implements qualitative identification of 'zero threshold', that is, the transgenosis products only need to be identified no matter the content of the transgenosis products which are included in the identification catalog.
With the rapid development of transgenic plant research, biotechnology is becoming the crop technology most rapidly applied worldwide. The average use of transgenic crops (average use of soybean, corn and oilseed rape) in five transgenic crop-growing countries has been approaching saturation, with 93.3% in the united states, 93% in brazil, 100% in argentina, 92.5% in canada, 95% in india. Rape is one of the main sources of edible oil for people, and the cultivation history is very long. China and india are the oldest countries in the world in which rape is cultivated. Typically, rapeseed has an oil content of about 40%. The rapeseed cultivated by the transgenic technology can greatly improve the oil yield and has better purity and quality of oil. However, there is much debate about the safety of transgenic crops. The method has important practical significance for detecting the transgenic rape in the face of impact of the biological genetic engineering technology on economic benefits of China and possible risks of foreign transgenic products on ecological environment and consumers.
At present, the detection method of the transgenic crops mainly comprises two types of detection methods based on protein and detection methods based on nucleic acid; the former method mainly comprises an immune test strip method, an enzyme-linked immunosorbent assay, a western blotting method and the like. Among the latter, the most widely used is the qualitative and quantitative PCR detection technique. The PCR detection method for the transgenic components mainly comprises a TaqMan real-time fluorescent PCR method and a common PCR method. The common PCR method is gradually replaced by the real-time fluorescence PCR detection method due to the defects of complex operation, environmental pollution, low sensitivity and the like. However, the single-channel TaqMan real-time fluorescence PCR method has high detection cost and complicated steps, and is not suitable for multi-line screening detection of transgenic products. Particularly, for the detection department with large business volume, a simple, quick, sensitive, accurate, time-saving and labor-saving efficient detection method needs to be developed.
In order to solve the problems, the PCR microfluidic chip detection technology is applied to the high-throughput screening detection of the transgenic rape products. In the process of one-time PCR amplification, screening and detection of 1 endogenous gene (CruA gene) and 10 commercial transgenic lines such as MO88302, 73496, Ms1, Rf2, Rf1, Topas19/2, GT73, Ms8, Rf3 and T45 are simultaneously completed. Compared with a single detection method, the method can simultaneously detect endogenous genes and a plurality of exogenous genes in one PCR amplification reaction, and can effectively reduce errors caused by experimental misoperation; can realize high-throughput screening detection of multiple samples and multiple gene targets.
Disclosure of Invention
The PCR microfluidic chip is prepared by using MEMS (Micro-Electro-Mechanical System) technology and microfluidic technology, and is an advanced in-vitro nucleic acid amplification technology. The micro-fluidic chip has the characteristics of small volume, large specific surface area, high integration level, high reaction speed, high heat transfer and the like. A series of micro-channels, micro-reaction chambers and various microcontrollers are processed on a substrate material such as silicon, glass, plastic, high polymer and the like, so that PCR can be rapidly amplified on a chip. Compared with the conventional PCR amplification technology, the PCR microchip has the advantages of high efficiency, high speed, less reagent consumption, easy carrying, high integration level and the like, is one of the hot spots of research in recent years, and has wide application in various fields of molecular biology, disease detection, biotechnology, immunology, genome engineering, clinical medicine, environmental detection and the like.
The invention aims to provide a micro-fluidic chip capable of screening multiple transgenic exogenous genes simultaneously and an application technology thereof, so that endogenous genes and multiple exogenous genes can be detected simultaneously in one PCR amplification reaction, and high-throughput screening detection of multiple gene targets is realized.
The invention provides a primer pair composition for identifying a transgenic rape line, which comprises one or more primer pairs as follows:
an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2 for detecting an endogenous gene Cru A;
an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.4 for detecting the rape MON 88302;
an upstream primer SEQ ID NO.5 and a downstream primer SEQ ID NO.6 for detecting the rape 73496;
an upstream primer SEQ ID NO.7 and a downstream primer SEQ ID NO.8 for detecting the rape Ms 1;
an upstream primer SEQ ID NO.9 and a downstream primer SEQ ID NO.10 for detecting the rape Rf 2;
an upstream primer SEQ ID NO.11 and a downstream primer SEQ ID NO.12 for detecting the rape Rf 1;
an upstream primer SEQ ID NO.13 and a downstream primer SEQ ID NO.14 for detecting the rape Topas 19/2;
an upstream primer SEQ ID NO.15 and a downstream primer SEQ ID NO.16 for detecting the rape GT 73;
an upstream primer SEQ ID NO.17 and a downstream primer SEQ ID NO.18 for detecting the rape Ms 8;
an upstream primer SEQ ID NO.19 and a downstream primer SEQ ID NO.20 for detecting the rape Rf 3;
an upstream primer SEQ ID NO.21 and a downstream primer SEQ ID NO.22 for detecting the rape T45.
Specific primer sequences are shown in table 1 below:
table 1: primer sequence Listing of this application
In research, the inventors of the present application found that the existing primer pair is not directly applicable to a microfluidic chip, and has the defects of difficulty in combining the primer pair to a reaction plate, low amplification efficiency on the reaction plate, and inaccurate result; based on the reasons, the primer pair combination is obtained by aiming at the detection design and adjustment of the applied microfluidic chip, and is more suitable for the detection of the microfluidic chip; and the primer is compared with the primer in the European Union CRL detection method to find that the primer can obtain the detection accuracy which is the same as that of the European Union CRL detection method.
Further, the invention provides a kit for identifying a transgenic rape line, which contains one or more primer pairs.
Preferably, the primer pair of the invention is pre-frozen on a PCR microfluidic chip, the primer is lyophilized in the form of a mixed solution with taurine, and the concentration of the primer and the taurine in the specific solution is as follows: PCR forward and reverse primers are respectively 10uM, and taurine is 20 mmol/L; the PCR microfluidic chip prepared by using the solution and pre-frozen with the PCR primers can be repeatedly frozen and thawed for up to 20 times, still can keep good amplification performance, and can also well keep the amplification performance after being stored at normal temperature for a long time.
Preferably, the kit of the present invention comprises a multi-channel microfluidic chip, and further preferably, the nucleic acid combination is pre-frozen in a pore channel of the chip, the primer is lyophilized in the form of a mixed solution with taurine, and the concentration of the primer and taurine in the specific solution is as follows: PCR forward and reverse primers are respectively 10uM, and taurine is 20 mmol/L; the chip is shown in fig. 1, in which fig. 1 is the channel.
More preferably, the kit further comprises a premix, and the premix comprises the following components: TTX enzyme, 2xBuffer, EVA Green and DEPC water; preferably, the premix consists of 1.6uL of TTX enzyme, 40uL of 2xBuffer, 4uL of EVA Green and 2.4uL of DEPC water.
The inventor of the application finds that the stability of the primer can be obviously improved by adding the taurine into the chip, so that the chip has good stability at normal temperature, and the primer can not be degraded basically in a week; meanwhile, the stability of the chip in a freeze-dried state is also obviously superior to that of the chip without taurine; the function of the freeze-thaw agent is not affected basically even after repeated 20 times of freeze thawing; and when the taurine solution is not added, the taurine solution is basically degraded when being frozen and thawed for 5 to 8 times, and the function of the taurine solution is lost.
Therefore, the invention provides the PCR microfluidic chip with the good stability and the PCR primers pre-frozen, wherein the chip has good stability at normal temperature and in a freeze-dried state, and can be repeatedly frozen and thawed for more than 20 times. The chip and the kit have good stability and are particularly suitable for commercial popularization.
Drawings
FIG. 1: schematic diagram of the microfluidic chip: in the figure, 1 is the detection pore channel, and PCR primers can be pre-frozen;
FIG. 2: transgenic rape MON88302 standard and sample detection curve: (1) a standard substance detection amplification curve diagram is obtained; (2) the detection curve is a transgenic rape MON88302 sample.
Detailed Description
Example 1: PCR detection method and sensitivity and specificity experiment
(1) Materials and methods
Transgenic standards were purchased from either the us standard AOCS or european standard ERM including: transgenic oilseed rape Topas19/2(AOCS0711-D4), transgenic oilseed rape RF2(AOCS0711-C3), transgenic oilseed rape Ms1(AOCS0711-A3), transgenic oilseed rape Ms8(AOCS0306-F7), transgenic oilseed rape Rf3(AOCS0306-G6), transgenic oilseed rape T45(AOCS0208-A6), transgenic oilseed rape Rf1(AOCS0711-B2), transgenic oilseed rape GT73(AOCS0304-B2), transgenic oilseed rape MON88302(AOCS1011-A), non-transgenic oilseed rape (AOCS 0304-A2), transgenic soybean MON89788 (CS 0906-B2), non-transgenic soybean (CS 0906-A), transgenic corn 21(AOCS 467-AOCS 0407), non-transgenic oilseed rape AOCS 0304-A2), transgenic soybean MON89788 (CS 0906-B2), non-transgenic soybean BF 2(AOCS 0403-BF 2), transgenic soybean BF 3-BF 3, transgenic rapeseed, Transgenic soybean DAS-81419-2(ERM-BF437e), transgenic corn DAS40278-9(ERM-BF433d) and other standard products. Samples of 16 transgenic rapes, transgenic soybeans, transgenic corn and the like were collected from positive samples obtained in daily tests.
(2) Extraction of template DNA
The test materials were ground in liquid nitrogen. The extraction of genomic DNA was carried out using a DNA extraction kit of Genetic Modified Organization (GMO) detection Ver2.0(No. D9093; TaKaRa, Dalian, China) kit of TaKaRa. The extracted genomic DNA was dissolved in 100. mu.L of Tris-EDTA (TE) solution. The concentration of the purified DNA sample was measured by an ultraviolet spectrophotometer (ND-1000; NanoDrop, Wilmington, DE).
(3) Design of ultrafast fluorescent PCR microfluidic chip
The ultra-fast fluorescence PCR detection system adopts a multi-channel micro-fluidic chip technology to realize the fast detection of a plurality of samples on a chip of a few centimeters, and all reactions can be completed within 20 minutes. Compared with the prior multiplex PCR technology, the multiplex diagnosis of 12 sample targets can be realized; because each reaction chamber is completely isolated from each other, each reaction is completely independent, and the primer competition problem typically existing in the multiplex PCR detection technology can not occur; the test procedure is simple-it is only necessary to mix reagents and DNA templates and then distribute them into the chip. A large number of experiments verify that the rapid detection system has high sensitivity, high specificity and high stability.
(4) Working process of micro-fluidic chip
Mixing the extracted sample nucleic acid solution 12uL (60-100ng/uL) with the freeze-dried PCR premix 48uL, then adding the mixture into a reaction pore channel of the microfluidic chip, wherein the total volume of the premix added into the chip and the nucleic acid is 55 uL; the premixed solution comprises 1.6uL of TTX enzyme, 40uL of 2xBuffer, 4uL of EVA Green and 2.4uL of DEPC water.
Amplifying after setting a program on a computer, and automatically analyzing and judging a result by software after 20-30 minutes; the specific reaction procedure is as follows:
(5) specificity of microfluidic chip detection
The method is characterized in that 15 transgenic standard products of transgenic rape Topas19/2, transgenic rape RF2, transgenic rape Ms1, transgenic rape Ms8, transgenic rape Rf3, transgenic rape T45, transgenic rape Rf1, transgenic rape GT73, transgenic rape MON88302, non-transgenic rape, non-transgenic soybean, non-transgenic corn, transgenic rape 73496, transgenic soybean GTS-40-3-2, transgenic corn 59122 and the like are subjected to inter-strain cross reaction tests, and the specificity of primers is verified. When the detection is carried out, the existence of the transgenic element can be judged only when the fluorescence threshold value (Ct value) and the melting temperature (Tm value) of the amplification product both accord with the positive judgment standard. As for the Tm value, when the Tm value of the test sample is within. + -. 1 ℃ of the Tm value of the positive sample, amplification positive is judged, and when the Tm value is outside the range, amplification negative is judged. With respect to the Ct value, amplification is determined to be positive when the test sample is exponentially amplified and the fluorescence signal is above a threshold value. In addition, other results can be judged as amplification negatives.
Taking MON88302 as an example (using primers SEQ ID NO.1-4), the detection result shows (as shown in FIG. 2), when the positive standard of transgenic rape MON88302(AOCS1011-A) is detected by the process in the step (4), the Tm value of the endogenous gene CruA is 79.2, and the Tm value of the standard MON88302 is 74.7; when the known transgenic rape MON88302 is detected, the Tm value of the endogenous gene CruA is 79.1, and the Tm value of the MON88302 is 74.1; as can be seen, Ct values also amplify exponentially, with fluorescence signals above the threshold. And according to the detection result, the judgment standard of a positive result is met. Endogenous positive indicates that the DNA of the sample meets the detection standard, and MON88302 detection gene positive indicates that a MON88302 specific strain is detected; while the other 14 samples were tested using primers against MON88302, both Tm and Ct values were negative. The other primer pairs (SEQ ID NO.5-22) of the present application were verified separately using the same method, and the results were similar to those of the above experiment, see Table 2 specifically.
Table 2: the application of the detection result of the microfluidic chip sample
As can be seen from the results shown in Table 2, the primers of the present application are all capable of detecting positive results for the corresponding transgenic varieties, but negative for other types of transgenic samples. The results of this example demonstrate that the microfluidic chip of the present application has good specificity, and can accurately detect whether a sample contains a transgenic component.
(6) Comparison of primers of the present application with existing primer sequences
By using the methods in the steps (4) to (5), primers in the European Union CRL detection method are respectively verified, and the detection results are found to be similar in accuracy and can achieve the same detection accuracy, but the detection chip can simultaneously complete detection of more than ten samples, and the efficiency is far higher than that of the original European Union CRL detection method. Meanwhile, the application also tries to combine the primers in the European Union CRL detection method into the chip and carries out detection according to the method, and the result shows that the chip combined by the primers can only achieve the detection results with the same precision on transgenic rape Topas19/2(AOCS0711-D4), transgenic rape Ms8(AOCS0306-F7), transgenic rape Rf1(AOCS0711-B2) and endogenous gene Cru, while the detection results on other transgenic rape RF2(AOCS0711-C3), transgenic rape Ms1(AOCS0711-A3), transgenic rape Rf3(AOCS0306-G6), transgenic T45(AOCS0208-A6), transgenic rape GT73(AOCS0304-B2), transgenic rape MON88302 (CS 1011-A) and transgenic rape 73496(ERM-BF 434) are negative. The reason for the analysis may be that the primers in the detection method of CRL of european union are not suitable for use in the microfluidic chip due to their length or for their location in detecting the gene target, and most of them cannot be used in the microfluidic chip.
(7) Sensitivity of microfluidic chip detection
10 standard products of refined extracted DNA of transgenic rape Topas19/2, transgenic rape RF2, transgenic rape Ms1, transgenic rape Ms8, transgenic rape Rf3, transgenic rape T45, transgenic rape Rf1, transgenic rape GT73, transgenic rape MON88302, transgenic rape 73496 and the like are respectively diluted to a plurality of gradients of 50ng/uL, 25ng/uL, 5ng/uL, 1ng/uL and 0.5ng/uL, 12 mu l of refined extracted DNA of each dilution is respectively taken for carrying out a microfluidic chip experiment, reaction is carried out according to a set program so as to determine the detection sensitivity, and meanwhile, the reaction time is determined according to the minimum sensitivity peak-out time and the false positive earliest peak-out time. The results are shown in the table. According to sensitivity test data, when the DNA concentration of the detection sample is diluted to 5ng/uL, the DNA can still be stably detected; the lowest sensitivity of the detection chip is determined to be 5 ng/uL.
Example 2: micro-fluidic chip stability verification
The stability of the microfluidic chip is verified, and the microfluidic chip which is not added with taurine and is freeze-dried is used as a reference to verify the detection performance of the chip under the conditions of normal temperature (25 ℃), -20 ℃ and repeated freeze thawing.
The CRUA, transgenic rape RF2 and transgenic rape T45 detecting chips are respectively placed at normal temperature (25 ℃) and-20 ℃ for 3 months, 6 months and 12 months for testing, and the stability of the kit is detected. A verification test was carried out using transgenic rapeseed Topas19/2(AOCS0711-D4) at a concentration of 5 ng/uL. The experimental results are shown in the following table 3, the micro-fluidic chip of the invention is placed at normal temperature (25 ℃) for 3 months, 6 months and 12 months, the Ct value and the Tm value have no obvious difference, and the detection sensitivity has no obvious difference; when the kit is placed at the temperature of minus 20 ℃ for 3 months, 6 months and 12 months, the Ct value and the Tm value are not obviously different, and the detection sensitivity is not obviously different. The control group had good stability at-20 ℃ and maintained good sensitivity even after 12 months of storage, but the Ct value and Tm value had changed greatly when the control group was left at room temperature (25 ℃) for 3 months, and the related detection was completely impossible at 6 months. The result shows that the microfluidic chip of the application has stability obviously superior to that of the contrast.
Table 3: experimental results on stability of primers
In addition, repeated freezing and thawing are respectively carried out on the microfluidic chip and the control group by CruA and RF2, and experiments show that the Ct value and the Tm value of the microfluidic chip are not obviously different when the microfluidic chip is repeatedly frozen and thawed for 20 times, but the result of the control group is obviously different after 5 times of freezing and thawing, and the effective Ct value and the Tm value cannot be detected after 10 times of freezing and thawing. The specific results are shown in Table 4.
Table 4: repeated freeze thawing stability detection result of PCR microfluidic chip
| Chip and method for manufacturing the same | Initiation of | 5 times (twice) | 10 times of | 15 times of |
| CruA (application) | 78.8 | 78.6 | 78.5 | 78.1 |
| RF2 (application) | 76.8 | 76.9 | 76.8 | 76.2 |
| CruA (control) | 78.7 | 52.6 | - | - |
| RF2(control) | 77.0 | 46.2 | - | - |
Claims (3)
1. A kit for identifying a transgenic rape line comprises a micro-fluidic chip primer pair composition and a micro-fluidic chip for identifying the transgenic rape line, wherein the primer pair is frozen in a detection pore channel of the chip, and the kit is characterized in that the primer pair composition comprises the following primer pairs:
an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2 for detecting an endogenous gene Cru A;
an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.4 for detecting the rape MON 88302;
an upstream primer SEQ ID NO.5 and a downstream primer SEQ ID NO.6 for detecting the rape 73496;
an upstream primer SEQ ID NO.7 and a downstream primer SEQ ID NO.8 for detecting the rape Ms 1;
an upstream primer SEQ ID NO.9 and a downstream primer SEQ ID NO.10 for detecting the rape Rf 2;
an upstream primer SEQ ID NO.11 and a downstream primer SEQ ID NO.12 for detecting the rape Rf 1;
an upstream primer SEQ ID NO.13 and a downstream primer SEQ ID NO.14 for detecting the rape Topas 19/2;
an upstream primer SEQ ID NO.15 and a downstream primer SEQ ID NO.16 for detecting the rape GT 73;
an upstream primer SEQ ID NO.17 and a downstream primer SEQ ID NO.18 for detecting the rape Ms 8;
an upstream primer SEQ ID NO.19 and a downstream primer SEQ ID NO.20 for detecting the rape Rf 3;
an upstream primer SEQ ID NO.21 and a downstream primer SEQ ID NO.22 for detecting the rape T45;
wherein, the primer is lyophilized in the form of mixed liquid with taurine, and the concentration of the primer and the taurine in the specific solution is as follows: PCR forward and reverse primers were 10uM each, and taurine was 20 mmol/L.
2. The kit of claim 1, wherein: the kit also contains a premix comprising TTX enzyme, 2xBuffer, EVA Green, and DEPC water.
3. The kit of claim 2, wherein: TTX enzyme 1.6uL, 2xBuffer40uL, EVA Green 4uL and DEPC water 2.4uL in the premix.
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