CN114622028B - Primer pair combination, kit and detection method for detecting transgenic papaya - Google Patents
Primer pair combination, kit and detection method for detecting transgenic papaya Download PDFInfo
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- 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
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a primer pair combination, a kit and a detection method for detecting transgenic papaya. The nucleotide sequence of the primer pair combination is shown in SEQ ID NO.1 to SEQ ID NO. 16; the qualitative and quantitative detection of multiple transgenic components is realized through one-time high-throughput detection, the condition that the existing Real-time technology needs to amplify and detect for multiple times to cover multiple target transgenic components in a sample is avoided, and meanwhile false positive or false negative results of the technology are avoided, so that the primer pair combination has the advantages of high efficiency, accuracy and sensitivity, and has a good application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer pair combination, a kit and a detection method for detecting transgenic papaya.
Background
Papaya is a crop generally planted worldwide, contains rich vegetable proteins and edible oil and fat, and is an important edible and feeding crop. Under the background that the planting area of papaya is difficult to be greatly increased at present, the method for cultivating a new variety of high-yield and high-quality transgenic papaya by using the modern biotechnology has great significance for promoting the happiness of papaya industry. However, with the increasing concern of the safety of transgenic products, which are produced directly or indirectly from a large amount of transgenic papaya, in international society, the detection of transgenic components in agricultural products has been brought into the detection projects of inspection and quarantine departments at home and abroad and gradually strengthened. Therefore, development of efficient and convenient transgenic food detection technology is very important.
The detection technology of the transgenic products mainly comprises a protein-based detection method and a nucleic acid-based detection method. The current PCR detection method based on nucleic acid is still the most common and accurate transgene detection technology at present, and mainly comprises the methods of common qualitative PCR, nested PCR, loop-mediated isothermal amplification (LAMP), fluorescent quantitative PCR multiplex PCR and the like. Compared with the common qualitative PCR method, the nested PCR has higher detection sensitivity and is easy to cause false positive. LAMP is simple to operate and high in specificity, however, primer design is complex, DNA pollution is easy to cause, and subsequent experiments are affected. The fluorescent quantitative PCR method has the advantages of good repeatability, high sensitivity and less nucleic acid cross contamination, but has high cost and needs a special detection instrument. The common multiplex PCR method can detect a plurality of genes simultaneously in one reaction, but the number of the genes is not more than six, otherwise, the interference among primers is large, and the detection effect is influenced. The gene chip and the digital PCR technology are also common transgenic product detection technologies, have the advantages of high flux, high sensitivity, strong specificity and the like, and can detect a plurality of genes in 1 transgenic crop in parallel or detect a plurality of transgenic crops simultaneously; however, the cost is high, special instruments and equipment are required, operators are required to have high professional quality, and the factors limit the wide application of the technology in detection.
Therefore, developing a high-efficiency, sensitive and high-flux transgenic product detection method becomes a key problem to be solved urgently.
Disclosure of Invention
The application provides a primer pair combination, a kit and a detection method for detecting papaya transformation, which are used for solving the technical problem of how to efficiently detect transgenic papaya and strains.
In a first aspect, the present application provides a primer pair combination for detecting transgenic papaya, the primer pair combination comprising:
a primer pair for specifically amplifying p35S, the nucleotide sequence of which is shown in SEQ ID NO.1 to SEQ ID NO. 2;
a primer pair for specifically amplifying t35S, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.3 to SEQ ID NO. 4;
a primer pair for specifically amplifying pNOS, the nucleotide sequence of which is shown in SEQ ID NO.5 to SEQ ID NO. 6;
a primer pair for specifically amplifying tNOS, the nucleotide sequence of which is shown in SEQ ID NO.7 to SEQ ID NO. 8;
a primer pair for specifically amplifying NPtII, the nucleotide sequence of which is shown in SEQ ID NO.9 to SEQ ID NO. 10;
a primer pair for specifically amplifying PRSV_CP, the nucleotide sequence of which is shown as SEQ ID NO.11 to SEQ ID NO. 12;
a primer pair for specifically amplifying the replicase, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.13 to SEQ ID NO. 14;
and/or a primer pair for specifically amplifying GUS, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO.15 to SEQ ID NO. 16.
Optionally, the primer pair combination includes:
a primer pair for specifically amplifying Rainbow-left, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.17 to SEQ ID NO. 18;
a primer pair for specifically amplifying Rainbow-right, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.19 to SEQ ID NO. 20;
and/or specifically amplifying primer pair of Huanong No.1, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.21 to SEQ ID NO. 22.
Optionally, the primer pair combination further comprises a primer pair for amplifying papaya reference gene cap_papain.
Optionally, a primer pair for amplifying the papaya reference gene Cap_papain has a nucleotide sequence shown in SEQ ID NO.23 to SEQ ID NO. 24.
Optionally, the primer pair combination includes:
primer pairs specifically amplifying papaya transgenic elements selected from the group consisting of: p35S, t35S, pNOS, tNOS, NPtII, PRSV _cp, replicase and GUS; the primer pair combination further comprises a primer pair for specifically amplifying a specific sequence of the papaya transgenic line selected from the group consisting of: rainbow-left, rainbow-right and Huanong No. 1.
In a second aspect, the present application provides a kit for detecting a transgene in papaya, said kit comprising said primer pair combination for detecting a transgene in papaya according to the first aspect.
Optionally, the kit includes a first container, and the first container contains the primer pair combination therein.
Optionally, the kit further comprises a multiplex PCR premix.
In a third aspect, the present application provides the use of the primer pair combination of the first aspect and the detection kit of the second aspect for detecting transgenic papaya and related products thereof.
In a fourth aspect, the present application provides a method of detecting transgenic papaya, the method comprising the steps of:
obtaining the DNA of papaya to be detected and the primer pair combination in the first aspect;
taking the DNA as a template, combining and adding the primer pair into a reaction system, and performing amplification reaction to obtain an amplification product;
carrying out high-throughput sequencing on the amplification product to obtain a high-throughput library;
and analyzing the gene sequence in the high-throughput library to obtain the result of detecting the transgenic papaya.
Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:
the nucleotide sequence of the primer pair combination provided by the embodiment of the application is shown in SEQ ID NO.1 to SEQ ID NO. 16; the qualitative and quantitative detection of multiple transgenic components is realized through one-time high-throughput detection, the condition that the existing Real-time technology needs to amplify and detect for multiple times to cover multiple target transgenic components in a sample is avoided, and meanwhile false positive or false negative results of the technology are avoided, so that the primer pair combination has the advantages of high efficiency, accuracy and sensitivity, and has a good application prospect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
Fig. 1 is a schematic flow chart of a method for detecting transgenic papaya according to an embodiment of the present application.
Detailed Description
For the purposes of making the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present application based on the embodiments herein.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention. For example, room temperature may refer to a temperature in the range of 10 to 35 ℃.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
according to an exemplary embodiment of the present invention, there is provided a primer pair combination for detecting transgenic papaya, the primer pair combination comprising: a primer pair for specifically amplifying p35S, the nucleotide sequence of which is shown in SEQ ID NO.1 to SEQ ID NO. 2;
a primer pair for specifically amplifying t35S, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.3 to SEQ ID NO. 4;
a primer pair for specifically amplifying pNOS, the nucleotide sequence of which is shown in SEQ ID NO.5 to SEQ ID NO. 6;
a primer pair for specifically amplifying tNOS, the nucleotide sequence of which is shown in SEQ ID NO.7 to SEQ ID NO. 8;
a primer pair for specifically amplifying NPtII, the nucleotide sequence of which is shown in SEQ ID NO.9 to SEQ ID NO. 10;
a primer pair for specifically amplifying PRSV_CP, the nucleotide sequence of which is shown as SEQ ID NO.11 to SEQ ID NO. 12;
a primer pair for specifically amplifying the replicase, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.13 to SEQ ID NO. 14;
and/or a primer pair for specifically amplifying GUS, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO.15 to SEQ ID NO. 16.
Specifically, transgenic papaya includes papaya comprising a transgenic element and a transgenic line.
In some embodiments, the primer pair combination comprises:
a primer pair for specifically amplifying Rainbow-left, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.17 to SEQ ID NO. 18;
a primer pair for specifically amplifying Rainbow-right, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.19 to SEQ ID NO. 20;
and/or specifically amplifying primer pair of Huanong No.1, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.21 to SEQ ID NO. 22.
Preferably, 12 pairs of primers are developed which do not affect each other and can be amplified efficiently by multiplex PCR. The multiplex PCR primer combination can be used for developing papaya transgenic elements and strain detection kits; meanwhile, each pair of primers consists of a forward primer and a reverse primer.
In the embodiment of the application, the amplification product combined by the primer pair can be subjected to one-time high-throughput sequencing and analysis to obtain a plurality of detection results, wherein the detection results comprise transgenic components, judging whether a sample to be detected contains target molecules or not, and determining the copy numbers of reference genes and target molecules in the sample to be detected so as to determine the content of exogenous genes; the method avoids that the existing Real-time technology can only detect one target molecule at a time, multiple times of amplification and detection are needed to cover multiple target transgene components in a sample, meanwhile, false positive or false negative results of the technology are avoided, more than 9 PCR reactions can be realized by one experiment, the detection efficiency, flux and sensitivity are improved, and the detection cost is also considered. In some embodiments, the primer pair combination comprises:
the specific nucleotide sequences of the primers and the papaya transgenic elements and strains amplified by the primers, the numbers of the corresponding primer pairs and the nucleotide sequences of the primer pairs are shown in table 1.
Table 1 the target molecules screened according to the invention and their primer sequences.
In the process of primer design, in order to enhance the applicability and sensitivity of the primers, the length of the primers is between 18 and 30bp, the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be normally amplified in one amplification reaction, and the use proves that the primers have high sensitivity and strong applicability.
In some embodiments, the primer pair combination further comprises a primer pair for amplifying the papaya reference gene cap_papain.
In order to realize the purpose of detecting the papaya transgenic elements and strains in the samples, when the papaya transgenic elements or strains are selected, detection primers for the papaya internal reference genes are added to realize quantitative detection of the transgenic component content.
In some embodiments, the primer pair for amplifying papaya reference gene cap_papain has a nucleotide sequence shown in SEQ ID No.23 to SEQ ID No. 24.
The reason for selecting 2 pairs of reference gene primer pairs is to avoid the instability of the reference gene and the failure of 1 pair of reference gene primers in the low-content sample to effectively detect the target reference gene.
In some embodiments, the primer pair number ranges are: the number of the pairs 1-12 is appropriately adjusted according to the specific detection sample. The later period can be increased periodically according to the newly collected transgenic elements or strains, the primer combination is tried for 3000 pairs, and the amplification effect is still good. To achieve detection of transgenic papaya, 8 common transgenic elements and 3 transgenic lines in papaya were collected, covering the common transgenic elements of most transgenic papaya lines on the market, the logarithmic range of the multiplex PCR primers was: 1-12 pairs including, but not limited to: 2. pairs 3, 4, 5, 6, 7, 8, 9, 10 and 11; compared with the conventional specific multiplex PCR with the number of less than 6, the method has the advantages of high detection flux and high sensitivity.
In some embodiments, the primer pair combination comprises a primer pair that specifically amplifies a papaya transgenic element selected from the group consisting of: p35S, t35S, pNOS, tNOS, NPtII, PRSV _cp, replicase and GUS; the primer pair combination further comprises a primer pair for specifically amplifying a specific sequence of the papaya transgenic line selected from the group consisting of: rainbow-left, rainbow-right and Huanong No. 1.
Based on the existing primer pair combination, the method can also comprise other primer pair logarithmic combinations for specifically amplifying the same papaya transgenic element, and can also comprise logarithmic groups for periodically adding multiple PCR primers according to the newly collected transgenic element in the later period, and the number of the primer pairs can reach 3000 pairs through verification.
In a second aspect, the present application provides a kit for detecting transgenic papaya, comprising the primer pair combination of the first aspect for detecting transgenic papaya.
In some embodiments, the kit comprises a first container containing the primer pair combination therein.
In some embodiments, the kit further comprises a multiplex PCR premix.
Specifically, when the components of the multiplex PCR premix include the primer sets for amplifying the transgenic papaya element, strain and reference gene, each primer is premixed in a ratio of 1:1, and the mixture of the primers is carried out according to different experimental purposes, and in a specific embodiment, the concentration of each primer is 2-2.5nM. Preferably, each primer is at a concentration of 2.1, 2.2 and 2.3nM.
In a third aspect, the present application provides the use of the primer pair combination of the first aspect and the detection kit of the second aspect for detecting transgenic papaya and related products thereof.
In a fourth aspect, the present application provides a method of detecting transgenic papaya, the method comprising the steps of:
obtaining the DNA of papaya to be detected and the primer pair combination in the first aspect;
taking the DNA as a template, combining and adding the primer pair into a reaction system, and performing amplification reaction to obtain an amplification product;
carrying out high-throughput sequencing on the amplification product to obtain a high-throughput library;
and analyzing the gene sequence in the high-throughput library to obtain the result of detecting the transgenic papaya.
In particular, the high throughput sequencing may be second generation sequencing or third generation sequencing, and the resulting high throughput library may analyze the transgenic components of papaya from multiple dimensions, including but not limited to, the transgenic elements or lines of papaya in the embodiments.
In some embodiments, the method can be used for detecting all target transgenic components of multiple samples at one time, has the advantages of high flux, high sensitivity, accuracy, rapidness and the like, and can be applied to qualitative and quantitative detection of the transgenic components of transgenic papaya strains and products thereof.
In the embodiments of the present application, the environment/procedure of the amplification reaction includes: pre-denaturation at 94 ℃ for 5 min; the first amplification reaction, denaturation at 94℃for 15s, annealing at 62℃to 56℃for 30s,12 TouchDown cycles, (0.5℃decrease in temperature for each cycle of annealing and extension); the second amplification step was 15s denatured at 94℃and 30S annealed at 57℃for 22 cycles.
In an embodiment of the present application, the reaction system includes: 40. Mu.l of the total system, 2.5. Mu.l of primer premix, 2 Xbuffer: 20 μl, multiplex PCR amplification enzyme: 0.5 μl; the rest is supplemented with water; the rest water is used for supplementing; the high throughput library is qualified at a concentration greater than 2 ng/ul.
Preferably, the environment/procedure of the amplification reaction of the method comprises: pre-denaturation at 94 ℃ for 15 min; the first amplification step, denaturation at 94℃for 20 seconds, annealing at 65℃to 57℃and extension for 60 seconds, 10 TouchDown cycles, (annealing and extension temperatures for each cycle reduced by 0.8 ℃); the second amplification step was performed by denaturation at 94℃for 20 seconds, annealing at 57℃and extension for 60 seconds, 26 cycles.
Still preferably, the reaction system of the method comprises: 30 μl of the total system, primer pair: 2 μl, 2 Xbuffer: 15ul, multiplex amplification enzyme: 0.5 μl; the rest water is used for supplementing; the high throughput library is qualified at a concentration greater than 2 ng/ul.
The kit provided by the invention can sensitively detect papaya transgenic products with transgenic content of 0.05% in samples.
In the reproducibility test of the invention, the reproducibility r=100% of detection results between different libraries and different library-building batches of each sample, and the accuracy a=98.6%.
The kit provided by the invention detects various transgenic elements of various papaya transgenic lines or samples in complex templates, and has high specificity.
The specific correspondence between the primers and their amplified nucleotide sequences of the papaya transgenic elements and lines, i.e., the numbers of the target molecules and corresponding primer pairs, and the nucleotide sequences of the primers are shown in Table 1.
The method of the present invention will be described in detail with reference to examples, comparative examples and experimental data.
Example 1 selection of target transgenic elements, lines and design of multiplex PCR amplification primers
In the embodiment of the application, the target transgenic component mainly refers to transgenic elements and strains, and papaya internal reference genes used in the embodiment and the transgenic elements and strains are mainly collected in a common transgenic database, a national standard, an industry standard or the existing literature, so that the specificity and the accuracy of detection are ensured as much as possible. The names of the transgenic elements, lines and internal reference genes screened are shown in Table 1.
In the embodiment of the application, primer3Plus is utilized to design multiple PCR primers, the length of the primers is between 18 and 30bp, the primers are not interfered with each other, the main evaluation is to evaluate the dimer among the primers, or the hairpin structure inside the primers, and the nonspecific amplification of a non-target sequence, all the evaluated primers can be combined into a Primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction. Specific primer sequences include: SEQ ID NO. 1-SEQ ID NO. 24.
Example 2 detection of papaya samples for transgenic Components
1. Experimental materials: transgenic material Huanong No. 1. The experimental material was transformed into p35S, NPtII, pNOS, tNOS, replicase, the transgene content of which was 10%, and was used as a study material.
Preparation of DNA templates: the extraction of plant genome adopts a high-efficiency plant genome DNA extraction kit (DP 350) of CTAB or Tiangen biochemical technology (Beijing) limited company. In this example, three biological replicates were performed for each sample of sample DNA extracted using the root DNA extraction kit.
PCR amplification, library construction and sequencing
Amplifying genomic DNA of the sample using 22 pairs of multiplex PCR amplification primers; connecting the amplified product of each sample with a sequencing joint and a specific sample DNA bar code, and then mixing to form a high-throughput sequencing library; and detecting the high-throughput sequencing library by using a high-throughput sequencing platform and performing quality control on the high-throughput sequencing data. The step is to research and adjust key parameters such as amplification cycle number, sequencing depth and the like according to the requirements of detection accuracy, sensitivity and the like; the step can also be connected with third generation sequencing to realize the complementary advantages between second generation sequencing and third generation sequencing.
4. Determination of results
1) Determining whether the contamination is acceptable based on the signal index S of the transgenic line in the test sample and the signal index P of the transgenic element or line in the blank, wherein:
the noise figure p=nc/Nc for the control, where Nc and Nc represent the number of sequenced fragments and total number of sequenced fragments, respectively, of the transgenic element or line in the control.
The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments and the total number of sequenced fragments, respectively, of the transgenic element or line in the test sample.
Signal to noise ratio = S/P
2) Determination of transgene outcome
And (3) distributing each sequencing fragment to each target position of each target species by utilizing the DNA bar code of the sample to be tested and homology comparison, wherein the targets comprise transgenic elements, strains and internal reference genes. Absolute quantification of transgenic elements or lines is achieved based on the number of sequenced sequences at each target position. When the sequencing sequences on the reference gene and the transgenic element or the line are compared and exceed a specified threshold value, qualitatively judging that the sample contains the transgenic component; when the sample contains the transgenic component, the content of the exogenous gene in the sample is quantitatively determined according to the ratio of the sequence of the transgenic component and the strain to the sequence of the internal reference gene. The calculation formula of the transgene content in this embodiment is shown in (a):
CtestDNA-transgenic content of test sample
tTi-number of sequencing sequences for each transgenic element and line in the test sample
tRi order of sequencing sequence of each internal reference gene fragment detected in the test sample
m-total number of internal Gene fragments detected in test sample
n-total number of transgenic elements and line fragments detected in Standard substance
According to this example, 2 samples, 1 transgenic line warrior 1 and one negative sample were tested, three biological replicates were made for each sample, and the results are shown in table 2 and fig. 1: promoters and terminators commonly used in negative samples also detect several sequences in negative papaya, requiring that fewer than 5 sequencing reads be filtered out. The invention provides that when the signal to noise ratio is greater than 10 times, it can be determined that the contamination in the detection system is acceptable. When the signal to noise ratio of the transgenic element or line in the sample is greater than 10, the nucleic acid of the transgenic element or line in the sample is determined to be detected.
Table 2 the transgene test results of the test sample of example 2.
As can be seen from the table, each corresponding transgenic element and strain in Huanong No.1 was effectively detected in three repeated experiments, and the content was close to the actual content thereof; from this table it is demonstrated that papaya transgene kits can be used to detect transgenic products.
Example 3 accuracy, specificity and sensitivity assessment
Transgenic papaya variety Huanong No.1 and YK1601 transgenic standards transgenic samples of different mass percentages were prepared to evaluate the accuracy, specificity and sensitivity of the developed technology. Specifically, the transgene content of each sample was diluted with mass percent, specifically 10%,1%,0.1%,0.05%,0.025% and 0.01% of the transgenic papaya of Huanong 1 and YK1601 were diluted with negative papaya, respectively, corresponding to the diluted sample numbers (A1, A2, A3, A4, A5, A6) of transgenic line Huanong 1 and the diluted sample numbers (B1, B2, B3, B4, B5, B6) of transgenic line YK1601, respectively. The accuracy of qualitative detection refers to the proportion of true positives to true negatives, and the quantitative accuracy refers to the degree of coincidence of the average value of multiple determinations with a true value, and is expressed by errors. The specificity is also called true negative rate, and the percentage of true negative detected by multiple detection is the percentage of all negative. Sensitivity refers to the lowest content of transgenic elements or lines that can be detected at 95% confidence, i.e., the lower detection limit. The assay was performed as in example 2, with three replicates per sample, and the results are shown in table 3.
TABLE 3 evaluation of accuracy and sensitivity of the methods of the invention
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Note that: + represents detected, -represents undetected, A1 and B1 represent transgene content of 10%, A2 and B2 represent transgene content of 1%, A3 and B3 represent transgene content of 0.1%, A4 and B4 represent transgene content of 0.05%, A5 and B5 represent transgene content of 0.025%, and A6 and B6 represent transgene content of 0.01%.
As can be seen from the table, the kit can stably detect each transgenic element or strain in a sample with the transgenic content of 0.05%, and can detect 1 transgenic component at most in a negative sample, so that the kit has strong specificity, can obviously distinguish the sample with the transgenic content of 0.05% from the negative sample, and has technical stability and detection sensitivity with the transgenic content of 0.05%.
Example 4
In order to verify the accuracy of the invention and the role in transgene detection of batch samples, a laboratory selects 143 samples of papaya leaves with unknown transgene components from a company for detection, the detection method of the embodiment 2 is adopted, the detection result is compared with the preservation type of the company, and the consistency of the result is counted. The analysis result shows that in 143 test samples, only 2 samples are inconsistent, and the consistency of the detection result is as high as 98.6%, so that the accuracy of the method is better demonstrated.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
1) The method is simple to operate, multiple transgenic components in multiple samples or one sample can be synchronously detected by single-tube PCR amplification, library construction and sequencing through primary sample pretreatment, and the method has the characteristics of parallel analysis and multiple judgment, so that the detection efficiency of transgenic products is greatly improved;
2) The test object is complete, comprises 8 transgenic elements and 3 transgenic lines which are common at present, can be conveniently added with new detection target sequences, avoids single target amplification failure, and improves the specificity, accuracy and sensitivity of detection;
3) The kit fuses the second generation sequencing platform to sequence the amplified product, so that the flux and the repeatability of a detection system are improved, the detection result can be directly digitized, and the kit is suitable for large-scale detection of transgenic papaya and products thereof.
Therefore, the method overcomes the defects of time and labor waste and high cost of the prior detection technology, and the provided papaya transgenic line detection kit is simple to operate, quick and sensitive, large in detection flux, good in repeatability of detection results, low in detection cost of multiple-sample multi-target sequences, and has important application to detection of transgenic products on entry and exit ports of seed stations, academy of agricultural sciences and customs.
It should be noted that in this document, relational terms such as "first" and "second" and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Jiang Handa science
<120> a primer set composition for detecting transgenic papaya, kit and detection method
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Claims (8)
1. A primer pair composition for detecting transgenic papaya, which is characterized by comprising
A primer pair for specifically amplifying p35S, the nucleotide sequence of which is shown in SEQ ID NO.1 to SEQ ID NO. 2;
a primer pair for specifically amplifying t35S, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.3 to SEQ ID NO. 4;
a primer pair for specifically amplifying pNOS, the nucleotide sequence of which is shown in SEQ ID NO.5 to SEQ ID NO. 6;
a primer pair for specifically amplifying tNOS, the nucleotide sequence of which is shown in SEQ ID NO.7 to SEQ ID NO. 8;
a primer pair for specifically amplifying NPtII, the nucleotide sequence of which is shown in SEQ ID NO.9 to SEQ ID NO. 10;
a primer pair for specifically amplifying PRSV_CP, the nucleotide sequence of which is shown as SEQ ID NO.11 to SEQ ID NO. 12;
a primer pair for specifically amplifying the replicase, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.13 to SEQ ID NO. 14;
a primer pair for specifically amplifying GUS, the nucleotide sequence of which is shown in SEQ ID NO.15 to SEQ ID NO. 16;
a primer pair for specifically amplifying Rainbow-left, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.17 to SEQ ID NO. 18;
a primer pair for specifically amplifying Rainbow-right, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.19 to SEQ ID NO. 20;
and a primer pair for specifically amplifying Huanong No.1, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO.21 to SEQ ID NO. 22.
2. The primer pair composition of claim 1, further comprising a primer pair for amplifying a papaya reference gene cap_pa in.
3. The primer pair composition according to claim 2, wherein the primer pair for amplifying the papaya reference gene cap_papain has a nucleotide sequence shown in SEQ ID No.23 to SEQ ID No. 24.
4. A kit for detecting transgenic papaya, comprising the primer pair composition for detecting transgenic papaya of any one of claims 1 to 3.
5. The kit of claim 4, wherein the kit comprises a first container containing the primer pair composition therein.
6. The kit of claim 4, further comprising a multiplex PCR premix.
7. Use of the primer set composition according to any one of claims 1 to 3, the detection kit according to any one of claims 4 to 6 for detecting papaya and related products thereof.
8. A method of detecting transgenic papaya, comprising the steps of:
obtaining the DNA of papaya to be tested and the primer pair composition of any one of claims 1-3;
adding the primer pair composition into a reaction system by taking the DNA as a template, and performing an amplification reaction to obtain an amplification product;
carrying out high-throughput sequencing on the amplification product to obtain a high-throughput library;
and analyzing the gene sequence in the high-throughput library to obtain the result of detecting the transgenic papaya.
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