阅读说明：本技术 Epsps ii微流控芯片检测的引物、方法和试剂盒 (Primer, method and kit for detecting EPSPS II microfluidic chip ) 是由 黄文胜 陈颖 韩建勋 邓婷婷 周杰 于 2019-04-25 设计创作，主要内容包括：本发明涉及用于农产品中转基因成分EPSPS II检测的寡核苷酸引物。本发明还涉及用于测定农产品中转基因成分EPSPS II的环介导等温扩增检测方法,所述方法包括使用针对农产品中转基因成分EPSPS II的特异性寡核苷酸引物。本发明还涉及用于快速检测农产品中转基因成分EPSPS II的环介导等温扩增微流控芯片检测试剂盒,所述试剂盒包括用于环介导等温扩增检测农产品中转基因成分EPSPS II的特异性寡核苷酸引物。本发明还涉及针对农产品中转基因成分EPSPS II的特异性寡核苷酸引物在检测农产品中转基因成分中的应用。使用本发明的环介导等温扩增检测方法和试剂盒,能够特异、灵敏、准确地测定大豆、玉米、大米、棉花、油菜及其加工品等样品中是否含有转基因成分EPSPS II。(The invention relates to an oligonucleotide primer for detecting a transgenic component EPSPS II in agricultural products. The invention also relates to a loop-mediated isothermal amplification detection method for determining the transgenic component EPSPS II in the agricultural product, which comprises using specific oligonucleotide primers aiming at the transgenic component EPSPS II in the agricultural product. The invention also relates to a loop-mediated isothermal amplification micro-fluidic chip detection kit for rapidly detecting the transgenic component EPSPS II in the agricultural products, wherein the kit comprises a specific oligonucleotide primer for loop-mediated isothermal amplification detection of the transgenic component EPSPS II in the agricultural products. The invention also relates to the application of the specific oligonucleotide primer aiming at the transgenic component EPSPS II in the agricultural product in detecting the transgenic component in the agricultural product. By using the loop-mediated isothermal amplification detection method and the kit, whether the samples such as soybean, corn, rice, cotton, rape and processed products thereof contain the EPSPS II can be specifically, sensitively and accurately determined.)

1. Specific oligonucleotide primer composition for detecting transgenic components in agricultural products by using a loop-mediated isothermal amplification method, wherein the primer composition comprises the following components in parts by weight:

2. a kit for detecting a transgenic component in an agricultural product by a loop-mediated isothermal amplification method, the kit comprising the primer composition of claims 1-2 and instructions for use.

3. A loop-mediated isothermal amplification microfluidic chip method for accurately detecting transgenic components in agricultural products, a primer embedding form, concentration and the like; the method comprises using the primer composition of claims 1-2 and the kit of claim 3.

4. Use of the primer composition according to claims 1-2 and the kit according to claim 3 for detecting transgenic components in samples such as soybean, corn, rice, cotton, rape and processed products thereof.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an oligonucleotide primer for detecting a transgenic component EPSPS II in an agricultural product, a loop-mediated isothermal amplification detection method for determining the transgenic component EPSPS II in the agricultural product, a loop-mediated isothermal amplification microfluidic chip detection kit for rapidly detecting the transgenic component EPSPS II in the agricultural product, and application of a specific oligonucleotide primer for detecting the transgenic component EPSPS II in the agricultural product.

Background

Transgenic crop:

the modern biotechnology analyzes the source and characteristics of raw materials and products from the gene level by the characteristics of convenience, rapidness, accuracy and the like, has strong specificity and high sensitivity, and is widely applied to the transgenic detection of agricultural products. However, the application of the loop-mediated isothermal amplification microfluidic chip detection technology in agricultural product transgene detection is less.

At present, no loop-mediated isothermal amplification micro-fluidic chip detection method and kit capable of quickly, simply, specifically and sensitively detecting transgenic components in soybeans, corns, rice, cotton, rapes and processed products thereof are reported at home and abroad.

Therefore, there is a need in the art for a rapid, highly specific, highly sensitive, microsystem, multi-index-flux method for detecting transgenic components in agricultural products, which can be used for detecting transgenic components in soybean, corn, rice, cotton, rape seeds and processed products thereof.

Disclosure of Invention

An object of the present invention is to provide specific oligonucleotide primers for accurately detecting the transgenic component EPSPS II in agricultural products.

The invention also aims to provide a loop-mediated isothermal amplification microfluidic chip detection method for accurately determining the transgenic component EPSPS II in agricultural products.

The invention also aims to provide a loop-mediated isothermal amplification microfluidic chip detection kit for accurately determining the transgenic component EPSPS II in agricultural products.

The invention also aims to provide the application of the specific oligonucleotide primer of the transgenic component EPSPS II in the agricultural products in accurately detecting the transgenic component in the agricultural products.

Aiming at the above purpose, the invention provides the following technical scheme:

the inventor designs an oligonucleotide primer group capable of specifically identifying a transgene element EPSPSII in agricultural products according to the gene EPSPS II, and can efficiently and specifically amplify a short section of EPSPS II specific gene fragment from sample DNA. According to one embodiment of the invention, the invention provides a specific oligonucleotide primer group for detecting a transgenic component in agricultural products by using a loop-mediated isothermal amplification method, wherein the primer group is designed according to the characteristic that EPSPS II gene sequences have difference in similar sequences. The primer group consists of an outer primer, an inner primer and a loop primer, wherein the outer primer 1 is CTATGCAAGCTATGGGTGCCAGA (SEQ ID No. 1), and the outer primer 2 is CGACCCATTGGACGCTTAGTGA (SEQ ID No. 2); the inner primer 1 is CGAGAGGAGCCTCAGGAGCAAGGTCCGTAAGGAAGGTGATACTTGGA (SEQ ID No. 3), and the inner primer 2 is GTAACGCTGCAACTGGTTGCCGTGCGTCACCAATGAAAGTGCTATCG (SEQ ID No. 4); the loop primer 1 is AGTCCACCGTTACCAACATCATCA (SEQ ID No. 5), and the loop primer 2 is TGGGTCTTGTTGGTGTTTACGA (SEQ ID No. 6). In one embodiment, the invention provides an EPSPS II specific detection composition comprising a specific oligonucleotide primer set. In a preferred embodiment, the invention provides a composition for qualitatively detecting a transgenic component in an agricultural product by using a loop-mediated isothermal amplification method, wherein the composition comprises an EPSPS II specific oligonucleotide primer set, wherein the EPSPS II specific primer set consists of an outer primer, an inner primer and a loop primer, the base sequences of the outer primer are SEQ ID No.1 and SEQ ID No.2, the base sequences of the inner primer are SEQ ID No.3 and SEQ ID No.4, and the base sequences of the loop primer are SEQ ID No.5 and SEQ ID No. 6. The loop-mediated isothermal amplification condition is 65 ℃ and 60 min.

According to another embodiment of the present invention, the present invention provides a kit for accurately and qualitatively detecting a transgenic component in an agricultural product, the kit comprising the specific oligonucleotide primer set for detecting the transgenic component in the agricultural product by the loop-mediated isothermal amplification method of the present invention, a probe and an instruction for use. In a preferred embodiment of the kit of the present invention, the oligonucleotide primer set for qualitative detection of transgenic component in agricultural product of the present invention is designed based on the feature that EPSPS II gene sequence has difference in similar sequence. In one embodiment, the EPSPS II-specific oligonucleotide primer set of the kit consists of an outer primer, an inner primer and a loop primer, wherein the base sequences of the outer primer are SEQ ID No.1 and SEQ ID No.2, the base sequences of the inner primer are SEQ ID No.3 and SEQ ID No.4, and the base sequences of the loop primer are SEQ ID No.5 and SEQ ID No. 6.

In a preferred embodiment, the instructions for use of the kit include a description of the loop-mediated isothermal amplification conditions for rapid detection of a transgenic component in an agricultural product. In a preferred embodiment, the PCR amplification conditions given in the instructions for the kit are 65 ℃ for 60 min. In a specific embodiment, the kit for qualitatively detecting the transgenic component in the agricultural product further comprises a reference substance. The control product comprises negative control product and positive control product. In one embodiment, the positive control is plasmid DNA containing the target gene sequence.

The invention takes EPSPS II DNA as the detection basis, and the EPSPS II gene sequence is compared and analyzed according to the characteristic that the EPSPS II gene sequence has difference in similar sequences. And designing primers according to the sequences, and qualitatively detecting the transgenic components in the agricultural products in the samples by using a loop-mediated isothermal amplification method.

The loop-mediated isothermal amplification detection method adopts complete closed-tube detection, does not need post-treatment of amplification products, and avoids cross contamination and false positive. The method skillfully utilizes the high-efficiency amplification of the DNA of the loop-mediated isothermal amplification technology, the rapidness and the sensitivity of the specificity and fluorescence detection technology, and has the advantages of simple operation, time and labor conservation, reliable result, accuracy and sensitivity and the like. The kit prepared according to the primer sequence is used for qualitative detection of the products, and has the advantages of high sensitivity, strong specificity, stable and reliable result and avoidance of false positive caused by cross contamination. The loop-mediated isothermal amplification detection method and the microfluidic chip detection kit can be used for qualitative detection, and have the characteristics of simplicity, rapidness, specificity and sensitivity, so that the loop-mediated isothermal amplification detection method and the microfluidic chip detection kit are suitable for detection of transgenic components in agricultural products in soybean, corn, rice, cotton, rape, processed products of the soybean, the corn, the rice, the cotton, the rape and other samples in domestic and foreign markets.

Drawings

FIG. 1 shows the results of specific detection of primers in tubular amplification, in which specific oligonucleotide primer sets SEQ ID Nos. 1-6 are used for detection, wherein the amplification curve of a positive sample is shown above the base line, and samples such as soybean, corn, wheat, rice, potato, tomato, black bean, broad bean, rape, cabbage, mung bean, sesame, taro, cucumber, mushroom, sweet potato (randomly purchased and prepared in the market) and blank controls (sterile double distilled water) are shown below the base line.

FIG. 2 shows the results of the detection of the sensitivity of the primers in the tubular amplification, which is the evaluation of the absolute sensitivity of the specific detection of the transgenic component in the loop-mediated isothermal amplification, by sequentially diluting the transgenic DNA in a gradient manner to final concentrations of 5 ng/. mu.L, 4 ng/. mu.L, 3 ng/. mu.L, 2 ng/. mu.L, 1 ng/. mu.L, 0.5 ng/. mu.L, 0.1 ng/. mu.L, 0.01 ng/. mu.L and a blank control (sterile double distilled water).

FIG. 3 is a graph showing the detection results of the detection limits of primers in the tubular amplification, which shows the evaluation of the relative sensitivity of the specific detection of the transgenic component in the loop-mediated isothermal amplification, using the purchased standard or the positive sample and the negative control mixed so that the mass ratios of the contents of the transgenic component are 100%, 50%, 10%, 5%, 1%, 0.5% and 0.1%, respectively.

FIG. 4 shows the characteristics of the chip amplification primer embedding concentration ratio with respect to the sensitivity of the primer amplification system, and the experiments were performed using two concentration ratios in combination with the requirements of the reaction reagents such as enzyme used for the primers and the biological characteristics of the chip itself, and the primer embedding ratio is shown in Table 1. Two groups in the chip repeatedly generate S-shaped amplification curves in corresponding reaction pools, and the peak time, the peak shape and the like are consistent, so that the concentration ratio of the embedded primers is suitable for screening and optimizing the EPSPS II primers (a is a disc I, and b is a disc II).

TABLE 1 embedding ratio of primers

FIG. 5 shows the results of primer specificity in amplification of chip a in the disc reaction for detecting EPSPS II specifically by loop-mediated isothermal amplification, in which the specific oligonucleotide primer sets SEQ ID No.1-6 are embedded and fixed in the disc reaction tank, and then the system is configured for detection. Wherein the EPSPS II gene and the positive control respectively correspond to the reaction tank to generate an S-shaped amplification curve, and a bottom normalized baseline is a corresponding equal sample and blank control (sterile double distilled water) of other reaction tanks; panel b shows the results of disc reactions with DNA templates of non-transgenic soybeans, corn, rice and wheat, where EPSPS II corresponding reaction pools showed "S" shaped amplification curves, and the bottom normalized baseline was equivalent samples and blank controls (sterile double distilled water) corresponding to other reaction pools.

The results of the primer sensitivity in the amplification of the chip in FIG. 6 show that the absolute sensitivity of the specific detection of the transgenic component in the LAMP is evaluated, and the concentration of the transgenic DNA is gradually diluted in a gradient manner, so that each reaction tank reaches 100 ng/. mu.L, 75 ng/. mu.L, 50 ng/. mu.L and 25 ng/. mu.L (which respectively correspond to the numbers a-d in the figures).

FIG. 7 shows the relative sensitivity of the PCR-specific detection of transgenic components, which is evaluated by purchasing standards or mixing positive samples with negative controls, in the amplification of the chip, so that the mass ratios of the contents of the transgenic components are 0.1%, 1% and 5%, respectively (corresponding to the numbers a-c in the figure).

Detailed Description

The present invention will be further described by way of examples, but the present invention is not limited to only the following examples.