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CN-121992132-A - Primer composition for simultaneously detecting multiple food transgenic elements, microfluidic chip, kit and method

CN121992132ACN 121992132 ACN121992132 ACN 121992132ACN-121992132-A

Abstract

The invention discloses a primer combination, a microfluidic chip, a kit and a method for simultaneously detecting multiple food transgenic elements, wherein the primer combination comprises a primer group for detecting a transgenic element pCaMV S, a primer group for detecting a transgenic element pUBI, a primer group for detecting a transgenic element pNOS, a primer group for detecting a transgenic element pRBCS4, a primer group for detecting a transgenic element pFMV S, a primer group for detecting a transgenic element tNOS and a primer group for detecting a transgenic element tpinII. The invention can detect a plurality of target transgenic elements of a plurality of samples simultaneously, can cover a plurality of transgenic crops, has the advantages of short detection time, high sensitivity and the like, has small volume and light weight, is convenient to carry, and can be applied to rapid field detection.

Inventors

  • HUANG JUN
  • GAO YUANLIANG
  • DU LEI
  • ZHAO QIAN

Assignees

  • 浙江科技大学

Dates

Publication Date
20260508
Application Date
20251230

Claims (10)

  1. 1. A primer combination for simultaneously detecting multiple food transgenic elements based on LAMP is characterized by comprising a primer group for detecting a transgenic element pCaMV S, a primer group for detecting a transgenic element pUBI, a primer group for detecting a transgenic element pNOS, a primer group for detecting a transgenic element pRBCS4, a primer group for detecting a transgenic element pFMV35S, a primer group for detecting a transgenic element tNOS and a primer group for detecting a transgenic element tpinII.
  2. 2. The primer composition for simultaneous detection of multiple food transgenic elements based on LAMP as claimed in claim 1, wherein the primer set for detection of transgenic element pCaMV S comprises: The nucleotide sequences of the outer primer pair F3 and B3 are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; The nucleotide sequences of the inner primer pair FIP and BIP are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4; the nucleotide sequences of the loop primer pair FIP and BIP are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6; A primer set for detecting a transgenic element puci, comprising: The nucleotide sequences of the outer primer pair F3 and B3 are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 8; the nucleotide sequences of the inner primer pair FIP and BIP are respectively shown as SEQ ID NO. 9 and SEQ ID NO. 10; the nucleotide sequence of the loop primer LF is shown as SEQ ID NO. 11; A primer set for detecting a transgenic element pNOS, comprising: The nucleotide sequences of the outer primer pair F3 and B3 are respectively shown as SEQ ID NO. 12 and SEQ ID NO. 13; the nucleotide sequences of the inner primer pair FIP and BIP are respectively shown as SEQ ID NO. 14 and SEQ ID NO. 15; the nucleotide sequences of the loop primer pair FIP and BIP are shown as SEQ ID NO. 16 and SEQ ID NO. 17 respectively; a primer set for detecting transgenic element pRBCS4, comprising: the nucleotide sequences of the outer primer pair F3 and B3 are respectively shown as SEQ ID NO. 18 and SEQ ID NO. 19; the nucleotide sequences of the inner primer pair FIP and BIP are respectively shown as SEQ ID NO. 20 and SEQ ID NO. 21; The nucleotide sequences of the loop primer pair FIP and BIP are shown as SEQ ID NO. 22 and SEQ ID NO. 23 respectively; a primer set for detecting transgenic element pFMV S comprising: the nucleotide sequences of the outer primer pair F3 and B3 are respectively shown as SEQ ID NO. 24 and SEQ ID NO. 25; The nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID NO. 26 and SEQ ID NO. 27 respectively; the nucleotide sequences of the loop primer pair FIP and BIP are shown as SEQ ID NO. 28 and SEQ ID NO. 29 respectively; a primer set for detecting a transgenic element tNOS comprising: The nucleotide sequences of the outer primer pair F3 and B3 are respectively shown as SEQ ID NO. 30 and SEQ ID NO. 31; the nucleotide sequences of the inner primer pair FIP and BIP are respectively shown as SEQ ID NO. 32 and SEQ ID NO. 33; The nucleotide sequences of the loop primer pair FIP and BIP are respectively shown as SEQ ID NO. 34 and SEQ ID NO. 35; a primer set for detecting a transgenic element tpinll comprising: The nucleotide sequences of the outer primer pair F3 and B3 are respectively shown as SEQ ID NO. 36 and SEQ ID NO. 37; The nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID NO. 38 and SEQ ID NO. 39 respectively; the nucleotide sequence of the loop primer LB is shown as SEQ ID NO. 40.
  3. 3. The primer composition for simultaneous detection of multiple food transgenic elements based on LAMP as claimed in claim 2, wherein the molar ratio of outer primer pair, inner primer pair, loop primer pair in the primer set for detection of transgenic element pCaMV S is (10-11): 1 (3-4); The molar ratio of the outer primer pair, the inner primer pair and the loop primer in the primer set for detecting the transgenic element pUBI is (9-10): 1 (3-4); The molar ratio of the outer primer pair, the inner primer pair and the loop primer in the primer group for detecting the transgenic element pNOS is (6-7): 1 (3-4); The molar ratio of the outer primer pair, the inner primer pair and the loop primer pair in the primer set for detecting the transgenic element pRBCS4 is (10-11): 1 (3-4); the molar ratio of the outer primer pair, the inner primer pair and the loop primer pair in the primer group for detecting the transgenic element pFMV S is (8-9): 1 (3-4); the molar ratio of the outer primer pair, the inner primer pair and the loop primer pair in the primer set for detecting the transgenic element tNOS is (6-7): 1 (2-3); The molar ratio of the outer primer pair, the inner primer pair and the loop primer in the primer set for detecting the transgenic element tpinll is (7.5-8.5): 1 (4-5).
  4. 4. A microfluidic chip for simultaneous detection of multiple food transgenic elements, comprising a plurality of independent units, each unit comprising a plurality of reaction wells, each primer component of the primer composition of any one of claims 1-3 being contained in a different reaction well of each unit.
  5. 5. The microfluidic chip for simultaneously detecting multiple food transgenic elements according to claim 4, further comprising a reaction well and a plurality of independent micro-channels in each unit, each reaction well being in communication with the reaction well through a corresponding micro-channel.
  6. 6. The microfluidic chip for simultaneously detecting multiple food transgenic elements according to claim 4, wherein each reaction well contains a corresponding primer set, DNA polymerase, deoxyribonucleoside triphosphate, mg 2+ .
  7. 7. The microfluidic chip for simultaneously detecting multiple food transgenic elements according to claim 6, wherein in the reaction well for detecting transgenic element pCaMV S, the inner primer pair, outer primer pair, loop primer pair, DNA polymerase, mg 2+ , deoxyribonucleoside triphosphate concentration is 1.50-1.60 μmol/L, 0.10-0.20 μmol/L, 0.50-0.60U/μl, 5-15mmol/L, 1.50-2.00mmol/L, respectively; In the reaction well for detecting the transgenic element pUBI, the concentrations of the inner primer pair, the outer primer pair, the loop primer pair, the DNA polymerase, the Mg 2 + and the deoxyribonucleoside triphosphate are respectively 1.60-1.70 mu mol/L, 0.15-0.20 mu mol/L, 0.65-0.75 mu mol/L, 0.60-0.70U/mu L, 10-15mmol/L and 1.00-1.50mmol/L; in the reaction hole for detecting the transgenic element pNOS, the concentration of the inner primer pair, the outer primer pair, the loop primer pair, the DNA polymerase, the Mg 2 + and the deoxyribonucleoside triphosphate is respectively 1.60-1.70 mu mol/L, 0.20-0.30 mu mol/L, 0.75-0.85 mu mol/L, 0.30-0.35U/mu L, 5-15mmol/L and 1.00-1.50mmol/L; In the reaction well for detecting the transgenic element pRBCS4, the concentrations of the inner primer pair, the outer primer pair, the loop primer pair, the DNA polymerase, the Mg 2+ and the deoxyribonucleoside triphosphate are respectively 1.50-1.60 mu mol/L, 0.10-0.20 mu mol/L, 0.50-0.60 mu mol/L, 0.30-0.35U/mu L, 10-15mmol/L and 1.00-1.50mmol/L; in the reaction well for detecting the transgenic element pFMV S, the concentration of the inner primer pair, the outer primer pair, the loop primer pair, the DNA polymerase, mg 2+ and deoxyribonucleoside triphosphates is respectively 1.50-1.60 mu mol/L, 0.15-0.20 mu mol/L, 0.50-0.70 mu mol/L, 0.60-0.65U/mu L, 5-15mmol/L and 1.00-1.50mmol/L; In the reaction well for detecting the transgenic element tNOS, the inner primer pair, the outer primer pair, the loop primer pair, the DNA polymerase, mg 2 + and deoxyribonucleoside triphosphates have the concentration of 1.60-1.65. Mu. Mol/L, 0.20-0.30. Mu. Mol/L, 0.50-0.60. Mu. Mol/L, 0.60-0.65U/. Mu.L, 5-15mmol/L and 1.50-2.00mmol/L respectively; In the reaction well for detecting the transgenic element tpinII, the concentrations of the inner primer pair, the outer primer pair, the loop primer pair, the DNA polymerase, mg 2+ and deoxyribonucleoside triphosphates are respectively 1.15-1.25 mu mol/L, 0.10-0.20 mu mol/L, 0.60-0.65U/mu L, 10-15mmol/L and 1.50-2.00mmol/L.
  8. 8. A kit for simultaneous detection of multiple food transgene elements, comprising a microfluidic chip according to any of claims 4-7.
  9. 9. A method for simultaneous detection of multiple food transgene elements, comprising detection using the microfluidic chip of any of claims 4-7 or the kit of claim 8.
  10. 10. The method for simultaneously detecting multiple transgenic elements of foods according to claim 9, which comprises performing a loop-mediated isothermal amplification reaction on a sample to be detected by using the microfluidic chip according to any one of claims 4 to 7 or the kit according to claim 8, and determining the presence of the transgenic elements in the sample to be detected according to the generated fluorescence intensity.

Description

Primer composition for simultaneously detecting multiple food transgenic elements, microfluidic chip, kit and method Technical Field The invention relates to the technical field of biology, in particular to a primer composition, a microfluidic chip, a kit and a method for simultaneously detecting various food transgenic elements. Background With the advancement of international trade freeform processes and the continual decrease of trade barriers, global food cross-border traffic has proliferated. Under the background, due to the obvious difference of the regulations of the transgenic food in each country, the detection of the transgenic ingredients in the cross-border food is efficiently and accurately completed, and the method has become an urgent task for the regulatory authorities of each country to guarantee the food safety and the awareness of consumers. The transgene detection is a detection method for identifying and verifying exogenous genes transferred into organisms through a molecular biology technology. The core principle is that the nucleic acid amplification technology or the protein detection technology is utilized to specifically identify transgenic elements (such as promoters, terminators and target genes). In recent years, detection methods based on molecular biology continue to evolve, with polymerase chain reaction (Polymerase Chain Reaction, PCR) and real-time quantitative techniques (Quantitative real time polymerase chain reaction, qPCR) still being the most widely used means of transgene detection. PCR utilizes the fact that DNA becomes single-stranded at a high temperature of 95 ℃ in vitro, primers and single-stranded are combined according to the base complementary pairing principle at a low temperature (usually about 60 ℃), the temperature is adjusted to the optimal reaction temperature (about 72 ℃) of DNA polymerase, and the DNA polymerase synthesizes complementary strands along the direction from phosphoric acid to pentose (5 '-3'). The PCR instrument based on polymerase is actually a temperature control device, and can well control the denaturation temperature, renaturation temperature and extension temperature. The basic principle of qPCR is to use the characteristic of DNA polymerase to synthesize new DNA chain in PCR process, and combine with a fluorescent marked probe or dye to measure the progress of PCR reaction by monitoring the increase of fluorescent signal in real time. However, PCR and qPCR techniques generally rely on a precise and strict laboratory environment, and the detection process is time-consuming, and their application is greatly restricted in situations requiring immediate results, such as rapid port clearance supervision, market field screening, and the like. Loop-mediated isothermal amplification (LAMP) has been studied intensively in the field of rapid field detection by virtue of its remarkable advantages of high sensitivity, high efficiency, low device dependence, etc. In the aspect of transgene detection, detection methods based on LAMP have been developed and optimized, and can realize high-efficiency detection of single and typical key exogenous gene elements (such as promoters, terminators and marker genes). However, current research hotspots and established method systems are primarily limited to detection of single targets or very few targets. In the practical scenario of cross-border food supervision, a variety of different transgenic elements possibly present in a sample are often screened and identified simultaneously in a limited time to comprehensively evaluate sample properties and compliance in the face of global flow-through foods with complex composition and diverse sources. Although each reaction of the existing standard single-target LAMP detection is quick and simple, if multi-target detection is needed, a plurality of independent reaction systems are needed to be arranged, so that the operation steps are complicated, the time consumption is long, the cost is increased, the sample consumption is increased, and higher requirements are put on the simplicity of field operation. Therefore, developing a new platform capable of conveniently and efficiently realizing multi-sample and multi-target synchronous detection under field conditions is a key challenge of breaking through the flux limit of the existing LAMP technology and fully applying the advantages of the LAMP technology to cross-border food complex transgene screening. Disclosure of Invention The invention provides a primer composition, a microfluidic chip, a kit and a method for simultaneously detecting multiple food transgenic elements, which can simultaneously detect multiple target transgenic elements of multiple samples, can cover multiple transgenic crops, have the advantages of short detection time, high sensitivity and the like, and the detection device has the advantages of small volume, light weight, convenient carrying and applicability to rapid field detection. The t