A primer set, detection method, kit and application thereof for identifying duck-derived components using LMTIA technology

Abstract

The present invention discloses a primer set, a detection method, a kit and an application thereof for identifying duck-derived components using LMTIA technology, and belongs to the technical field of molecular biology nucleic acid detection. The present invention designs an LMTIA primer set for identifying duck-derived components, including primers Ya‑ITS1‑1F2, Ya‑ITS1‑1B2, Ya‑ITS1‑1LF, Ya‑ITS1‑LB and a probe Ya‑ITS1‑LBPr, and constructs a detection method for identifying duck-derived components based on LMTIA technology, which has the advantages of fast detection speed, high sensitivity, and strong specificity. This detection method can be used to quickly, efficiently and accurately identify duck-derived components in food, providing a technical means for authenticity identification of food.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology nucleic acid detection, and in particular to a primer set, a detection method, a kit and applications thereof for identifying duck-derived components using LMTIA technology. Background Art

[0002] DNA has high genetic stability, and different organisms contain different specific DNA. DNA-based detection methods have become the most important and effective technology for identifying species-derived ingredients in food authenticity verification.

[0003] Meat products are an important part of people's healthy diet. However, there are also many problems of adulteration, such as passing cheap chicken and duck meat as expensive beef and mutton. Authenticity testing of meat products is an effective guarantee for combating adulteration and smuggling and maintaining market order. For adulteration of meat products, commonly used detection methods include sensory identification, immunological technology, molecular biology technology, etc. In recent years, the application of nucleic acid detection technology in the food field has become a major research hotspot due to its advantages of sensitivity, rapidity, and low equipment requirements.

[0004] According to the research in recent years, rolling circle amplification (RCA), recombinase polymerase amplification (RPA), cross primer isothermal amplification (CPA), loop mediated isothermal amplification (LAMP) have been applied to the adulteration detection of meat products. However, according to the research reports and our previous research, although the reaction mechanisms of these methods are well designed, they still have different disadvantages.

[0005] The disadvantages of RCA technology are: cumbersome pre-treatment, the reaction template is required to be single-stranded circular DNA, and samples that do not meet the requirements must be annealed and cyclized. Therefore, it has not been actually commercialized and applied 29 years after its establishment. RCA technology is also constantly developing. Hu Xuejia and others have constructed a saltatory rolling circle isothermal amplification technology (Saltatory Rolling Circle Amplification, SRCA) for detecting pork DNA. The sensitivity of SRCA fluorescence visualization method for detecting pork DNA is 6.5fg / μL. Afterwards, Hu Xuejia and others selected the mitochondrial Cyt b gene of mice as the target gene to verify the accuracy of the SRCA method. In simulated adulterated samples with artificially added rat meat, the SRCA method can detect rat meat with a content of 0.01%. The practicality of the new technology SRCA remains to be verified.

[0006] The disadvantages of RPA technology are: (1) The required primers and probes are long and have high requirements, and it is impossible to detect short-sequence nucleic acids; (2) The reaction temperature is around 37°C and the reaction time is short. When testing a large number of samples at the same time, it is difficult to control the reaction at the same time; (3) The design of primers and probes is immature and requires multiple attempts to optimize the conditions. In addition, there is currently no design software available; (4) The cost of basic reagents per unit of RPA detection is relatively high, about 30 times that of PCR, so it is difficult to promote and apply commercially.

[0007] CPA technology has good commercial application. Hangzhou Yousida Company has developed more than ten commercial test kits, mainly for foodborne pathogen detection, and there is little research in the field of meat products. It has been reported that CPA technology should be combined with other technologies in the identification of meat products. Feng et al. established a CPA combined with colloidal gold nucleic acid test strip technology to distinguish cattle, sheep, Arctic fox and pork, but the sensitivity is low, with a detection limit of 1%. Feng et al. also established a rapid detection method for CPA combined with nucleic acid test strips, with a detection limit of 1%. Studies have shown that CPA technology is lower in sensitivity than LAMP technology.

[0008] In 2000, Japanese scholar Notomi and others invented the LAMP technology, which cleverly achieves amplification through multiple pairs of primers. The LAMP reaction efficiency is very high, and there is no time-consuming temperature change. Therefore, in theory, LAMP should be better than PCR in terms of reaction speed, specificity, and sensitivity. However, the LAMP technology, which claims to replace PCR, has not been commercialized and applied in practice for 24 years. The core obstacle is the high false positive rate. The root of the problem is that the LAMP technology is only an ideal model, and its founders failed to figure out the single-stranded template denaturation mechanism of the LAMP technology. The scientific community believes that aerosol pollution is the main cause of false positives, but our previous research found that the LAMP system uses 2-3 pairs of primers, which makes the reaction system too complicated during amplification, and primers are prone to form primer dimers or hairpin structures; in addition, the concentration of dNTP in the LAMP reaction system is several times higher than that in the PCR reaction, and the concentration of magnesium ions and enzymes are higher than that in the PCR reaction. In the study of Real-time PCR, in order to avoid nonspecific amplification, the concentration of these three factors must be strictly controlled. High concentrations make the amplification speed too fast. The above reasons produce nonspecific amplification, which is the main cause of false positives. The problem of LAMP false positives caused by nonspecific amplification has gradually been confirmed by the scientific community.

[0009] LMTIA is an isothermal amplification technique based on a ladder melting curve, which is based on the difference between the melting temperatures (Tm) of the primers and the target sequence. Compared with traditional PCR, the LMTIA method has the following advantages:

[0010] (1) No thermal cycler is required; (2) Suitable for shorter target sequences, the shortest can be up to 60 bp; Fast reaction speed, the amplification reaction can be completed within 20 minutes; (3) High sensitivity, 50 times higher than LAMP technology; (4) Wide applicability, the Bst enzyme used has reverse transcriptase activity and can be used to detect DNA or RNA.

[0011] At present, LMTIA technology has been successfully applied to the detection of cassava ingredients in sweet potato starch; detection of soybean-derived ingredients in edible oils and dairy products; detection of Listeria monocytogenes contamination in food; detection of chicken and pork-derived ingredients in beef; identification of ginseng and American ginseng, etc. At present, there is no public report on the method of rapid identification of duck-derived ingredients using LMTIA technology. Summary of the invention

[0012] The purpose of the present invention is to provide a primer set, a detection method, a kit and its application for identifying duck-derived components using LMTIA technology, so as to solve the problems existing in the above-mentioned prior art. By designing LMTIA primers, a rapid detection method for duck-derived components is established, which provides technical support for the identification of duck-derived components in food and provides a detection means for food adulteration and fraud.

[0013] To achieve the above object, the present invention provides the following solutions:

[0014] The present invention provides a primer set for identifying duck-derived components using the LMTIA technique, comprising the following primers and probes:

[0015] Ya-ITS1-1F2: 5'-CAGCCCCGGTAATGATTTTAGTCGTAACAAGGTTTCCGT-3' (SEQ IDNO.1);

[0016] Ya-ITS1-1B2: 5'-ACCGGGGCTGGGCCTTTGGGCTCCGTCGCTCAC-3' (SEQ ID NO. 2);

[0017] Ya-ITS1-1LF: 5'-CTTCCGCAGGTTCACCT-3' (SEQ ID NO.3);

[0018] Ya-ITS1-LB: 5'-GGCTTGGGCGTCGCA-3' (SEQ ID NO. 4);

[0019] Ya-ITS1-LBPr: 5'-BHQ2-GGCTTGGGCGTCT-JOE-3' (SEQ ID NO. 5).

[0020] The invention also provides a kit for identifying duck-derived components by using the LMTIA technology, comprising the primer set.

[0021] The present invention also provides the use of the primer set or the kit in identifying duck-derived components in food.

[0022] The present invention also provides a detection method for identifying duck-derived components based on LMTIA technology, comprising the following steps:

[0023] The genomic DNA of the sample to be tested is obtained, and the genomic DNA is used as a template to perform an LMTIA reaction using the primer set, and whether the sample to be tested contains duck-derived components is determined based on the presence or absence of an amplification curve.

[0024] Preferably, the LMTIA reaction system includes: 0.4 μL of primers Ya-ITS1-1F2, Ya-ITS1-1B2, Ya-ITS1-1LF, Ya-ITS1-LB, 0.1 μL of probe Ya-ITS1-LBPr, 5 μL of reaction premix, 0.4 μL of DNA polymerase, 2.1 μL of DEPC water and 2 μL of DNA template.

[0025] More preferably, in the LMTIA reaction system, the molar ratio of Ya-ITS1-1F2, Ya-ITS1-1B2, Ya-ITS1-1LF, Ya-ITS1-LB and Ya-ITS1-LBPr is 8:8:2:2:5.

[0026] Preferably, the procedure of the LMTIA reaction is: isothermal amplification at 66°C for 20 minutes.

[0027] Preferably, the judgment method is: if an exponential amplification curve appears in the amplification result, the sample to be tested contains duck-derived components; if no amplification curve appears in the amplification result, the sample to be tested does not contain duck-derived components.

[0028] The present invention discloses the following technical effects:

[0029] The present invention designs a LMTIA specific primer set for detecting duck-derived ingredients. The primer set has good specificity and sensitivity, and the absolute sensitivity can reach 1pg / μL. It can detect adulteration of 0.1% by mass content, and the detection speed is fast. The detection of duck-derived ingredients in samples can be achieved under constant temperature conditions. The present invention establishes a method for rapid identification of duck-derived ingredients by LMTIA technology based on a specific primer set, provides a new method for authenticity identification of duck-derived ingredients, and can provide a detection means for food adulteration and fraud. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The results of the optimization of the reaction temperature of duck-derived ITS gene LMTIA; A, 65°C; B, 66°C; C, 67°C; D, 68°C; the negative control is DEPC water, and RFU is the relative fluorescence unit;

[0032] Figure 2 This is the result of the LMTIA reaction specificity test of the duck-derived ingredient ITS gene; the negative control is DEPC water, and RFU is the relative fluorescence unit;

[0033] Figure 3 This is the result of the LMTIA reaction sensitivity test of the duck-derived ingredient ITS gene; the negative control is DEPC water, and RFU is the relative fluorescence unit;

[0034] Figure 4 This is the result of the adulteration identification test of duck-derived ingredients using the ITS gene LMTIA method; the negative control is DEPC water, RFU is the relative fluorescence unit, and the percentage content is the mass percentage of duck meat adulterated in the food. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1LMTIA primer set design and temperature optimization

[0041] 1.LMTIA Primer Set Design

[0042] Nuclear genes are expressed in stable and constant copy numbers in different species and tissues, avoiding the interference of species diversity, and are mostly used for quantitative detection. The internal transcribed spacer (ITS) has multiple copy numbers and is mostly used for qualitative detection. Multiple copies of ITS genes are suitable as targets for designing and screening primers. The present invention is based on LMTIA technology and performs primer design and screening for duck-derived component ITS genes.

[0043] Taking the duck-derived component ITS gene as the target sequence, according to the principle of LMTIA screening target sequence, DNAMANv7.0.2 software was used to compare these sequences to find specific sites, Oligo 7 software was used to analyze and screen out ITS gene fragments with a ladder structure, and primer design software Primer 3plus (http: / / www.primer3plus.com / ) was used to design duck-derived component LMTIA primers. After a large number of screening, the primer set with the following sequence was obtained, which was synthesized by General Biosystems (Anhui) Co., Ltd. after design. The specific primer sequences are shown in Table 1.

[0044] Table 1 Primer sets and sequences of duck-derived LMTIA components

[0045]

[0046] 2. Extraction of Template DNA

[0047] The duck-derived DNA was extracted using a blood / cell / tissue genomic DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The specific operation steps are shown in the instructions of the kit.

[0048] 3. Temperature optimization of LMTIA reaction system

[0049] A 10 μL reaction system was used, specifically: 0.4 μL of primers Ya-ITS1-1F2, Ya-ITS1-1B2, Ya-ITS1-1LF and Ya-ITS1-LB, 0.1 μL of probe Ya-ITS1-LBPr, 5 μL of universal LMTIA reaction premix (purchased from Shandong Dege Biotechnology Co., Ltd.), 0.4 μL DNA polymerase, 2.1 μL DEPC-water and 2 μL duck-derived genomic DNA. The reaction temperatures of the Gentier 96E fully automatic medical PCR system were set to 65°C, 66°C, 67°C, and 68°C, respectively; the number of cycles was set to 40, and the fluorescence signal was collected every 30 seconds, for a total of 40 fluorescence signals.

[0050] Observe the temperature optimization amplification curve of duck-derived components, such as Figure 1 As shown in the figure, it can be seen that the duck-derived component primers designed with ITS as the target are significantly affected by temperature changes. DEPC-water was used as a negative control and no amplification was performed. However, the duck-derived component genomic DNA showed amplification at all tested temperatures, with the highest amplification efficiency and the strongest signal at 66°C. Therefore, the optimal reaction temperature was determined to be 66°C, and subsequent measurements were performed.

[0051] Example 2 Specificity detection of LMTIA primers

[0052] For the specificity test of duck-derived primers, genomic DNA extracted from chicken, pork, mutton, beef, horse meat, etc. was used as a reference, DEPC water was used as a negative control, and duck genomic DNA was used as a positive control. According to the optimized experimental plan, three parallel tests were performed in each experimental group, and the results were analyzed using the Gentier 96E fully automatic medical PCR analysis system.

[0053] The results are as follows Figure 2 As shown, only when duck-derived component DNA is added, the LMTIA reaction has a good amplification curve, and its LMTIA reaction is positive; the others have no amplification curve, and the reaction is negative. Therefore, the LMTIA detection method for duck-derived component detection established by the present invention has high specificity.

[0054] Example 3 Sensitivity Detection of LMTIA Primers

[0055] The extracted duck-derived genomic DNA was diluted to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL and 100 fg / μL. DEPC-water was used as a negative control, and two parallel reactions were set up for each reaction to determine the sensitivity of the LMTIA method. The Gentier 96E fully automatic medical PCR system was used, the temperature was set to 66°C, and the fluorescence signal was collected every 30 seconds, for a total of 40 fluorescence signals.

[0056] The results are as follows Figure 3 As shown, the duck-derived genomic DNA at concentrations of 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, and 1pg / μL were amplified, while the duck-derived genomic DNA at 100fg / μL was not amplified. Therefore, the established duck-derived ITS gene LMTIA system can detect duck-derived genomic DNA as low as 1pg / μL. Therefore, the sensitivity of this method can reach 1pg / μL.

[0057] Example 4 LMTIA simulated adulteration detection experiment

[0058] The established duck-derived ingredient LMTIA method was used to conduct a simulated adulteration detection experiment: duck meat was mixed with other meat genomic DNA to prepare mixed DNA samples, and the LMTIA reaction system was used for detection. The proportion of duck DNA in the mixed DNA samples was 0.01%, 0.1%, 1.0%, 5.0%, 10.0%, and 20.0%, respectively. The mixed meat DNA with different duck DNA mass fractions was added to the LMTIA reaction system after the optimized temperature, and DEPC water was used as a negative control. Two parallels were set for each test. The Gentier 96E fully automatic medical PCR system was used, the temperature was set to 66℃, and the fluorescence signal was collected every 30 seconds, and a total of 40 fluorescence signals were collected.

[0059] The results are as follows Figure 4 As shown, when the duck-derived component genomic DNA was mixed with other meat genomic DNA at a mass ratio of 20.0%, 10.0%, 5.0%, 1.0% and 0.1%, all samples could be successfully amplified, and the minimum detection limit of duck-derived components was 0.1%. The negative control and 0.01% mixed DNA were not amplified. Only when the duck-derived component DNA was added, the LMTIA reaction had an amplification curve, and the amplification curve had good repeatability and high stability. Therefore, the established LMTIA detection method for duck-derived component detection has good repeatability and high stability, and can be used for adulteration detection.

[0060] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer set for identifying duck-derived components using LMTIA technology, characterized in that: The following primers and probes are included: Ya-ITS1-1F2: 5'-CAGCCCCGGTAATGATTTTAGTCGTAACAAGGTTTCCGT-3'; Ya-ITS1-1B2: 5'-ACCGGGGCTGGGCCTTTGGGCTCCGTCGCTCAC-3'; Ya-ITS1-1LF: 5'-CTTCCGCAGGTTCACCT-3'; Ya-ITS1-LB: 5'-GGCTTGGGCGTCGCA-3'; Ya-ITS1-LBPr:5'-BHQ2-GGCTTGGGCGTCT-JOE-3'.

2. A kit for identifying duck-derived ingredients using LMTIA technology, characterized in that: Comprising the primer set of claim 1.

3. Use of the primer set according to claim 1 or the kit according to claim 2 in identifying duck-derived ingredients in food.

4. A detection method for identifying duck-derived ingredients based on LMTIA technology, characterized in that: The following steps are involved: Obtain genomic DNA of the sample to be tested, use the genomic DNA as a template, and perform LMTIA reaction using the primer set described in claim 1, and determine whether the sample to be tested contains duck-derived components based on the presence or absence of an amplification curve.

5. The detection method according to claim 4, characterized in that: The LMTIA reaction system includes: 0.4 μL of primers Ya-ITS1-1F2, Ya-ITS1-1B2, Ya-ITS1-1LF, and Ya-ITS1-LB, 0.1 μL of probe Ya-ITS1-LBPr, 5 μL of reaction premix, 0.4 μL of DNA polymerase, 2.1 μL of DEPC water, and 2 μL of DNA template.

6. The detection method according to claim 5, characterized in that In the LMTIA reaction system, the molar ratio of Ya-ITS1-1F2, Ya-ITS1-1B2, Ya-ITS1-1LF, Ya-ITS1-LB and Ya-ITS1-LBPr is 8:8:2:2:

5.

7. The detection method according to claim 4, characterized in that: The procedure of the LMTIA reaction is: isothermal amplification at 66°C for 20 minutes.

8. The detection method according to claim 4, characterized in that: The judgment method is: if an exponential amplification curve appears in the amplification result, the sample to be tested contains duck-derived components; if no amplification curve appears in the amplification result, the sample to be tested does not contain duck-derived components.

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