A primer and probe combination, detection method, kit and application thereof for identifying chicken-derived components using LMTIA technology

Abstract

The present invention discloses a primer and probe combination, a detection method, a kit and its application for identifying chicken-derived components by LMTIA technology, and relates to the field of molecular biology nucleic acid detection technology. The primer and probe combination includes primer JI-ITS-F1, primer JI-ITS-B1, primer JI-ITS-LF, primer JI-ITS-LB and probe Ji-ITS-LFPr, whose nucleotide sequences are respectively shown in SEQ ID NO.1-5. The present invention designs a LMTIA specific primer and probe combination for detecting chicken-derived components, which has good specificity and sensitivity, and the absolute sensitivity can reach 1pg / μL, and can detect adulteration of 0.1% by mass content, and the detection speed is fast, and the detection of chicken-derived components in the sample can be realized under constant temperature conditions. The present invention provides a new method for authenticity identification of chicken-derived components, and can provide a detection means for identifying food adulteration and fraud.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biological nucleic acid detection, and in particular to a primer and probe combination, a detection method, a kit and applications thereof for identifying chicken-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. For meat product adulteration, 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 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 technology based on a trapezoidal melting curve, which is based on the difference between the melting temperatures (Tm) of the primer and the target sequence. Compared with traditional PCR, the LMTIA method has the following advantages: no thermal cycler is required; it is suitable for shorter target sequences, the shortest of which can reach 60bp; it has a fast reaction speed, and the amplification reaction can be completed within 20 minutes; it has high sensitivity, which is 50 times more sensitive than the LAMP technology; it has wide applicability, and the Bst enzyme used has reverse transcriptase activity and can be used to detect DNA or RNA. LMTIA technology has been successfully applied to the detection of cassava components in sweet potato starch; the detection of soybean-derived components in edible oils and dairy products; the detection of Listeria monocytogenes contamination in food; the detection of chicken and pork-derived components in beef; the identification of ginseng and American ginseng, and other fields. The present invention intends to develop a new LMTIA method for identifying chicken-derived components, thereby providing technical support for the rapid identification of chicken-derived components in food. Summary of the invention

[0010] The purpose of the present invention is to provide a primer and probe combination, a detection method, a kit and its application for identifying chicken-derived components by LMTIA technology to solve the problems existing in the above-mentioned prior art. The present invention establishes a rapid detection method for chicken-derived components by designing LMTIA primers and probes, providing technical support for the identification of chicken-derived components in food.

[0011] To achieve the above object, the present invention provides the following solutions:

[0012] The invention provides a primer and probe combination for identifying chicken-derived components by LMTIA technology, comprising primer JI-ITS-F1, primer JI-ITS-B1, primer JI-ITS-LF, primer JI-ITS-LB and probe Ji-ITS-LFPr;

[0013] The nucleotide sequence of the primer JI-ITS-F1 is shown in SEQ ID NO.1;

[0014] The nucleotide sequence of the primer JI-ITS-B1 is shown in SEQ ID NO.2;

[0015] The nucleotide sequence of the primer JI-ITS-LF is shown in SEQ ID NO.3;

[0016] The nucleotide sequence of the primer JI-ITS-LB is shown in SEQ ID NO.4;

[0017] The nucleotide sequence of the probe Ji-ITS-LFPr is shown in SEQ ID NO.5.

[0018] Furthermore, the 5' end of the probe Ji-ITS-LFPr is labeled with a BHQ2 group, and the 3' end is labeled with a 6-FAM group.

[0019] The present invention also provides an application of the primer and probe combination in preparing an LMTIA kit for identifying chicken-derived components.

[0020] The present invention also provides a LMTIA kit for identifying chicken-derived components, comprising the above primer and probe combination.

[0021] Furthermore, the LMTIA kit also includes LMTIA reaction premix, DNA polymerase and DEPC water.

[0022] The present invention also provides the use of the primer and probe combination or the LMTIA kit in identifying chicken-derived components.

[0023] The present invention also provides a method for identifying chicken-derived ingredients, characterized in that it comprises the following steps:

[0024] Obtain the genomic DNA of the sample to be tested, use the genomic DNA of the sample to be tested as a template, use the above-mentioned primer and probe combination to perform LMTIA reaction, and judge whether the sample to be tested contains chicken-derived components according to the amplification result: if an exponential amplification curve appears in the amplification result, the sample to be tested contains chicken-derived components; if no exponential amplification curve appears in the amplification result, the sample to be tested does not contain chicken-derived components.

[0025] Furthermore, the reaction system of the LMTIA reaction is: 0.4 μL of primer mixture, 0.2 μL of probe, 5 μL of LMTIA reaction premix, 0.4 μL of DNA polymerase, 2 μL of DEPC water and 2 μL of template.

[0026] Furthermore, the temperature of the LMTIA reaction is 61-65° C., and the time is 20 min.

[0027] Furthermore, the temperature of the LMTIA reaction is 62°C.

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

[0029] The present invention designs a LMTIA specific primer and probe combination for detecting chicken-derived ingredients, which has good specificity and sensitivity, an absolute sensitivity of up to 1pg / μL, can detect adulteration of 0.1% by mass content, has a fast detection speed, and can detect chicken-derived ingredients in samples under constant temperature conditions. The present invention establishes a method for quickly identifying chicken-derived ingredients by LMTIA technology based on a specific primer group, provides a new method for identifying the authenticity of chicken-derived ingredients, and can provide a detection means for identifying 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 LMTIA reaction temperature optimization experiment for the ITS gene of chicken-derived ingredients; the negative control was DEPC-water; AD were the amplification curves at reaction temperatures of 61, 62, 63, and 64°C, respectively;

[0032] Figure 2 This is the specificity test result of LMTIA reaction of ITS gene of chicken-derived ingredients; the negative control is DEPC-water;

[0033] Figure 3 This is the result of the LMTIA reaction sensitivity test of the ITS gene of chicken-derived ingredients; the negative control is DEPC-water;

[0034] Figure 4 This is the result of the adulteration identification test of chicken-derived ingredients ITS gene LMTIA method; the negative control is DEPC-water: the percentage content is the mass percentage content of chicken mixed into 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) Design of LMTIA primer set

[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. Multi-copy 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 ITS genes of chicken-derived ingredients.

[0043] Taking the chicken-derived ingredient 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 chicken-derived ingredient LMTIA primers. After a large number of screening, primers and probes with the following sequences were obtained (synthesized by General Biosystems (Anhui) Co., Ltd. after design), as shown in Table 1.

[0044] Table 1 Primers and probes for LMTIA of chicken-derived ingredients

[0045]

[0046] (2) Extraction of template DNA

[0047] The chicken-derived component genomic 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 JI-ITS-F1, JI-ITS-B1, JI-ITS-LF and JI-ITS-LB, 0.2 μL of probe Ji-ITS-LFPr, 5 μL of universal LMTIA reaction premix (purchased from Shandong Dege Biotechnology Co., Ltd.), 0.4 μL DNA polymerase, 2 μL DEPC water (DEPC-water) and 2 μL chicken-derived genomic DNA. Among them, the molar ratio of JI-ITS-F1, JI-ITS-B1, JI-ITS-LF, JI-ITS-LB and Ji-ITS-LFPr was 4:4:1:1:5.

[0050] The reaction temperatures of the Gentier 96E fully automatic medical PCR system were set to 61°C, 62°C, 63°C, and 64°C, respectively; the number of cycles was set to 40 (20 min in total), and the fluorescence signal was collected every 30 seconds, for a total of 40 fluorescence signals.

[0051] Observe the temperature optimization amplification curve of chicken-derived ingredients, such as Figure 1 As shown in the figure, it can be seen that the chicken-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 chicken-derived component genomic DNA showed amplification at all tested temperatures, with the highest amplification efficiency and the strongest signal at 62°C. Therefore, the optimal reaction temperature was determined to be 62°C, and subsequent measurements were performed.

[0052] Example 2 Specificity detection of LMTIA primers

[0053] For the specificity test of chicken-derived primers, genomic DNA extracted from duck, pork, mutton, beef, horse meat, etc. was used as a reference, DEPC water was used as a negative control, and chicken genomic DNA was used as a positive control. According to the experimental method after temperature optimization in Example 1, three parallel tests were performed in each experimental group, and the results were analyzed using the Gentier 96E fully automatic medical PCR analysis system.

[0054] The results are as follows Figure 2As shown, only when chicken-derived component DNA is added, the LMTIA reaction has a good amplification curve, and its LMTIA reaction is positive; the others do not have an amplification curve, and the reaction is negative. Therefore, the LMTIA detection method for chicken-derived component detection established by the present invention has high specificity.

[0055] Example 3 Sensitivity Detection of LMTIA Primers

[0056] The extracted chicken-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. According to the experimental method after temperature optimization in Example 1, two parallels were set for each reaction to determine the sensitivity of the LMTIA method. A Gentier 96E fully automatic medical PCR system was used, the temperature was set to 62°C, and the fluorescence signal was collected every 30 seconds, for a total of 40 fluorescence signals.

[0057] The results are as follows Figure 3 As shown, the genomic DNA of chicken-derived components at concentrations of 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, and 1pg / μL were amplified, while the genomic DNA of chicken-derived components at 100fg / μL was not amplified. Therefore, the established chicken-derived component ITS gene LMTIA system can detect chicken-derived component genomic DNA as low as 1pg / μL. Therefore, the sensitivity of this method can reach 1pg / μL.

[0058] Example 4 LMTIA simulated adulteration detection experiment

[0059] The chicken-derived ingredient LMTIA detection method established in Example 1 was used to conduct a simulated adulteration detection experiment: chicken and other meat genomic DNA were mixed to prepare a mixed DNA sample, and the LMTIA reaction system was used for detection. The proportion of chicken DNA in the mixed DNA sample was 0.01%, 0.1%, 1.0%, 5.0%, 10.0%, and 20.0%, respectively. The mixed meat sample DNAs with different chicken DNA mass fractions were 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 62°C, and the fluorescence signal was collected every 30 seconds, and a total of 40 fluorescence signals were collected.

[0060] The results are as follows Figure 4As shown, when the chicken-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 the chicken-derived component was 0.1%. The negative control and 0.01% mixed DNA were not amplified. Only when the chicken-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 chicken-derived component detection has good repeatability and high stability.

[0061] 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 method for identifying chicken-derived ingredients, characterized in that: The following steps are involved: Obtaining genomic DNA of the sample to be tested, using the genomic DNA of the sample to be tested as a template, performing an LMTIA reaction using a primer and probe combination for identifying chicken-derived components using an LMTIA technique, and judging whether the sample to be tested contains chicken-derived components based on the amplification result: if an exponential amplification curve appears in the amplification result, the sample to be tested contains chicken-derived components; If no exponential amplification curve appears in the amplification result, the sample to be tested does not contain chicken-derived components; The primer and probe combination includes primer JI-ITS-F1, primer JI-ITS-B1, primer JI-ITS-LF, primer JI-ITS-LB and probe Ji-ITS-LFPr; The nucleotide sequence of the primer JI-ITS-F1 is shown in SEQ ID NO.1; The nucleotide sequence of the primer JI-ITS-B1 is shown in SEQ ID NO.2; The nucleotide sequence of the primer JI-ITS-LF is shown in SEQ ID NO.3; The nucleotide sequence of the primer JI-ITS-LB is shown in SEQ ID NO.4; The nucleotide sequence of the probe Ji-ITS-LFPr is shown in SEQ ID NO.5; The 5' end of the probe Ji-ITS-LFPr is labeled with a BHQ2 group, and the 3' end is labeled with a 6-FAM group; The reaction system of the LMTIA reaction is: 0.4 μL of primer mixture, 0.2 μL of probe, 5 μL of LMTIA reaction premix, 0.4 μL of DNA polymerase, 2 μL of DEPC water and 2 μL of template.

2. The method according to claim 1, characterized in that The temperature of the LMTIA reaction is 61-65° C. and the reaction time is 20 min.

3. The method according to claim 2, characterized in that The temperature of the LMTIA reaction was 62°C.

4. Use of a primer and probe combination in the preparation of an LMTIA kit for identifying chicken-derived ingredients, characterized in that: The primer and probe combination includes primer JI-ITS-F1, primer JI-ITS-B1, primer JI-ITS-LF, primer JI-ITS-LB and probe Ji-ITS-LFPr; The nucleotide sequence of the primer JI-ITS-F1 is shown in SEQ ID NO.1; The nucleotide sequence of the primer JI-ITS-B1 is shown in SEQ ID NO.2; The nucleotide sequence of the primer JI-ITS-LF is shown in SEQ ID NO.3; The nucleotide sequence of the primer JI-ITS-LB is shown in SEQ ID NO.4; The nucleotide sequence of the probe Ji-ITS-LFPr is shown in SEQ ID NO.5; The 5' end of the probe Ji-ITS-LFPr is labeled with a BHQ2 group, and the 3' end is labeled with a 6-FAM group.

5. A LMTIA kit for identifying chicken-derived ingredients, characterized in that: A primer and probe combination is included, characterized in that the primer and probe combination includes primer JI-ITS-F1, primer JI-ITS-B1, primer JI-ITS-LF, primer JI-ITS-LB and probe Ji-ITS-LFPr; The nucleotide sequence of the primer JI-ITS-F1 is shown in SEQ ID NO.1; The nucleotide sequence of the primer JI-ITS-B1 is shown in SEQ ID NO.2; The nucleotide sequence of the primer JI-ITS-LF is shown in SEQ ID NO.3; The nucleotide sequence of the primer JI-ITS-LB is shown in SEQ ID NO.4; The nucleotide sequence of the probe Ji-ITS-LFPr is shown in SEQ ID NO.5; The 5' end of the probe Ji-ITS-LFPr is labeled with a BHQ2 group, and the 3' end is labeled with a 6-FAM group.

6. The LMTIA kit according to claim 5, characterized in that The LMTIA kit also includes LMTIA reaction premix, DNA polymerase and DEPC water.

7. Use of a primer and probe combination or the LMTIA kit according to claim 5 or 6 in identifying chicken-derived ingredients, characterized in that: The primer and probe combination includes primer JI-ITS-F1, primer JI-ITS-B1, primer JI-ITS-LF, primer JI-ITS-LB and probe Ji-ITS-LFPr; The nucleotide sequence of the primer JI-ITS-F1 is shown in SEQ ID NO.1; The nucleotide sequence of the primer JI-ITS-B1 is shown in SEQ ID NO.2; The nucleotide sequence of the primer JI-ITS-LF is shown in SEQ ID NO.3; The nucleotide sequence of the primer JI-ITS-LB is shown in SEQ ID NO.4; The nucleotide sequence of the probe Ji-ITS-LFPr is shown in SEQ ID NO.5; The 5' end of the probe Ji-ITS-LFPr is labeled with a BHQ2 group, and the 3' end is labeled with a 6-FAM group.

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