LMTIA primer group for wheat source component detection, kit, detection method and application

Abstract

The invention belongs to the technical field of biomolecule detection, and particularly relates to an LMTIA primer group, a probe and a kit for wheat source component detection and application of the LMTIA primer group, the probe and the kit. The invention discloses an LMTIA primer group and a probe for wheat source component detection, and the LMTIA primer group and the probe comprise a wheat source general F primer, a wheat source general B primer, a wheat source general LF primer and a wheat source Pr probe, the LMTIA primer group and the probe can be used for wheat source component detection, the detection speed is high, the sensitivity is high, and the specificity is strong; the wheat source component in the sample can be quickly, efficiently and accurately identified, and an effective technical means is provided for authenticity identification of food.

Description

Technical Field

[0001] The invention belongs to the technical field of biomolecule detection, and specifically relates to LMTIA primers and Proofman probes for detecting wheat-derived components, a kit for rapidly detecting wheat-derived components, and the application of the kit in rapidly detecting wheat-derived components added to food. Background Art

[0002] Wheat (scientific name: Triticum aestivum L.) is an annual or biennial herbaceous plant of the Poaceae family and the genus Triticeae. As one of the most important food crops, it is rich in nutrients such as starch, protein, and fat and is widely consumed. In the supply chain of starch products, adulteration is common, and the means of adulteration are diverse. It is common to use low-cost wheat starch raw materials and false labels in expensive starch products. It is imperative to study and develop efficient wheat source identification methods.

[0003] There are various methods for identifying wheat sources, among which the gene detection method has the characteristics of short time, strong specificity and high sensitivity, and is of great research value. CN111394506B discloses a competitive allele-specific PCR wheat molecular marker method, and provides an application of a primer set to identify wheat grain weight traits; CN106011301B discloses primers, kits and methods for PCR for flux identification of major dormancy genes in wheat seeds. The above is based on the PCR method, which is complicated to operate and takes a long time to detect. Ladders-shape melting temperature isothermal amplification (LMTIA) is a new type of nucleic acid isothermal amplification technology that can achieve stable amplification of nucleic acids within 20 minutes, with high sensitivity and specificity, and there is no report on the use of LMTIA technology in the detection of wheat-derived components. It is of great practical significance to apply LMTIA technology to the identification of wheat-derived components and establish a LMTIA detection method for wheat-derived components. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a primer set, a kit, a detection method and its application for identifying wheat-derived components using the LMTIA technology. By designing the LMTIA primer set and the Proofman probe, the LMTIA technology is established to detect wheat-derived components, thereby providing a means for identifying wheat sources and detecting food adulteration wheat starch.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first aspect of the present invention provides a LMTIA primer set and probe for single-plex detection of wheat-derived components, the sequences of which are as follows:

[0007] XM-F: 5'-AGCAAGCTAGACTTTTCGAAGGCGTCAAGGAACACT-3'

[0008] XM-B: 5'-TAGCTTGCTAGCTTTTTAGCTTTGCAACACGAGGGA-3'

[0009] XM-LF: 5'-ATGCCCCCGGGTTAG-3'

[0010] XM-Pr: 5'BHQ2-ATGCCCCCGGGTTAA-3'6-FAM.

[0011] The second aspect of the present invention provides a kit for single-plex detection of wheat-derived components, comprising the above-mentioned LMTIA primer set and probe.

[0012] Preferably, the probe is a proofreading enzyme-mediated probe, with a fluorescent group and a quenching group connected to both ends respectively.

[0013] Use of the LMTIA primer set and probe described in the first aspect of the present invention or the kit described in the second aspect in the detection of wheat-derived components.

[0014] The third aspect of the present invention provides a method for detecting wheat-derived components, comprising the following steps: taking a sample to be tested, extracting sample DNA, preparing an LMTIA reaction system using the LMTIA primer set and probe described in the first aspect or the kit described in the second aspect, and placing the prepared LMTIA reaction system in a real-time fluorescence PCR instrument for constant temperature amplification; to detect whether wheat-derived components are present in the sample.

[0015] Preferably, the method for determining whether wheat-derived components are present in the test sample is as follows: if an exponential amplification curve appears in the amplification result, the sample contains wheat-derived components; if no exponential amplification curve appears in the amplification result, the sample does not contain wheat-derived components.

[0016] Preferably, the method for extracting sample DNA refers to a plant DNA extraction kit (Zhengzhou Beibei Biotechnology Co., Ltd.).

[0017] Preferably, the temperature of the isothermal amplification is 65° C., and the time of the isothermal amplification is 20 minutes.

[0018] Preferably, the molar ratio of XM-F:XM-B:XM-LF:XM-Pr in the LMTIA reaction system is 8:8:2:5.

[0019] The present invention also provides the use of the LMTIA primer set and probe for detecting wheat-derived components described in the first aspect or the kit described in the second aspect in detecting wheat-derived components in food.

[0020] The present invention also provides the use of the LMTIA primer set and probe for detecting wheat-derived components described in the first aspect or the kit described in the second aspect in detecting wheat-derived components in starch or starch-processed foods.

[0021] The beneficial effects of the present invention are:

[0022] The LMTIA primer set and probe for detecting wheat-derived components of the present invention have good specificity and sensitivity, a fast detection speed, and can detect wheat-derived components in a sample under a constant temperature condition.

[0023] The detection result can be obtained by reacting the LMTIA primer set and probe at 65° C. for 20 minutes.

[0024] The sensitivity of the LMTIA primer set and probe of the present invention can reach 10 fg / μL, and the detection limit can reach 0.1%.

[0025] The LMTIA primer set and probe of the present invention can be used to detect wheat-derived components in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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 or the description of the prior art 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.

[0027] Figure 1 This is a ladder sequence curve diagram of the wheat-derived nucleic acid target sequence in Example 1 of the present invention;

[0028] Figure 2 This is a graph showing the amplification result of the wheat-derived LMTIA primer amplification temperature in Example 2 of the present invention;

[0029] Figure 3 This is a graph showing the specific amplification results of wheat-derived LMTIA primers in Example 3 of the present invention;

[0030] Figure 4 This is a graph showing the amplification results of wheat-derived LMTIA primer sensitivity in Example 4 of the present invention;

[0031] Figure 5 This is a graph showing the amplification result of the detection limit of wheat-derived LMTIA primers in Example 5 of the present invention;

[0032] Figure 6 This is a diagram showing the amplification results of different actual market samples detected by wheat-derived LMTIA primers in Example 6 of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] All documents cited in the present invention are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with the present invention, the description of the present invention shall prevail. Various terms and phrases used in the present invention have the general meanings known to those skilled in the art.

[0035] Unless otherwise defined, all professional terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention; unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0036] The sources of the reagents used in the present invention are shown in Table 1.

[0037] Table 1 Experimental reagents and sources

[0038]

[0039]

[0040] Example 1

[0041] LMTIA primer set, probe design and temperature optimization

[0042] (1) Design of LMTIA primers and probes: DNA of wheat standard was extracted using a plant DNA extraction kit (Beibei Biotechnology). Plasmids were extracted from the extracted DNA. The bacterial solution that met the bands was screened through electrophoresis, gel cutting, gel recovery, transformation of competent cells, plasmid extraction, enzyme cutting and electrophoresis verification, etc. One part was sent for sequencing, and the other part was stored as glycerol bacteria. The bacterial solution can be stored for a long time at -80°C. The sequencing results were analyzed using BLAST sequence alignment, with the internal transcribed spacer (ITS) as the target sequence, and the sequence with a ladder-shaped melting temperature (such as Figure 1 As shown in the figure), Primer3Plus was used to design wheat-derived LMTIA primers and probes. After extensive screening, the following primer sets and probes were obtained (synthesized by Universal Biosystems (Anhui) Co., Ltd.). The specific sequences are shown in Table 2.

[0043] Table 2LMTIA primer set and probe sequence

[0044]

[0045] (2) DNA extraction: A plant DNA extraction kit purchased from Zhengzhou Beibei Biotechnology Co., Ltd. was used. For specific procedures, please refer to the kit manual.

[0046] (3) LMTIA reaction system of the present invention:

[0047] The reaction system of LMTIA is shown in Table 3.

[0048] Table 3LMTIA reaction system (10 μL)

[0049]

[0050] Example 2

[0051] Temperature optimization of LMTIA reaction system

[0052] Take eight PCR tubes, add the reaction reagents prepared in Table 3 to four tubes respectively (the system does not include template DNA, 8 μL in each tube), add 2 μL ddH2O to two tubes as negative control, and add 2 μL wheat standard DNA to the other two tubes. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature gradient to 59°C, 61°C, 63°C and 65°C, set the fluorescence signal to be collected every 30 seconds, and collect 40 fluorescence signals in total to screen the temperature.

[0053] Observe the temperature amplification curve of wheat standard ( Figure 2 ) It can be seen that at 59°C, 61°C, 63°C and 65°C, there is no nonspecific amplification caused by primer dimers. According to the amplification efficiency and reproducibility of the wheat standard amplification curve at different temperatures, 65°C was selected as the optimal temperature for LMTIA and subsequent determinations were performed.

[0054] Example 3

[0055] Specificity of the LMTIA primers of the present invention

[0056] For LMTIA wheat-source specific detection, 3 ng / μL wheat-source standard was used as positive control, ddH2O as negative control, and potatoes, peas, cassava, sweet potatoes, mung beans, corn, and lotus root powder were selected as samples to be tested. According to the LMTIA reaction system in Table 3, 2 μL of positive control, negative control, and sample to be tested were added to a single tube, and two parallels were set for each reaction. The Gentier 96E fully automatic medical PCR analysis system was used to set the temperature to 65°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 in the figure, only when the positive control was added did an amplification curve appear, and the amplification curve had good repeatability and high stability. The LMTIA reaction was positive, and the other reactions were negative. Therefore, the LMTIA detection method for wheat source component detection established had good repeatability and high stability.

[0058] Example 4

[0059] Sensitivity of the LMTIA primers of the present invention

[0060] For LMTIA wheat source sensitivity detection, 1 ng / μL wheat standard was used as the positive control and diluted to 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, and 10 fg / μL. ddH2O was used as the negative control. Two parallel reactions were set for each reaction. The temperature was set to 65°C using the Gentier 96E fully automatic medical PCR analysis system. Fluorescence signals were collected every 30 seconds for a total of 40 times.

[0061] The results are as follows Figure 4 As shown, when the concentrations of the positive control and wheat standard DNA were 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, and 10fg / μL, the LMTIA amplification curves were more obvious, and no amplification curve appeared in the negative control. Therefore, the absolute sensitivity of this method can reach 10fg / μL. In a 10μL reaction system, wheat DNA extracted from the sample can be detected when it reaches 10fg.

[0062] Example 5

[0063] The detection limit of the LMTIA primers of the present invention is

[0064] For the LMTIA wheat source detection limit test, wheat starch was added to corn starch at volume ratios of 20.0%, 10.0%, 5.0%, 1.0% and 0.1%, respectively, and mixed evenly. DNA was extracted using an automatic nucleic acid extraction kit (magnetic bead method). The extracted DNA was used as the sample to be tested, ddH2O was used as a negative control, and the wheat standard was used as a positive control. Two parallel reactions were set for each reaction. The temperature was set at 65°C using a Gentier 96E fully automatic medical PCR analysis system. Fluorescence signals were collected every 30 seconds, and a total of 40 fluorescence signals were collected.

[0065] The results are as follows Figure 5 As shown, when wheat starch was mixed with corn starch at a volume ratio of 20.0%, 10.0%, 5.0%, 1.0% and 0.1%, all positive controls were amplified, and negative controls were not amplified. All samples to be tested were successfully amplified, and the minimum detection limit of wheat source was 0.1%. Only when wheat source DNA was added, the LMTIA reaction had an amplification curve, and the amplification curve had good reproducibility and high stability.

[0066] Example 6

[0067] Detection of actual market samples by the LMTIA primer set and probe of the present invention

[0068] The actual samples No. 1-12 in Table 1 were used for testing, among which the ingredient list of sample No. 8 was marked with wheat ingredients, and the other ingredient lists were not marked with wheat ingredients. DNA was extracted using a nucleic acid manual extraction kit, and the LMTIA primer set and probe of the present invention were used to amplify and detect the above-extracted DNA, and the detection of different actual samples was compared by amplification efficiency. A wheat standard was used as a positive control, ddH2O as a negative control, and the DNA extracted from the actual sample was used as the sample to be tested. Two parallels were set for each reaction, and the temperature was set to 65°C using the Gentier 96E fully automatic medical PCR analysis system. Fluorescence signals were collected every 30 seconds, and a total of 40 fluorescence signals were collected.

[0069] The results are as follows Figure 6 All positive controls were amplified, negative controls were not amplified, samples No. 2 and No. 12 were amplified, and other samples were not amplified, indicating that samples No. 2 and No. 12 contained wheat-derived ingredients, and other samples did not contain wheat-derived ingredients.

[0070] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. LMTIA primer set and probe for single-plex detection of wheat-derived components, It is characterized in that The sequences of the primer set and probe are: XM-F: 5'-AGCAAGCTAGACTTTTCGAAGGCGTCAAGGAACACT-3' XM-B: 5'-TAGCTTGCTAGCTTTTTAGCTTTGCAACACGAGGGA-3' XM-LF: 5'-ATGCCCCCGGGTTAG-3' XM-Pr: 5'BHQ2-ATGCCCCCGGGTTAA-3'6-FAM.

2. The LMTIA primer set and probe for single-plex detection of wheat-derived components according to claim 1, It is characterized in that The probe is a proofreading enzyme-mediated probe, with a fluorescent group and a quenching group connected to both ends respectively.

3. Kit for single-plex detection of wheat-derived ingredients, It is characterized in that It comprises the LMTIA primer set and probe as described in claim 1 or 2.

4. Use of the LMTIA primer set or probe according to claim 1 or 2 or the kit according to claim 3 in detecting wheat-derived components.

5. Use of the LMTIA primer set or probe according to claim 1 or 2 or the kit according to claim 3 in detecting wheat-derived ingredients in food.

6. Use of the LMTIA primer set or probe according to claim 1 or 2 or the kit according to claim 3 in detecting wheat-derived components in starch or starch-processed foods.

7. A method for detecting wheat-derived ingredients, It is characterized in that The following steps are involved: Take the sample to be tested, extract the sample DNA, prepare the LMTIA reaction system using the LMTIA primer set and probe described in claim 1 or 2 or the kit described in claim 3, and place the prepared LMTIA reaction system in a real-time fluorescence PCR instrument for constant temperature amplification to detect whether there is wheat-derived component in the sample.

8. The method for detecting wheat-derived components according to claim 7, It is characterized in that The method for determining whether wheat-derived components are present in the test sample is as follows: if an exponential amplification curve appears in the amplification result, the sample contains wheat-derived components; if no exponential amplification curve appears in the amplification result, the sample does not contain wheat-derived components.

9. The method for detecting wheat-derived components according to claim 7, It is characterized in that The temperature of the isothermal amplification is 65° C., and the time of the isothermal amplification is 20 minutes.

10. The method for detecting wheat-derived components according to claim 7, It is characterized in that The molar ratio of XM-F:XM-B:XM-LF:XM-Pr in the LMTIA reaction system is 8:8:2:5.

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