Fluorescent PCR (Polymerase Chain Reaction) amplification primer probe group capable of detecting goose-derived components and detection kit

Abstract

The invention relates to the technical field of animal-derived detection, in particular to a fluorescent PCR (polymerase chain reaction) amplification primer probe group and a detection kit capable of detecting goose-derived ingredients, the primer probe group comprises a primer pair and a probe, the primer pair comprises an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2, and the probe is a probe as shown in SEQ ID NO.3. The primer probe group disclosed by the invention can be used for effectively detecting goose-derived components, and comprises a primer combination and a probe which are high in specificity and sensitivity. The kit is sensitive, convenient, high in specificity, high in sensitivity and good in reliability, whether a sample contains the goose-derived component or not can be judged only through one-time detection, large-batch sample analysis can be conducted at the same time, powerful technical support is provided for goose-derived component detection and product quality detection, and the kit has high practical value and good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of animal-derived detection, and in particular to a fluorescent PCR amplification primer probe set and a detection kit capable of detecting goose-derived components. Background Art

[0002] In recent years, my country has frequently seen vicious incidents of counterfeiting ingredients in food, products, and supplies. There has been a phenomenon of replacing ingredients that should be in advertisements or ingredient lists with similar ingredients that are cheaper and more easily available. At present, the identification of animal-derived ingredients has become a common concern of testing departments and consumers. Therefore, the detection of animal-derived ingredients and raw materials can no longer rely solely on sensory testing and verification. For regulatory departments and testing departments, it is very necessary to establish a rapid and effective method for detecting animal-derived ingredients and quickly identify the quality of animal-derived products. For goose-derived ingredients, such as goose meat, goose down, and goose liver, because of their high value, both their direct products and related products have the problem of counterfeiting ingredients. Therefore, the rapid detection of goose (Anseranser)-derived ingredients is of great significance. Summary of the invention

[0003] In view of this, the purpose of the present invention is to propose a fluorescent PCR amplification primer probe set and a detection kit that can detect goose-derived components, so as to effectively detect goose-derived components with high sensitivity and good specificity.

[0004] Based on the above purpose, the present invention provides a fluorescent PCR amplification primer probe group that can detect goose-derived components, the primer probe group includes a primer pair and a probe, the primer pair includes an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2, and the probe is a probe as shown in SEQ ID NO.3.

[0005] The present invention also provides a fluorescent PCR detection kit capable of detecting goose-derived components, wherein the kit comprises the fluorescent PCR amplification primer probe group capable of detecting goose-derived components.

[0006] Preferably, the kit also includes a goose-derived component qPCR positive control. To increase product stability and avoid the spread of infectious pathogens, this product does not provide a live sample as a positive control, but only provides a non-infectious DNA fragment as a positive control. The positive control is 1E7 copies / μL, which is used to standardize curve construction and distinguish false negative samples.

[0007] The PCR reaction system used in the kit is 20 μL, consisting of 10 μL of 2×Probe qPCR MasterMix, 3 μL of probe primer mixture, and 7 μL of DNA template to be tested.

[0008] Preferably, the amplification reaction program adopted by the kit is 95°C for 10 min; 45 cycles, each cycle being 95°C for 15 sec and 60°C for 1 min; the fluorescence signal is collected in the second step of each cycle.

[0009] Preferably, the 5' end of the probe is labeled with a FAM fluorophore and the 3' end is labeled with an Eclipse fluorophore.

[0010] Preferably, the positive control is a recombinant plasmid containing the gene fragment of the C-terminal of 16S rRNA of goose origin, and the positive control is 1E7 copies / μL, which is used for standard curve construction and identification of false-negative samples. The kit also includes a negative control. The negative control is distilled water.

[0011] When detecting with the kit, when the Ct value ≤ 40 and a specific amplification curve appears in the channel corresponding to the probe, it is determined that the sample contains goose-derived components.

[0012] In the present invention, the primers and probes are designed for the 16S rRNA of goose.

[0013] Advantages of the present invention: The primer-probe group of the present invention can effectively detect goose-derived components, including a primer combination and a probe with strong specificity and high sensitivity. The kit of the present invention is sensitive, convenient, highly specific, highly sensitive and reliable. It can determine whether the sample contains goose-derived components with only one detection, and can analyze a large number of samples simultaneously, providing strong technical support for the detection of goose-derived components and the detection of product quality, and having strong practical value and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0015] Figure 1 It is a schematic diagram for verifying the feasibility of the primers-probes of the present invention;

[0016] Figure 2 It is a schematic diagram for verifying the sensitivity of the primers-probes of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] In order to make the purpose, technical solutions and advantages of the present invention clearer, the following will further describe the present invention in detail with reference to specific embodiments.

[0018] It should be noted that unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those with ordinary skills in the field to which the present invention belongs. Words such as "including" or "comprising" and the like mean that the elements or objects appearing before this word cover the elements or objects listed after this word and their equivalents, without excluding other elements or objects.

[0019] 1. Design of Fluorescent PCR Amplification Primers and Probes for Detecting Goose-derived Components

[0020] Download the genomic sequence with the gene number AF363031 from NCBI. To ensure the detection effect and improve the detection rate of goose-derived components, primers and probes are designed according to the highly conserved region of 16S rRNA of goose-derived components (the primer and probe sequences are shown in Table 1). Among them: F represents the upstream primer; P represents the probe; R represents the downstream primer.

[0021] Table 1 Design of Fluorescent PCR Primers / Probes

[0022]

[0023] 2. Verification of the Feasibility and Sensitivity of Fluorescent PCR Amplification Primers and Probes for Detecting Goose-derived Components

[0024] First, dilute the standard curve samples. Using the provided positive control of 1E7 / μL as the mother liquor, perform 10-fold serial dilutions with the special template diluent for fluorescent PCR to obtain six 10-fold dilution gradient standard curve samples of 1E1 - 1E6 copies / μL. Use the Q1600 real-time fluorescence quantitative PCR instrument from Hangzhou Baiheng Technology Co., Ltd. for detection. The total volume of a single reaction for detection is 20 μL. Among them, the components of the six standard curve sample tubes include 10 μL of 2×Probe qPCR MasterMix each, 3 μL of the qPCR primer-probe mixture for goose-derived components each, and 7 μL of the standard curve sample diluent each; the components of the PCR negative control include 10 μL of 2×Probe qPCR MasterMix each, 3 μL of the qPCR primer-probe mixture for goose-derived components each, and 7 μL of ultrapure water. The reaction program is in a fixed mode, that is, pre-denaturation at 95°C for 10 min; 45 cycles of PCR reaction, each cycle being 95°C for 15 sec and 60°C for 1 min; collect the fluorescence signal of the FAM channel in the second step of each cycle. Construct a standard curve with the log value of the positive control concentration as the abscissa and the Ct value as the ordinate and perform linear fitting.

[0025] According to the results ( Figure 1 and Figure 2 ), it can be seen that the primers and probes have good performance and high sensitivity. The lowest detection limit tested with the positive control is not less than 100 copies / reaction, and the linear relationship is good with a high fitting degree R2 Not less than 0.95, and the linear range can reach at least 6 orders of magnitude during quantitative detection (where the numbers ① - ⑥ correspond to the positive control concentrations of E6 - E1).

[0026] 3. Specificity verification of a fluorescence PCR amplification primer and probe for detecting goose - derived components

[0027] Design to amplify positive samples of chicken and duck using the fluorescence PCR method established in this patent, and the results are all negative.

[0028] 4. Repeatability verification of a fluorescence PCR amplification primer - probe set and a detection kit for detecting goose - derived components

[0029] Use plasmids with known concentrations (1E6 copies, 1E4 copies, 1E2 copies) as templates, and let an operator repeat the detection on the same day to verify the intra - group repeatability of the method; let two operators operate at different times and calculate the inter - group repeatability. As shown in Table 2 below, the results show that the intra - group and inter - group repeatabilities of this method are good, and the coefficients of variation are respectively in the range of 0.45% - 0.85% and 0.48% - 1.43%.

[0030] Table 2 Repeatability verification of the fluorescence PCR method

[0031]

[0032]

[0033] 5. Detection of different types of samples by a fluorescence PCR amplification primer - probe set and a detection kit for detecting goose - derived components

[0034] Take DNA of different types of positive samples for detection and compare it with the PCR method for detecting goose components in the national standard. As shown in Table 3 below, the results show that all the detection results of the national standard are positive; the methods in the present invention are also all positive and can be quantitatively detected.

[0035] Table 3 Verification and comparison of the detection methods of the present invention and the national standard method for different types of positive DNA

[0036]

[0037] Those of ordinary skill in the art should understand that: The discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of the present invention is limited to these examples; Under the concept of the present invention, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, and they are not provided in detail for the sake of brevity. Any omission, modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A fluorescent PCR amplification primer probe set capable of detecting goose-derived components, characterized in that: The primer-probe set includes a primer pair and a probe, wherein the primer pair includes an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2, and the probe includes a probe as shown in SEQ ID NO.

3.

2. A fluorescent PCR detection kit for detecting goose-derived components, characterized in that: The kit comprises the fluorescent PCR amplification primer probe set capable of detecting goose-derived components as described in claim 1.

3. The fluorescent PCR detection kit for detecting goose-derived components according to claim 2, characterized in that: The kit also includes a special diluent for fluorescent PCR template and a goose-derived component qPCR positive control.

4. The fluorescent PCR detection kit for detecting goose-derived components according to claim 3, characterized in that: The goose-derived component qPCR positive control is a recombinant plasmid containing a gene fragment of the C-terminus of the goose-derived component 16S rRNA.

5. The fluorescent PCR detection kit for detecting goose-derived components according to claim 3, characterized in that: The goose-derived component qPCR positive control is an artificially synthesized DNA sequence, which can be used for marker construction and subsequent quantitative detection.

6. The fluorescent PCR detection kit for detecting goose-derived components according to claim 3, characterized in that: The kit also includes a negative control.

7. The fluorescent PCR detection kit for detecting goose-derived components according to claim 2, characterized in that: The amplification reaction program adopted by the kit is 95° C. for 10 min; 45 cycles, each cycle is 95° C. for 15 sec, 60° C. for 1 min; and the fluorescence signal is collected in the second step of each cycle.

8. The fluorescent PCR detection kit for detecting goose-derived components according to claim 2, characterized in that: The 5' end of the probe is labeled with a FAM fluorescent group, and the 3' end is labeled with an Eclipse fluorescent group.

9. The fluorescent PCR detection kit for detecting goose-derived components according to claim 2, characterized in that: When the kit is used for detection, when the Ct value is ≤40 and a specific amplification curve appears in the channel corresponding to the probe, it is determined that goose-derived components are contained.

Images