A fluorescent PCR probe method detection kit for chlamydia trachomatis

Abstract

The application belongs to the technical field of biological detection and identification, and discloses a fluorescence PCR probe method detection kit for chameesiphanes grayanus, which comprises a pair of specific fluorescence primers CADQF and CADQR, a probe CADQP, a real-time fluorescence reaction mixture of 2xPremix Ex TaqTM PCR and ROXII, a positive control, a negative control and nuclease-free water; the sequence of the specific fluorescence primer CADQF is SEQ ID NO:1; and the sequence of the specific fluorescence primer CADQR is SEQ ID NO:2. The fluorescence PCR probe method detection kit can quickly and efficiently determine whether the sample to be tested is chameesiphanes grayanus, and can determine the type and content of raw materials in products that cannot be determined from morphology or use chameesiphanes grayanus as raw materials through the fluorescence PCR probe method, thereby providing a more convenient and efficient method for commodity quality and marine sample ecology.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection and identification, and particularly relates to a fluorescent PCR probe method detection kit for Coelomactra antiquata.

[0002] The present application provides a fluorescent PCR probe method detection kit for Coelomactra antiquata. The content of the present application relates to a pair of fluorescent quantitative PCR primers and probes that can be used to detect and identify Coelomactra antiquata, and a set of fluorescent PCR probe method detection kits comprising real-time fluorescent PCR reaction mixture I, real-time fluorescent PCR reaction mixture II (containing primers and probes), positive control, negative control, nuclease-free water, and corresponding detection operation procedures. BACKGROUND

[0003] At present, Coelomactra antiquata (Spengler, 1802) is a valuable shellfish with high economic and nutritional value, and it is difficult to be distinguished from other species with similar shell morphologies.

[0004] Coelomactra antiquata (Spengler, 1802), also known as sea clam or "royal concubine clam," belongs to the phylum Mollusca, class Lamellibranchia, order Veneroida, family Mactridae, and genus Coelomactra. Its shell is large, approximately 40 to 100 mm long, slightly triangular in shape, and thin, with a pale yellow or yellowish-white surface. The apex is smooth and purple, while the rest of the shell has concentric growth rings and is covered with a silky, earthy-yellow rind. The interior of the shell is off-white, with a pale purple apex; the mantle sinus is shallow and semicircular. It lives on the fine-grained beaches of the middle and lower intertidal zones. Coelomactra antiquata is not only large, tender, and delicious, but also contains a variety of nutrients. It also grows quickly and has a beautiful appearance. It is a valuable shellfish with both edible and medicinal properties, and a rare and excellent species in my country's shellfish aquaculture industry. The scalloped clams are found in the western Pacific Ocean, including my country, Japan, Vietnam, and the coasts of the Indian Peninsula. They are also found in the Yellow Sea, East China Sea, and South China Sea, with Jiaonan, Shandong, Nantong, Jiangsu, Taizhou, Zhejiang, Changle, Fujian, and Beihai, Guangxi being the primary distribution areas. However, in recent years, due to the impact of the marine environment and human factors, the natural resources of the scalloped clams have been declining. Due to regional differences, farming methods, and feeding habits, the morphology of the scalloped clams varies, making it difficult for the average person to identify them. They can also be easily confused with similar-looking shellfish such as white clams and Chinese clams. Professionals with extensive experience in the scalloped clams industry may be able to distinguish them based on their morphology, but processed products are difficult to distinguish based on morphology. Applying molecular biology techniques to the identification of scalloped clams could effectively alleviate this problem by reducing the professional requirements for identification. Therefore, a sensitive and specific identification method is urgently needed.

[0005] The fluorescent PCR probe method combines probes with real-time fluorescent PCR, enabling qualitative detection and quantitative analysis of target DNA fragments without requiring additional steps after PCR. This method, unaffected by the organism's growth and developmental state, offers high identification accuracy and is currently widely used in numerous areas of life science research, including species identification and genotyping. This technology, first applied to the identification of the species of the Chinese tongue, not only provides a means of identifying the species, which is difficult to identify due to its morphology, but also has applications in identifying raw materials used in processed Chinese tongue products, both commercially and in scientific research, including ecological research.

[0006] Through the above analysis, the problems and defects of the existing technology are: the existing identification method of Xishi tongue still requires professionals to analyze through professional software, which is not intuitive enough, and the identification method is not sensitive, the detection efficiency is not high, and there is no specific identification method for Xishi tongue. Summary of the Invention

[0007] In view of the problems in the prior art, the application provides a chamelea gallus fluorescent PCR probe detection kit.

[0008] The application is achieved as follows: a chamelea gallus fluorescent PCR probe detection kit, which comprises:

[0009] a pair of specific fluorescent primers CADQF and CADQR, a probe CADQP, a real-time fluorescent reaction mixture of 2xPremix Ex TaqTM PCR and ROXII, a positive control, a negative control and nuclease-free water;

[0010] The sequence of the specific fluorescent primer CADQF is SEQ ID NO: 1, and the sequence of the specific fluorescent primer CADQR is SEQ ID NO: 2.

[0011] Further, the sequence of the probe CADQP is SEQ ID NO: 3.

[0012] Further, the positive control is a chamelea gallus DNA extract solution with a concentration of 100 ng / μL, and the negative control is a clam DNA extract solution with a concentration of 100 ng / μL.

[0013] Another object of the application is to provide a chamelea gallus rapid detection and identification method using the chamelea gallus fluorescent PCR probe detection kit, which comprises:

[0014] Step one, obtaining DNA of a to-be-detected object as a to-be-detected sample, and performing fluorescent PCR based on the to-be-detected sample;

[0015] Step two, obtaining a fluorescent PCR detection result, and determining whether the to-be-detected object is a chamelea gallus based on a fluorescent amplification curve and a threshold value.

[0016] Further, in step one, performing fluorescent PCR based on the to-be-detected sample comprises:

[0017] Firstly, 13 μL of a real-time fluorescent PCR reaction mixture I, 3 μL of a real-time fluorescent PCR reaction mixture II and 6 μL of nuclease-free water are respectively added into three EP tubes;

[0018] Secondly, 3 μL of the to-be-detected sample, the positive control and the negative control are respectively added into the EP tubes, and after mixing, amplification is performed on a fluorescent quantitative PCR instrument.

[0019] Further, the amplification reaction condition is: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 40 s, 40 cycles.

[0020] Furthermore, in step 2, obtaining the fluorescence PCR test results includes:

[0021] To obtain the fluorescence PCR test results, the baseline is based on the threshold line just exceeding the highest point of the normal negative control amplification curve;

[0022] The negative control has no threshold and no amplification curve; the positive control threshold is less than or equal to 35, and a typical amplification curve appears;

[0023] If any of the positive control and negative control do not meet the above conditions, return to the fluorescence PCR test again.

[0024] Furthermore, in step 2, judging whether the subject is a cyperus thunbergii based on the fluorescence amplification curve and the threshold value includes:

[0025] If the threshold value is less than or equal to 35 and the fluorescence amplification curve presents a typical amplification curve, the subject is judged to be Xishi tongue; if the threshold value is greater than 35 and less than or equal to 40, the test is performed again. If the threshold value is less than 40 and the fluorescence amplification curve presents a typical amplification curve, the subject is judged to be Xishi tongue;

[0026] Otherwise, it is determined that the subject is not Xishi tongue or the nucleic acid concentration in the subject is too low.

[0027] Another object of the present invention is to provide an application of the Xishi Tongue fluorescent PCR probe detection kit in the detection of Xishi Tongue processed products.

[0028] Another object of the present invention is to provide a use of the Xishi tongue fluorescent PCR probe method detection kit in the preparation of products for detecting Xishi tongue or Xishi tongue processed products.

[0029] In combination with the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solutions to be protected by the present invention from the following aspects:

[0030] First, in view of the technical problems existing in the above-mentioned prior art and the difficulty of solving these problems, this paper closely combines the technical solutions to be protected by the present invention and the results and data during the research and development process, and analyzes in detail and in depth how the technical solutions of the present invention solve the technical problems and some creative technical effects brought about by solving the problems. The specific description is as follows:

[0031] The technical problems existing in the prior art and the difficulty of solving the problems: the existing technology mainly uses 16S, ITS2 or COI sequence markers to distinguish Radula marginata, and through designing primers, PCR amplification, product purification and bidirectional sequencing, the Radula marginata is distinguished by sequence alignment. Or distinguish different geographical populations of Radula marginata by the above sequence markers. The above method requires the identification personnel to perform sequence alignment on the sample, and the conclusion is obtained after analyzing the special point marker, and the practitioner needs to have professional molecular biology theoretical knowledge. It has high requirements for personnel, equipment and software application.

[0032] The fluorescence PCR probe method of the application distinguishes the molecular characteristics of Radula marginata by designing specific primers, which cannot be achieved by existing conventional or published primers. The fluorescence PCR probe method of the application is first used in Radula marginata or its close relatives, and there is no technical reference for related species identification method, and it is difficult to screen and design the specific primers. The specific primers obtained by screening need to verify the important parameters of the complementarity of the primers themselves, the complementarity with the DNA template, the complementarity between the two primers, the GC content, the melting temperature, and most importantly, the verification experiment needs to be scientifically designed according to the species relationship to ensure the uniqueness of the primers to Radula marginata. The primers used have creativity, which is not obvious to those skilled in the art. The detection kit provided by the application can quickly and efficiently determine whether the sample to be tested is Radula marginata, and the fluorescence PCR probe method can be used to determine the raw material species and content in the processed products that cannot be determined from the morphology or use Radula marginata as raw materials, which provides a more convenient and efficient method for commodity quality and marine ecology.

[0033] The kit provided by the application has high specificity, only the typical amplification curve appears in Radula marginata and its products, and no amplification curve appears in other species; the detection is time-saving, the result is accurate, and no post-PCR treatment is required, which can effectively avoid false positives and cross contamination, and provides a new method for rapid and accurate identification in species resource protection, fishery production, consumption and trade.

[0034] Secondly, the technical solution is regarded as a whole or from the perspective of the product, the technical effects and advantages of the technical solution to be protected by the application are described as follows:

[0035] The application is not affected by individual morphology, size and other characteristics and integrity, and the limitations existing in morphological identification are avoided, rich identification basis can be provided directly from the gene level, and the differences between species can be effectively distinguished.

[0036] The fluorescence quantitative PCR detection method established by the present application provides a new technical means for accurately identifying chama sinica, and also provides a quick and simple detection and identification method for identification personnel who are not professional in soft-bodied animal classification. In actual work, the identification personnel who are not professional in soft-bodied animal classification can not only preliminarily identify according to the shell morphology, but also use the fluorescence quantitative PCR method for confirmation, so as to ensure that the identification result is accurate and reliable.

[0037] The present application first applies PCR fluorescence detection to the species identification of chama sinica, not only provides the identification of chama sinica which is difficult to identify in morphology, but also has application value in commercial and ecological scientific research for identifying the raw materials of chama sinica processing products.

[0038] Thirdly, the creativity of the present application as the claim is also embodied in the following important aspects:

[0039] (1) The expected income and commercial value of the technical scheme of the present application after transformation are as follows: the present application first establishes the fluorescence PCR probe method of chama sinica, and provides a kit for detecting chama sinica, which provides a new technical means for accurately identifying chama sinica, and provides a quick and accurate detection method for identification and authenticity identification of chama sinica which is difficult to identify in morphology or processing products. It has great significance for protecting marine species resources, fishery production and processing, developing and utilizing medicinal value, and further maintaining national ecological safety and consumer rights and interests.

[0040] (2) The technical scheme of the present application fills the domestic and foreign technical blank: the kit provided by the present application first applies the fluorescence PCR probe method to the species identification of chama sinica in the Arcidae, which fills the blank of this technology in marine biological identification in China, and also provides a research basis for protecting more marine biological species resources in the future. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 It is a flow chart of the rapid detection and identification method of chama sinica provided by the embodiment of the present application.

[0042] Figure 2 It is a fluorescence PCR probe detection result schematic diagram of suspected chama sinica fresh shell provided by the embodiment of the present application; in the diagram, the Ct values of positive samples and suspected samples are all less than or equal to 35, and typical amplification curves appear, and no amplification curve appears in the negative sample;

[0043] Figure 3 It is a fluorescence quantitative PCR probe detection result schematic diagram of suspected chama sinica frozen mussel slices provided by the embodiment of the present application; in the diagram, the Ct values of positive samples and suspected samples are all less than or equal to 35, and typical amplification curves appear, and no amplification curve appears in the negative sample;

[0044] Figure 4This is an amplification curve diagram of the sensitivity detection test of Xishi tongue fluorescent PCR probe method provided by an embodiment of the present invention; in the figure, the positive samples with Ct values ​​≤35 for the detection sample DNA concentrations of 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL and 10fg / μL respectively show typical amplification curves, and the negative samples have no amplification curve.

[0045] Figure 5 This is an amplification curve diagram of a sensitivity test for the fluorescent PCR probe method for the detection of tamarisk (Ctenophora truncatula) according to an embodiment of the present invention. The test samples in the figure are tamarisk (Ctenophora truncatula), white mussel, Chinese clam, Meretrix meretrix, Meretrix brevis, Trigonella ternatea, and Cynanchum cylindrica, and ddH2O. Tamarisk (Ctenophora truncatula) samples with a Ct value ≤ 35 exhibit a typical amplification curve, while the remaining samples exhibit no amplification curve. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0047] 1. Explanatory Examples In order to enable those skilled in the art to fully understand how to implement the present invention, this section provides an illustrative example that expands upon the technical solutions of the claims.

[0048] like Figure 1 As shown, the rapid detection and identification method of Xishi tongue provided by the embodiment of the present invention includes:

[0049] S101, obtaining DNA from a subject to be tested as a sample to be tested, and performing fluorescent PCR based on the sample to be tested;

[0050] S102, obtaining a fluorescence PCR test result, and judging whether the tested object is a Herpes simplex virus (Hymenoptera: Hy ...

[0051] The embodiment of the present invention provides a fluorescent PCR probe method detection kit for scutellaria baicalensis, comprising: (1) a real-time fluorescent PCR reaction mixture I, comprising 2×Premix Ex TaqTM PCR and ROXII; (2) a real-time fluorescent PCR reaction mixture II, comprising primers CADQF and CADQR, and a probe CADQP; (3) a positive control (containing a scutellaria baicalensis DNA extract); (4) a negative control (a clam DNA extract) and nuclease-free water.

[0052] The sequence of the primer CADQF provided in the embodiment of the present invention is: SEQ ID NO: 1: 5'-CCGTCTTCACTGACGGTTTG-3'.

[0053] The sequence of the primer CADQR provided by the embodiment of the application is: SEQ ID NO: 2: 5'-GGGTAGTCTCGTCTGATCC-3'.

[0054] The sequence of the fluorescent probe CADQP provided by the embodiment of the application is: SEQ ID NO: 3: 5'-FAM-ACGTGCGAGAGCCCGCTCCT-BHQ1-3'; the fluorescent reporter group labeled at the 5' end is FAM, and the fluorescent quenching group labeled at the 3' end is BHQ1.

[0055] The detection method of the fluorescent PCR probe method detection kit for Mytilopsis sallei provided by the embodiment of the application comprises the following steps.

[0056] (1) Obtain the DNA of the subject. Extract the DNA of the sample according to the commercial extraction kit or the conventional method, and store it at -20℃ for standby;

[0057] (2) Fluorescent PCR. The reaction system is 25 μL. In three 0.2ml EP tubes, 13 μL of real-time fluorescent PCR reaction mixture I, 3 μL of real-time fluorescent PCR reaction mixture II and 6 μL of nuclease-free water are added respectively, and then 3 μL of sample, positive control and negative control are added into the EP tubes, mixed and placed in a fluorescent quantitative PCR instrument for amplification. The amplification reaction conditions are as follows: 95℃ pre-denaturation for 5min; 95℃ denaturation for 10s, 60℃ annealing and extension for 40s, 40 cycles;

[0058] (3) Fluorescent PCR result analysis. The detection result is directly read, and the baseline is taken as the highest point of the amplification curve of the normal negative control just above the threshold line. The negative control has no Ct value and no amplification curve; the Ct value of the positive control should be ≤35, and a typical amplification curve appears; if any of the positive control and the negative control does not meet the above conditions, this experiment is invalid.

[0059] (4) Result determination. The data quality is detected by the fluorescent amplification curve and the threshold value (Ct). If the Ct value is ≤35 and a typical amplification curve appears, the sample is Mytilopsis sallei; if 35 < Ct value ≤40, repeat once, if the Ct value is less than 40 and a typical amplification curve appears, the sample is Mytilopsis sallei, otherwise the sample is not Mytilopsis sallei or the nucleic acid concentration of the sample is too low.

[0060] The technical solutions of the application will be further described in combination with specific embodiments.

[0061] Embodiment 1

[0062] Take a fresh scallop suspected to be a scallop of Xishi tongue, take a small amount of muscle tissue, extract DNA according to the instructions of the tissue genomic DNA kit, and use the scallop fluorescent PCR probe detection kit provided by the embodiment of the present invention to detect it. The results are shown in the attached figure. Figure 2 The test subjects and positive control samples showed typical amplification curves when the Ct value was <35, while the negative samples showed no amplification curve, which was consistent with the PCR test results.

[0063] Example 2

[0064] Take frozen clam slices (Xishi tongue), take a small amount of muscle tissue, extract DNA according to the instructions of the tissue genomic DNA kit, and use the Xishi tongue fluorescent PCR probe detection kit provided by the embodiment of the present invention to detect the results as shown in the attached Figure 3 The test subjects and positive control samples showed typical amplification curves when the Ct value was <35, while the negative samples showed no amplification curve, which was consistent with the PCR test results.

[0065] Example 3

[0066] A small amount of muscle tissue of fresh scallops was taken, and DNA was extracted according to the instructions of the tissue genomic DNA kit. The DNA concentration was determined using an ultra-micro UV spectrophotometer, and then the DNA was diluted to 100 ng / μL with TE solution. The scallop DNA stock solution (100 ng / μL) was diluted to different concentration gradients of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL and 10 fg / μL using a 10-fold concentration serial dilution method. The scallop DNA was detected using the scallop fluorescent PCR probe detection kit provided in an embodiment of the present invention. The results are shown in the attached figure. Figure 4 Typical amplification curves were observed on the fluorescence quantitative PCR instrument at DNA concentrations of 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL, indicating that DNA could still be detected at a concentration of 1 pg / μL, and that the kit of the present invention has high sensitivity.

[0067] 2. Application Examples: In order to demonstrate the creativity and technical value of the technical solution of the present invention, this section provides application examples of the claimed technical solution on specific products or related technologies.

[0068] The fluorescence PCR probe method detection kit of the chamelea gallina of the embodiment of the present application is applied to the detection of chamelea gallina, and chamelea gallina can be identified from samples containing chamelea gallina, white bivalve, chino clam, hard clam, short hard clam, triangle clam, and green clam. The DNA of chamelea gallina, white bivalve, chino clam, hard clam, short hard clam, triangle clam, and green clam is respectively amplified by real-time fluorescence quantitative PCR with primers CADQF, CADQR, and probe CADQP. Only the target species chamelea gallina presents a typical amplification curve on the fluorescence quantitative PCR instrument, and the negative control samples white bivalve, chino clam, hard clam, short hard clam, triangle clam, and green clam and the blank control ddH2O have no amplification curve.

[0069] The fluorescence PCR probe method detection kit of the chamelea gallina of the embodiment of the present application is applied to the detection of chamelea gallina processed products.

[0070] III. Evidence of the effects of the embodiment. The embodiment has achieved some positive effects in the development or use process, and indeed has great advantages compared with the prior art. The following content is described in combination with the data and graphs of the test process.

[0071] 1. Materials and methods

[0072] 1.1. Materials

[0073] 1.1.1. Test chamelea gallina and similar species

[0074] The test materials are chamelea gallina, white bivalve, chino clam, hard clam, short hard clam, triangle clam, and green clam. The above samples are collected through domestic investigation or purchased from the market, and are confirmed by the Fuzhou Customs State Mollusk Quarantine Identification Key Laboratory and stored at -20 DEG C for standby use.

[0075] 1.1.2. Consumables and instruments

[0076] Mortar, PCR tube, Eppendorf tube (1.5 mL), pipette (1000 μL adjustable, 200 μL adjustable, 20 μL adjustable, 2 μL adjustable), electronic balance, constant temperature water bath, low temperature refrigerator, high pressure sterilization pot, table type high speed centrifuge, micro centrifuge, vortex shaker, eight connected row fluorescence PCR reaction tube, ultramicro ultraviolet spectrophotometer, super clean bench, real-time fluorescence quantitative PCR instrument.

[0077] 1.2. Main reagents

[0078] TE solution, sterilized ddH2O, anhydrous ethanol, DNA marker, DNA extraction kit, fluorescence PCR probe method detection kit.

[0079] 1.3. Methods

[0080] 1.3.1. DNA extraction

[0081] About 25 g of the soft body part of the test chama, white clam, chino chama, clam, short clam, triangle clam, and green clam was taken, and the TIANGEN tissue genomic DNA extraction kit was used for extraction, and the DNA was extracted according to the operation instruction. The extracted DNA was determined for DNA concentration by ultramicro UV spectrophotometer, and then the DNA was diluted to 100 ng / μL with TE solution, and stored at -20℃ for standby.

[0082] 1.3.2 Primer and probe

[0083] The extracted chama DNA was amplified and sequenced by using the ITS2 universal primer to obtain the ITS2 gene sequence, and the primer and probe were designed and analyzed with the aid of PrimerExpress3 software, and were screened through repeated experiments. All the primers and probes were synthesized by Shanghai Sangon Biotech Service Co., Ltd.

[0084] 1.3.3 Fluorescent PCR probe method detection reaction system

[0085] The total volume of the reaction system was 25 μL, wherein 2xPremix Ex TaqTM PCR was 12.5 μL, the upstream and downstream primers and the probe were each 1 μL, the 100 ng / μL DNA template was 3 μL, the ROXII was 0.5 μL, and the rest was supplemented with sterilized ddH2O, and then mixed and placed in a fluorescent quantitative PCR amplifier for amplification. The amplification reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 40 s, 40 cycles; and end reaction.

[0086] 1.3.4 Specific detection of fluorescent PCR probe method

[0087] The chama was used as a positive control, and the white clam, chino chama, clam, short clam, triangle clam, and green clam identified by morphology were used as negative controls, and the blank control was sterilized ddH2O. The real-time fluorescent quantitative PCR specificity test was carried out according to the reaction system and procedure of 1.3.3.

[0088] 1.3.5 Sensitivity detection of fluorescent PCR probe method

[0089] The 10-fold concentration series dilution method was used to dilute the chama DNA stock solution (100 ng / μL) into different concentration gradients of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, and 10 fg / μL, and then the sensitivity test of the real-time fluorescent quantitative PCR method was carried out according to the reaction system and procedure of 1.3.3.

[0090] 2 Results and analysis

[0091] 2.1 Primer and probe

[0092] Based on the sequence of ITS2 of Xishi Tongue, after repeated experiments and comparison tests, the optimal primers and probes for the real-time fluorescence quantitative PCR detection and identification method of Xishi Tongue were finally determined as follows:

[0093] The upstream primer is CADQF: 5'-CCGTCTTCACTGACGGTTTG-3',

[0094] The downstream primer is CADQR: 5'-CGGGTAGTCTCGTCTGATCC-3',

[0095] Fluorescent probe CADQP: 5'-FAM-ACGTGCGAGAGCCCGCTCCT-BHQ1-3', the fluorescent reporter group labeled at the 5' end is FAM, and the fluorescent quencher group labeled at the 3' end is BHQ1.

[0096] 2.2 Primer specificity test results

[0097] Primers CADQF and CADQR and probe CADQP were used to amplify the genes of clams from Coleus truncatus, white clams, Chinese clams, Meretrix meretrix, Meretrix brevis, and Cynoglossum cyprinus by real-time fluorescence quantitative PCR. ddH2O was used as a blank control. Only Coleus truncatus showed a typical amplification curve on the fluorescence quantitative PCR instrument, with a Ct value of less than 35. No amplification curves were found in other samples. The experimental results were consistent with the expectations ( Figure 5 ), only the Chinese tongue can be detected, while other species are not detected. This shows that the primers have good specificity.

[0098] Figure 5 In the chart, 1 represents the Chinese tongue; 2 represents the white clam; 3 represents the Chinese clam; 4 represents the Manila clam; 5 represents the short clam; 6 represents the triangular clam; 7 represents the green clam; and 8 represents ddH2O.

[0099] 2.3 Primer sensitivity test results

[0100] The concentration of the extracted DNA from Xishi tongue was determined by UV spectrophotometry and diluted to 100 ng / μL as template DNA. The DNA was diluted into 8 different concentration gradients using the method in 1.3.5. ddH2O was used as a blank control. The primers CADQF and CADQR and the probe CADQP were used for real-time fluorescence quantitative PCR amplification. Typical amplification curves were observed on the fluorescence quantitative PCR instrument when the DNA concentrations were 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL and 1 pg / μL (see Figure 5). Figure 4 ), indicating that DNA concentration of 1 pg / μL can still be detected, and this method has high sensitivity.

[0101] Figure 4In the table, 1 represents 100 ng / μL; 2 represents 10 ng / μL; 3 represents 1 ng / μL; 4 represents 100 pg / μL; 5 represents 10 pg / μL; 6 represents 1 pg / μL; 7: 100 fg / μL; 8: 10 fg / μL)

[0102] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention. Sequence Listing <110> Fuzhou Customs Technology Center <120> A fluorescent PCR probe detection kit for Xishi tongue <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 ccgtcttcac tgacggtttg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 cgggtagtct cgtctgatcc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 acgtgcgaga gcccgctcct 20

Claims

1. A kit for detecting syringa sutsugamushi, characterized in that: The Xishi tongue detection kit comprises: A pair of specific fluorescent primers CADQF and CADQR, probe CADQP, 2×Premix Ex TaqTM PCR and ROXII real-time fluorescent reaction mixture, positive control, negative control and nuclease-free water; The sequence of the specific fluorescent primer CADQF is: SEQ ID NO: 1; the sequence of the specific fluorescent primer CADQR is: SEQ ID NO: 2; The sequence of the probe CADQP is: SEQ ID NO:

3.

2. The Xishi tongue detection kit according to claim 1, characterized in that The positive control is a DNA extract of Coleus truncatus with a concentration of 100 ng / μL; the negative control is a DNA extract of Meretrix meretrix with a concentration of 100 ng / μL.

3. A method for rapid detection of shishi tongue using the shishi tongue detection kit according to claim 1, characterized in that: The method comprises: Step 1: obtaining DNA from a subject to be tested as a sample to be tested, and performing fluorescent PCR based on the sample to be tested; Step 2: Obtain the fluorescence PCR test results, and determine whether the subject is Xishi tongue based on the fluorescence amplification curve and threshold.

4. The rapid detection method for Xishi tongue as claimed in claim 3, characterized in that: In the step 1, performing fluorescent PCR based on the sample to be detected includes: First, add 13 μL of real-time fluorescence PCR reaction mixture I, 3 μL of real-time fluorescence PCR reaction mixture II, and 6 μL of nuclease-free water to three EP tubes respectively; Secondly, 3 μL of the sample to be tested, positive control, and negative control were added to the EP tube respectively, mixed and then placed in a fluorescent quantitative PCR instrument for amplification.

5. The rapid detection method for Xishi tongue according to claim 4, characterized in that: The amplification reaction conditions were as follows: pre-denaturation at 95° C. for 5 min, denaturation at 95° C. for 10 s, annealing and extension at 60° C. for 40 s, and 40 cycles.

6. The rapid detection method for Xishi tongue as claimed in claim 3, characterized in that: In the second step, obtaining the fluorescence PCR test results includes: To obtain the fluorescence PCR test results, the baseline is based on the threshold line just exceeding the highest point of the normal negative control amplification curve; The negative control has no threshold and no amplification curve; the positive control threshold is less than or equal to 35, and a typical amplification curve appears; If any of the positive control and negative control do not meet the above conditions, return to the fluorescence PCR test again.

7. The rapid detection method for Xishi tongue as claimed in claim 3, characterized in that: In the step 2, judging whether the subject is a cyperus thunbergii based on the fluorescence amplification curve and the threshold value includes: If the threshold value is less than or equal to 35 and the fluorescence amplification curve presents a typical amplification curve, the subject is judged to be Xishi tongue; if the threshold value is greater than 35 and less than or equal to 40, the test is performed again; if the threshold value is less than 40 and the fluorescence amplification curve presents a typical amplification curve, the subject is judged to be Xishi tongue; Otherwise, it is determined that the subject is not Xishi tongue or the nucleic acid concentration in the subject is too low.

8. Use of the Xishi tongue detection kit according to claim 1 in the detection of Xishi tongue processed products.

9. Use of the Xishi tongue detection kit according to claim 1 in preparing a product for detecting Xishi tongue or Xishi tongue processed products.

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