Loop-mediated isothermal amplification detection primer and kit for inclined tube worms and application of loop-mediated isothermal amplification detection primer and kit

Abstract

The invention discloses an inclined tube worm loop-mediated isothermal amplification detection primer, a kit and application thereof, belongs to the technical field of gene detection, and provides an inclined tube worm loop-mediated isothermal amplification detection primer which comprises an outer primer F3, an outer primer B3, an inner primer FIP and an inner primer BIP. The nucleotide sequence of the outer primer F3 is as shown in SEQ ID NO. 1; the nucleotide sequence of the outer primer B3 is as shown in SEQ ID NO. 2; the nucleotide sequence of the inner primer FIP is as shown in SEQ ID NO.3; and the nucleotide sequence of the inner primer BIP is as shown in SEQ ID NO. 4. The invention provides an application of the primer in preparation of a reagent for detecting Inclinonus obliquus. The primer provided by the invention is designed for a conserved sequence 18S rRNA (ribosomal Ribose Nucleic Acid) of Inonotus obliquus, the Inonotus obliquus can be specifically recognized, the specificity is strong, and a loop-mediated isothermal amplification detection method established by utilizing the primer can be used for fish body tissue detection.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection, and in particular relates to a primer and a kit for detecting loop-mediated isothermal amplification of a sphenodontid, and applications thereof. Background Art

[0002] Parasitic diseases are closely related to human health and the development of animal husbandry. Rapid and effective detection of parasites is the key to disease prevention and control. Morphological identification has always been a traditional and highly practical technology in the field of parasite research and is one of the commonly used methods for diagnosing parasitic diseases. However, the morphological identification method is time-consuming and labor-intensive, and requires professional personnel. When the infection rate is investigated in large quantities or when the parasite individuals are extremely small, this method becomes very cumbersome and difficult, which is not conducive to the rapid diagnosis and rapid prevention of fish parasitic diseases.

[0003] In recent years, molecular biology technology has developed rapidly, and methods such as polymerase chain reaction are increasingly used in parasite detection. Molecular detection of Obliquus is mainly based on ordinary PCR. PCR technology is a method of identifying species through DNA molecular comparison. It can not only quickly detect the presence of pathogens, but also has a high accuracy rate. However, this type of technology has high requirements for facilities, equipment and personnel, making it difficult to implement detection on-site in aquaculture.

[0004] Loop-mediated isothermal amplification (LAMP) is a new type of nucleic acid amplification method, which is characterized by designing 4 specific primers for 6 regions of the target gene, and performing constant temperature amplification at 60-65°C under the action of strand displacement DNA polymerase. DNA can be amplified more than 1 million times in about 5-30 minutes. Its method is similar to polymerase chain reaction. The LAMP method is relatively simple and can provide fast, efficient and high-precision detection, and is more suitable for on-site detection of miniaturized instruments. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes primers, a kit and an application of loop-mediated isothermal amplification detection of Obliquus. The primers of the present invention are designed for the conserved sequence 18S rRNA of Obliquus, can specifically identify Obliquus, and have strong specificity. The loop-mediated isothermal amplification detection method established using the primers can be used for fish tissue detection.

[0006] To achieve the above object, the present invention provides primers for detecting the loop-mediated isothermal amplification of Obliquus sphaeroides, wherein the primers include an outer primer F3, an outer primer B3, an inner primer FIP and an inner primer BIP;

[0007] The nucleotide sequence of the external primer F3 is shown in SEQ ID NO.1;

[0008] The nucleotide sequence of the external primer B3 is shown in SEQ ID NO.2;

[0009] The nucleotide sequence of the inner primer FIP is shown in SEQ ID NO.3;

[0010] The nucleotide sequence of the inner primer BIP is shown in SEQ ID NO.4.

[0011] The invention also provides application of the primer in preparing a reagent for detecting Obliquus sphaeroides.

[0012] The invention also provides a kit for detecting acanthocephalus containing the primers.

[0013] Preferably, the kit further comprises Bst DNA polymerase, dNTPs, MgSO4, calcein, template DNA, ddH2O and 10× reaction buffer.

[0014] Preferably, the concentration of the outer primer F3 in the kit is 10 μmol / L, the concentration of the outer primer B3 is 10 μmol / L, the concentration of the inner primer FIP is 10 μmol / L, the concentration of the inner primer BIP is 10 μmol / L, and the ratio of the total volume of the outer primer F3 and the outer primer B3 to the total volume of the inner primer FIP and the inner primer BIP is 1:8.

[0015] Preferably, the concentration of Bst DNA polymerase in the kit is 8 U / μL, and the concentration of dNTPs in the kit is 10 mmol / L.

[0016] Preferably, the concentration of MgSO4 in the kit is 100 mmol / L.

[0017] The present invention also provides a method for detecting the worm using the primers, comprising the following steps:

[0018] (1) extracting genomic DNA of the sample to be tested;

[0019] (2) Using the genomic DNA of the sample to be tested extracted in step (1) as the template DNA, LAMP amplification was performed using the outer primers F3 and B3 and the inner primers FIP and BIP. The total volume of the LAMP detection reaction system was 25 μL.

[0020] Preferably, the LAMP detection reaction system in step (2) is: 1 μL of 8U / μL Bst DNA polymerase, 2.5 μL of 10× reaction buffer, 1 μL of MgSO4 at a concentration of 100 mmol / L, 3.5 μL of dNTPs at a concentration of 10 mmol / L, 0.5 μL of each of an outer primer F3 at a concentration of 10 μmol / L and an outer primer B3 at a concentration of 10 μmol / L, 4 μL of each of an inner primer FIP at a concentration of 10 μmol / L and an inner primer BIP at a concentration of 10 μmol / L, 1 μL of calcein, 1 μL of template DNA, and ddH2O is added to 25 μL.

[0021] Preferably, the LAMP amplification in step (2) is performed in a water bath, the temperature of the LAMP amplification is 65° C., and the time of the LAMP amplification is 45 min.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects:

[0023] The present invention utilizes loop-mediated isothermal amplification technology, avoids the complex processes such as temperature cycling and thermal denaturation of templates required in conventional PCR technology, does not rely on temperature cycle control equipment, and can complete the process of melting and cyclic amplification under constant temperature conditions, reducing the requirements for expensive and precise experimental instruments. By designing four specific primers for six regions of the target gene, six different regions on the target sequence can be identified, the specificity of amplification is improved, and efficient nucleic acid amplification can be achieved in a short time for detecting clinical samples. The detection results of the detection of Oblique Tubularia by the loop-mediated isothermal amplification of the present invention are consistent with PCR, providing a more simple, rapid, accurate and economical Oblique Tubularia nucleic acid detection method. By observing the color change of the reaction system with the naked eye, it is possible to quickly determine whether the target gene exists, greatly improving the efficiency and accuracy of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 Mg in the LAMP reaction system of the present invention 2+ Determination of optimum concentration;

[0026] Figure 2 Determination of the optimum concentration of dNTPs in the LAMP reaction system of the present invention;

[0027] Figure 3Determination of the most suitable internal and external primer ratio in the LAMP reaction system of the present invention;

[0028] Figure 4 The optimum reaction temperature for the LAMP reaction system of the present invention is determined, wherein a is 61°C, b is 62°C, c is 63°C, d is 64°C, e is 65°C, f is 66°C, and g is 67°C;

[0029] Figure 5 The results of using the LAMP reaction system of the present invention to detect Bagworm, Ichthyophthirius, Trichodina and Obliquus;

[0030] Figure 6 Results of PCR amplification for detection of Bagworm, Ichthyophthirius, Trichodina, and Trichodina. In the figure, M represents DNAmarkerDL2000, N represents negative control, 1 represents Trichodina, 2 represents Bagworm, 3 represents Ichthyophthirius, and 4 represents Trichodina.

[0031] Figure 7 It is the sensitivity determination of the LAMP reaction system of the present invention;

[0032] Figure 8 The minimum detection amount of PCR amplification is determined. In the figure, M represents DNAmarker DL2000, N represents negative control, 1 represents 20.4 ng / μL, 2 represents 2.04 ng / μL, and 3 represents 2.04×10 -1 ng / μL, 4 represents 2.04×10 -2 ng / μL, 5 represents 2.04×10 -3 ng / μL, 6 represents 2.04×10 -4 ng / μL, 7 represents 2.04×10 -5 ng / μL, 8 represents 2.04×10 -6 ng / μL, 9 represents 2.04×10 -7 ng / μL;

[0033] Fig. 9 The results of the LAMP visualization test of the present invention, in which 1 represents the negative control group, and 2 to 8 represent the positive control group;

[0034] Fig.10 The results of the LAMP reaction system of the present invention detecting fish tissue samples, where 1 to 10 represent different fish tissue samples respectively;

[0035] Fig.11 This is the result of PCR amplification detection of fish tissue samples. In the figure, M represents DNA marker DL2000, N represents negative control, and 1 to 10 represent different fish tissue samples. DETAILED DESCRIPTION

[0036] 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.

[0037] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0038] 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.

[0039] 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 the skilled artisan. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example

[0042] 1.1 Primer design and synthesis.

[0043] Using the LAMP primer online design software PrimerExplorer, according to the 18S rRNA gene of Obliquus in NCBI (No.: KY476314.1), the outer primers F3 / B3 and the inner primers FIP / BIP were designed, as shown in Table 1 below.

[0044] Table 1 LAMP amplification primers

[0045]

[0046] 1.2 Optimization and establishment of LAMP reaction conditions.

[0047] The LAMP reaction solution was prepared according to the BstⅡ DNA polymerase instruction manual. The reaction system was 25 μL: 2.5 μL 10× reaction buffer, 1 μL 8U / μL Bst DNA polymerase, 1 μL 100 mmol / LMgSO4, 3.5 μL 10 mmol / L dNTPs, 1 μL calcein, 0.5 μL 10 μmol / L F3, 0.5 μL 10 μmol / L B3, 4 μL 10 μmol / L FIP, 4 μL 10 μmol / L BIP, 1 μL template DNA, and the volume was filled to 25 μL with deionized water.

[0048] Based on this system, Mg 2+ The final concentration gradient in the 25 μL system is 0, 2, 4, 6, 8, 10, 12, 14 mmol / L; Figure 1 As shown, Mg 2+ The optimal reaction concentration was 4mmol / L.

[0049] Based on this system, the final concentrations of dNTP Mix in a 25 μL system were set to 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mmol / L, respectively; Figure 2 As shown, the optimal reaction concentration of dNTPs is 1.4mmol / L.

[0050] Based on this system, the final concentration of the outer primers F3 and B3 in the 25 μL system was fixed to 0.2 μmol / L, and the concentration of the inner primer was adjusted. The concentration ratio of the outer primer to the inner primer was set to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 and 1:8; the results are as follows Figure 3 As shown, the optimal final concentration of the inner primer FIP and the inner primer BIP in the 25 μL system is 1.6 μmol / L, and the amplification effect is best when the ratio of inner and outer primers is 8:1.

[0051] Based on this system, the reaction temperature was set at 61-67°C with an interval of 1°C to determine the optimal reaction conditions. Figure 4 As shown, the optimal reaction temperature of LAMP was determined to be 65°C.

[0052] 1.3 Specificity experiment.

[0053] According to the optimal reaction system optimized in 1.2, DNA of Bagnacles, Ichthyophthirius, Trichodina, and Obliquus were used as templates to test the specificity of the LAMP method. PCR verification was also performed.

[0054] The results are as follows Figure 5 As shown, only the sample of Obliquus spp. showed positive amplification, while the other three ciliate samples did not show amplification, e.g. Figure 6As shown, ordinary PCR verified this result, indicating that the LAMP primers of the present invention have good specificity.

[0055] 1.4 Sensitivity experiment.

[0056] The plasmid was tested three times using an ultra-micro spectrophotometer. The plasmid concentration was 20.4 ng / μL. It was diluted 10 times with ddH2O to a concentration of 20.4, 2.04, and 2.04×10 -1 , 2.04×10 -2 , 2.04×10 -3 , 2.04×10 -4 , 2.04×10 -5 , 2.04×10 -6 , 2.04×10 -7 ng / μL, and 1 μL was taken as template for LAMP reaction. At the same time, the minimum detection amount was compared with that of conventional PCR to evaluate the sensitivity of this method.

[0057] The results are as follows Figure 7 As shown, the detection method of the present invention can detect concentrations not less than 2.04×10 -7 ng / μL of DNA of the oblique tubeworm, with high sensitivity. Figure 8 As shown, compared with ordinary PCR, the amplification efficiency of LAMP is 1000 times that of ordinary PCR.

[0058] 1.5LAMP visualization detection.

[0059] Before the reaction, calcein with a final concentration of 0.05 mM and manganese chloride solution with a final concentration of 0.4 mM were added to the prepared standard system, and the mixture was reacted at 65°C for 45 min.

[0060] like Fig. 9 As shown, visual judgment can be made by observing the color change of the reaction tube, with negative being light orange and positive being light green.

[0061] 1.6 Sample testing.

[0062] LAMP and PCR methods for detecting clinical samples

[0063] Ten sturgeons were collected from a cold-water fish farm and their gill tissue samples were collected for testing.

[0064] The gill tissue samples were ground and DNA was extracted and detected using the established LAMP and PCR methods.

[0065] like Fig.10 and Fig.11As shown in the figure, the results of the two methods were consistent, showing that 1, 2, 4 and 5 were positive, and the rest were negative. This indicates that a total of 4 fish were infected with Obliquus, and the method established in this study can be applied to fish tissue detection.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer for detecting the loop-mediated isothermal amplification of the tubeworm, characterized in that: The primers include outer primer F3, outer primer B3, inner primer FIP and inner primer BIP; The nucleotide sequence of the external primer F3 is shown in SEQ ID NO.1; The nucleotide sequence of the external primer B3 is shown in SEQ ID NO.2; The nucleotide sequence of the inner primer FIP is shown in SEQ ID NO.3; The nucleotide sequence of the inner primer BIP is shown in SEQ ID NO.

4.

2. Use of the primers as claimed in claim 1 in preparing a reagent for detecting Obliquus spp.

3. A kit for detecting Obliquus sphaeroides containing the primers according to claim 1.

4. The kit according to claim 3, characterized in that: The kit also includes Bst DNA polymerase, dNTPs, MgSO4, calcein, template DNA, ddH2O and 10× reaction buffer.

5. The kit according to claim 3, characterized in that: The concentration of the outer primer F3 in the kit is 10 μmol / L, the concentration of the outer primer B3 is 10 μmol / L, the concentration of the inner primer FIP is 10 μmol / L, the concentration of the inner primer BIP is 10 μmol / L, and the ratio of the total volume of the outer primer F3 and the outer primer B3 to the total volume of the inner primer FIP and the inner primer BIP is 1:

8.

6. The kit according to claim 4, characterized in that: The concentration of Bst DNA polymerase in the kit is 8 U / μL, and the concentration of dNTPs in the kit is 10 mmol / L.

7. The kit according to claim 4, characterized in that: The concentration of MgSO4 in the kit is 100 mmol / L.

8. A method for detecting a sphaerocephalus using the primers of claim 1, characterized in that: The following steps are involved: (1) extracting genomic DNA of the sample to be tested; (2) Using the genomic DNA of the sample to be tested extracted in step (1) as the template DNA, LAMP amplification is performed using the outer primers F3 and B3 described in claim 1 and the inner primers FIP and BIP. The total volume of the LAMP detection reaction system is 25 μL.

9. The method for detecting Obliquus tubeworm according to claim 8, characterized in that: The LAMP detection reaction system in step (2) is: 1 μL of 8U / μL Bst DNA polymerase, 2.5 μL of 10× reaction buffer, 1 μL of 100 mmol / L MgSO4, 3.5 μL of 10 mmol / L dNTPs, 0.5 μL of 10 μmol / L outer primer F3 and 10 μmol / L outer primer B3, 4 μL of 10 μmol / L inner primer FIP and 10 μmol / L inner primer BIP, 1 μL of calcein, 1 μL of template DNA, and ddH2O to 25 μL.

10. The method for detecting Obliquus tubeworm according to claim 8, characterized in that: In step (2), LAMP amplification is performed in a water bath, the temperature of LAMP amplification is 65° C., and the time of LAMP amplification is 45 min.

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