Primer probe combination, method and application of RPA-LFD rapid detection of Ayu sutsugamushi

Abstract

The present invention discloses a primer-probe combination, method and application for rapid detection of sweetfish larvae by RPA-LFD, belonging to the field of biotechnology. The primer-probe combination consists of an upstream primer with a sequence as shown in SEQ ID NO.3, a downstream primer with a sequence as shown in SEQ ID NO.4 and a probe with a sequence as shown in SEQ ID NO.7. The RPA-LFD detection method for sweetfish larvae established based on the primer-probe combination has the advantages of high sensitivity, good specificity, simple operation, and visualized results. It can accurately detect sweetfish larvae and effectively reduce the economic losses caused by sweetfish larvae disease. The RPA-LFD rapid detection method for sweetfish larvae provided by the present invention does not require special instruments, has simple sample processing, rapid amplification, and intuitive results. The minimum detection limit of the sample can reach 9.38×10 ‑6 ng / μL, 1000 times higher than traditional PCR methods.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer-probe combination, method and application thereof for rapid detection of Glechoma ayutans by RPA-LFD. Background Art

[0002] Sweetfish (Plecoglossus altivelis) is widely distributed in the east coast of China, the Korean Peninsula, the Japanese archipelago and northern Vietnam. It has high nutritional value and good economic benefits. It is currently an important freshwater aquaculture economic fish in Zhejiang Province. Glugea plecoglossi is a type of Glugea microsporidia. In nature, it specifically infects sweetfish. The high infection stage is especially concentrated in the growth period of juveniles and the spawning period of adult fish. It is an important cause of economic losses in farmed sweetfish. Severely infected sweetfish can be detected by observing external signs, but most infected sweetfish do not have any symptoms on the outside. The body size of Glugea plecoglossi is only a few microns and can only be found after dissection.

[0003] At present, the detection methods for G. ayuensis include naked eye observation, microscopic tissue section examination and loop-mediated isothermal amplification (LAMP). Among them, naked eye observation and microscopic tissue section examination require the detection personnel to have considerable professional knowledge background and operation skills, and are also prone to missed detection, which is not suitable for rapid screening and timely identification of the worm. LAMP has the advantages of convenient operation, high sensitivity and high specificity, and is playing an important role in pathogen diagnosis, but it requires a water bath for reaction, and the entire detection process is relatively long, which is not suitable for on-site rapid detection.

[0004] Recombinase polymerase amplification (RPA) is a rapid, specific and highly sensitive DNA amplification method. RPA reactions can be performed at a constant ambient temperature, thus avoiding the need for a high-cost thermal cycler. This simple method for detecting aquatic pathogens without expensive instruments is of great significance to aquatic biosafety. So far, there have been no reports on the use of RPA technology to detect G. aquaticus. Summary of the invention

[0005] The purpose of the present invention is to provide a primer-probe combination, method and application of RPA-LFD rapid detection of sweetfish larvae to solve the problems existing in the above-mentioned prior art. The present invention designs a set of primer-probe combinations based on the Beta-tubulin gene. The RPA-LFD detection method of sweetfish larvae established based on the primers and probes has obvious advantages such as high sensitivity, good specificity, simple operation and visualization of results, and can accurately detect sweetfish larvae and effectively reduce the economic losses caused by sweetfish larvae disease.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a primer-probe combination for detecting Glechoma ayuensis based on RPA-LFD. The primer-probe combination consists of an upstream primer with a sequence as shown in SEQ ID NO.3, a downstream primer with a sequence as shown in SEQ ID NO.4 and a probe with a sequence as shown in SEQ ID NO.7.

[0008] Furthermore, the 5' end of the probe is modified with an antigen label, a base is replaced by dSpacer in the middle from the 5' end to the 3' end as a recognition site of nfo, and a modification group is labeled at the 3' end.

[0009] Furthermore, the middle from the 5' end to the 3' end is the 31st position from the 5' end to the 3' end.

[0010] Furthermore, the antigen label includes a FAM fluorescent group, the dSpacer includes tetrahydrofuran, and the modification group includes a C3-Spacer.

[0011] The present invention also provides a kit for rapid detection of Glechoma ayutans by RPA-LFD, comprising the primer-probe combination.

[0012] Furthermore, it also includes lateral flow chromatography test strips.

[0013] The present invention also provides application of the primer-probe combination or the kit in the rapid detection of Glechoma ayutans by RPA-LFD.

[0014] The present invention also provides a method for rapid detection of Glechoma ayutans by RPA-LFD, comprising the steps of using the DNA of a sample to be detected as a template and performing an RPA reaction using the primer-probe combination.

[0015] Furthermore, the RPA reaction system includes 1 μL upstream primer, 1 μL downstream primer, 0.3 μL probe, 4.75 μL ddH2O, 2 μL DNA template and 1.25 μL magnesium acetate.

[0016] Furthermore, the RPA reaction conditions are 39° C., 10 min.

[0017] The present invention discloses the following technical effects:

[0018] The present invention designs a group of primer-probe combinations according to the Beta-tubulin gene. The RPA-LFD detection method of the sweetfish larvae established based on the primers and probes has obvious advantages such as high sensitivity, good specificity, simple operation, and visualized results. It can accurately detect the sweetfish larvae and effectively reduce the economic losses caused by the sweetfish larvae disease.

[0019] The RPA-LFD rapid detection method for Ayu sutsugamushi provided by the present invention does not require special instruments, has simple sample processing, rapid amplification, and intuitive results, and the minimum detection limit of the sample can reach 9.38×10 -6 ng / μL, 1000 times higher than traditional PCR methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 The matching positions of the three pairs of designed primers and probes on the target gene sequence (A) and the screening results of lateral flow chromatography test strips (B);

[0022] Figure 2 The results of RPA-LFD reaction temperature optimization, where 1-5 are reaction temperatures of 30°C, 35°C, 39°C, 42°C and negative control, respectively;

[0023] Figure 3 The results of RPA-LFD reaction time optimization, where 1-5 are reaction times of 5 minutes, 8 minutes, 10 minutes, 12 minutes and negative control;

[0024] Figure 4 is the specificity test result, where 1-6 are G. plecoglossi, Z. ophiocephali, C. hongtzensis, M. drjagini, M. carassii and negative control;

[0025] Figure 5 The sensitivity test results of the RPA-LFD reaction of the present invention (A) and the sensitivity test results of the common PCR reaction (B), wherein 1-8 are DNA concentrations of 9.38, 9.38×10-1 ,9.38×10 -2 ,9.38×10 -3 ,9.38×10 -4 ,9.38×10 -5 ,9.38×10 -6 ng / μL and negative control. DETAILED DESCRIPTION

[0026] 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.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] 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.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example

[0032] 1. Experimental Materials

[0033] DNA samples of Glechoma saury, Zschokkellaophiocephali, Ceratomyxahongtzensis, Myxobolus drjagini and Myxobilatuscarassii. SteadyPure Universal Genomic DNA Extraction Kit (Acori Biotech, China), DNA Constant Temperature Rapid Amplification Kit (Colloidal Gold Test Strip) (Anpu Future, Changzhou, China), Hybridetect Colloidal Gold Test Strip (Anpu Future, Changzhou, China).

[0034] 2. Experimental Methods

[0035] 1. Parasite strains and genomic DNA extraction

[0036] The DNA samples of G. ayutans were provided by the College of Marine Science and Engineering, Qingdao Agricultural University, and the samples were stored at −20°C until further use.

[0037] The tissue samples of Channa chukla, Tetrapolaris hongzei, Cynomorium iodineum and Pseudocercospora crucianum used in the specific detection were obtained from the College of Marine Science and Engineering, Qingdao Agricultural University. Genomic DNA samples were extracted using the SteadyPure Universal Genomic DNA Extraction Kit (Acori Biotech, China), and all DNA extractions were processed according to the manufacturer's extraction protocol and stored at -20°C until further use.

[0038] 2. Primer and probe design

[0039] Beta-tubulin was selected as the target gene. According to the multiple sequence alignment of the genome in the National Center for Biotechnology Information (NCBI) database, three sets of primers were designed to meet the requirements of highly homologous genes of Gracilariae. Primers were designed in some conservative fragments of the gene using the TwistAmp kit according to the instructions. Probe design principles: between the upstream and downstream primers, a sequence with a length of 46-52nt complementary to the target fragment was designed, an antigen marker (typically FAM) was modified at the 5' end, a dSpacer (tetrahydrofuran, THF) was marked in the middle of the 5' end and the 3' end as the recognition site of nfo, and a modification group, such as C3-Spacer, was marked at the 3' end. All primers and probes were synthesized by Beijing Ruibo Xingke Biotechnology Co., Ltd.

[0040] 3. Establishment of RPA-LFD system

[0041] The recombinase polymerase amplification (RPA) reaction system was established according to the instructions of the DNA Constant Temperature Rapid Amplification Kit (Colloidal Gold Test Strip Type) (Anpu Future, Changzhou, China). Take out the required components of the kit 30 minutes in advance, melt at room temperature, and shake to mix. Add 29.4μL of buffer to each dry powder reaction tube and divide it evenly into two 0.2mL centrifuge tubes. The buffer needs to be completely melted and mixed, otherwise it will affect the experimental effect. Add 1μL of upstream primer, 1μL of downstream primer and 0.3μL of probe to each reaction tube, add 4.75μL of ddH2O and 2μL of nucleic acid template to the reaction tube in turn, and finally add 1.25μL of magnesium acetate to the reaction tube and mix thoroughly. After mixing, the reaction solution is quickly centrifuged to the bottom of the tube, and then the reaction tube is immediately placed in a constant temperature device and incubated at 39°C for 10 minutes. After the reaction is completed, take 10 μL of the reaction product and add it to a centrifuge tube containing 190 μL of ddH2O. After mixing evenly, take 50 μL of the product and add it dropwise to the sample well of the lateral flow chromatography test strip. Observe the quality control line and the detection line within 5 minutes to read the results.

[0042] 4. Primer screening

[0043] Multiple sequence alignment was performed from the NCBI database to analyze highly conserved regions, probes were designed based on the conserved regions, and three pairs of candidate primers were designed. The three pairs of candidate primer combinations (F1 / R1, F2 / R2, F3 / R3) designed for the conserved regions were amplified at 39°C for 10 minutes, and the most suitable primer combination was screened using the lateral flow chromatography test strip results.

[0044] 5. RPA-LFD temperature optimization

[0045] A recombinase polymerase amplification (RPA) reaction system was established, and RPA assays were performed at different reaction temperatures (30°C, 35°C, 39°C, and 42°C). The optimal reaction conditions were selected based on the LFD results. The RPA assay products were detected using Hybridetect colloidal gold test strips (Anpu Future, Changzhou, China).

[0046] 6. RPA-LFD time optimization

[0047] A recombinase polymerase amplification (RPA) reaction system was established, and RPA assays were performed at different reaction times (5 min, 8 min, 10 min, and 12 min) to select the optimal reaction conditions. The RPA assay product was detected using lateral flow chromatography test strips.

[0048] 7. RPA-LFD specificity detection

[0049] In order to determine the specificity of the RPA-LFD method established in the present invention, a series of DNA samples of different parasites, including Channa chukla, Tetrapolaris hongzei, Cynomorium iodineum and Pseudocercospora crucianum, were used as templates for RPA reactions, and different RPA products were directly analyzed by lateral flow chromatography test strips. DNA of G. ayutans was used as a positive control, and sterile water was used as a negative control.

[0050] 8. RPA-LFD sensitivity detection

[0051] To detect the concentration of the DNA sample of the sweetfish, the DNA was diluted to 9.38×10 -6 ng / μL was used as the template of RPA to evaluate the sensitivity of RPA. -1 ,9.38×10 -2 ,9.38×10 -3 ,9.38×10 -4 ,9.38×10 -5 and 9.38×10 -6 ng / μL) DNA samples were subjected to RPA and conventional PCR detection to evaluate the sensitivity of RPA and conventional PCR.

[0052] 9. Conventional PCR

[0053] The present invention compares the sensitivity of the conventional PCR method with the RPA-LFD method. The total volume of the PCR reaction system is 25 μL, consisting of 12.5 μL of Taq enzyme, 2 μL of DNA template, 9.5 μL of ddH2O, 0.5 μL of forward and 0.5 μL of reverse primers. The PCR amplifier is set to perform PCR reaction conditions at 94°C for 5 minutes, 34 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute, and finally extended at 72°C for 8 minutes. After the amplification, 5 μL of PCR product was taken for 2% agarose gel electrophoresis, and the brightness of the DNA band was observed by the electrophoresis gel imaging system.

[0054] Conclusion

[0055] 1. Best RPA Primers

[0056] Three pairs of primers (F1 / R1, F2 / R2, F3 / R3) and a probe (Table 1 and Figure 1 A). Amplify at 39°C for 10 minutes and detect the LFD results. The results show that the detection line of F2 / R2 primers and probes is the most obvious, while other primer and probe combinations can also successfully amplify the target sequence, but the amplification products are less ( Figure 1 B). Therefore, the F2 / R2 primer and probe combination was used for subsequent detection.

[0057] Table 1 Primer and probe sequences

[0058]

[0059]

[0060] Note: In actual detection, the bold and underlined base "c" in the probe sequence is marked as dSpacer (tetrahydrofuran, THF), which serves as the recognition site of nfo.

[0061] 2. Optimization of reaction temperature

[0062] In order to select the optimal temperature for the RPA-LFD method, RPA analysis was performed at different temperatures of 30°C, 35°C, 39°C and 42°C for 10 minutes, and a negative control was set at 39°C, and then the amplification product was detected by LFD. The results showed that in all temperature ranges, positive samples could obtain both the test line and the quality control line, while negative samples could only obtain the quality control line. The test line was clearest at 39°C ( Figure 2 ), so the optimal reaction temperature is 39°C.

[0063] 3. Optimization of response time

[0064] In order to determine the optimal reaction time of the RPA-LFD method, the RPA method was performed multiple times (5 minutes, 8 minutes, 10 minutes, and 12 minutes), and the negative control was set to 10 minutes. Figure 3 As shown in the figure, the test line can be detected within the time range of 5-12 minutes, among which the bands at 5 minutes and 8 minutes are weak, and the bands at 10 minutes and 12 minutes are obvious. No bands were detected in the negative control. Therefore, the optimal reaction time for the LFD test is 10 minutes.

[0065] 4. Specificity of RPA-LFD detection

[0066] To evaluate the specificity of RPA-LFD, genomic DNA from four additional parasites was used for RPA amplification under optimal conditions. Figure 4 As shown in the figure, the DNA of other parasites (Z.ophiocephali, C.hongtzensis, M.drjagini and M.carassii) and negative control (sterile water) did not produce detection lines, and only the genomic DNA of G.plecoglossi was positive. This result shows that RPA-LFD has excellent specificity.

[0067] 5. Sensitivity of RPA-LFD detection

[0068] Template DNA was mixed at different concentrations (9.38, 9.38×10 -1 ,9.38×10 -2 ,9.38×10 -3 ,9.38×10 -4 ,9.38×10 -5 ,9.38×10 -6 ng / μL) were serially diluted, and a negative control (sterile water) was set. The RPA-LFD results showed that as the template DNA concentration decreased, the brightness of the detection line decreased, and the negative sample detection line could not be detected. The detection limit of RPA-LFD was 9.38×10 -6 ng / μL( Figure 5 A). At the same time, the RPA-LFD method was compared with the traditional PCR method. The detection limit of the traditional PCR method was 9.38×10 -3 ng / μL( Figure 5 This result shows that RPA-LFD has good sensitivity, which is 1000 times that of traditional PCR method.

[0069] 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-probe combination for detecting Glechoma ayuensis based on RPA-LFD, characterized in that: The primer-probe combination consists of an upstream primer with a sequence as shown in SEQ ID NO.3, a downstream primer with a sequence as shown in SEQ ID NO.4, and a probe with a sequence as shown in SEQ ID NO.

7.

2. The primer-probe combination according to claim 1, characterized in that: The 5' end of the probe is modified with an antigen label, the 31st base from the 5' end to the 3' end is replaced by dSpacer as the recognition site of nfo, and the 3' end is labeled with a modification group.

3. The primer-probe combination according to claim 2, characterized in that: The antigen marker is a FAM fluorescent group, the dSpacer is tetrahydrofuran, and the modification group is a C3-Spacer.

4. A kit for rapid detection of Ayu sutsugamushi by RPA-LFD, characterized in that: The invention comprises the primer-probe combination according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that Also included are lateral flow chromatography test strips.

6. Use of the primer-probe combination according to any one of claims 1 to 3 or the kit according to claim 4 or 5 in the RPA-LFD rapid detection of Glechoma sutsugamushi for non-disease diagnosis purposes.

7. A method for rapid detection of Glechoma sutsugamushi by RPA-LFD for non-disease diagnosis purposes, characterized in that: The method comprises the steps of using the sample DNA to be tested as a template and performing an RPA reaction using the primer-probe combination described in any one of claims 1 to 3.

8. The method according to claim 7, characterized in that The RPA reaction system includes 1 μL of upstream primer, 1 μL of downstream primer, 0.3 μL of probe, 4.75 μL of ddH2O, 2 μL of DNA template and 1.25 μL of magnesium acetate.

9. The method according to claim 7, characterized in that: The RPA reaction was carried out at 39° C. for 10 min.

Images