Primer probe combination, kit and detection method for monitoring echinochal based on MIRA and eDNA (enhanced deoxyribonucleic acid)

Abstract

The invention relates to the technical field of molecular biological identification and wild animal ecology. The invention provides a primer and probe combination, a kit and a detection method for monitoring spinomill based on MIRA and eDNA. The primer and probe combination comprises a forward primer, a reverse primer and a probe, the nucleotide sequence of the forward primer is as shown in SEQ ID NO. 1; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO. 2; and the nucleotide sequence of the probe is as shown in SEQ ID NO. 3. According to the invention, through the design of the specific multi-enzyme isothermal amplification (MIRA) primers and probes of the Phenalihaiganl and combinatorial optimization, the MIRA reaction is combined with a lateral flow chromatography test strip (LFD) detection technology, so that the Phenalihaiganl can be rapidly and conveniently detected and identified from eDNA (enhanced deoxyribonucleic acid). The method has the advantages of high sensitivity and strong specificity, and has a huge advantage in field survey of Anghai Echevron.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biological identification and wildlife ecology, and in particular to a primer-probe combination, a kit and a detection method for monitoring Zhenhai Acanthus zhenhaiensis based on MIRA and eDNA. Background Art

[0002] The Zhenhai Acanthus zhenhaiensis is a very ancient amphibian, listed as Critically Endangered by the International Union for Conservation of Nature (IUCN) and a Class I protected species on the National List of Key Protected Wildlife. The Zhenhai Acanthus zhenhaiensis not only holds significant scientific and ecological value, but also offers broad potential for development in medicine and healthcare. However, due to habitat loss, climate change, and human activities, the population of the species has declined dramatically, and it faces serious threats. To protect this species, researchers have conducted extensive research. However, since its egg-laying and hatching period is limited to late March to early mid-May each year, spawning in leaf litter and microhabitats with low soil moisture (33.51% ± 1.87%), often located beneath dead leaves or vegetation near semi-permanent stagnant pools or very slow-flowing waterways, direct observation is difficult, requiring significant time and labor to search, greatly hindering field surveys and often making it difficult to locate.

[0003] Organisms release their own tissue cells or DNA into their environment through excretions such as feces and urine, as well as shed skin and surface cells. Upon death, genomic DNA is released into the environment from damaged, degraded tissues. This DNA is known as environmental DNA (eDNA). Currently, eDNA technology can effectively detect rare and unknown species in water or soil, improving the accuracy and convenience of survey results. It is primarily combined with real-time quantitative PCR (qPCR) or high-throughput sequencing technologies and has been applied in surveys and detection of numerous rare, endangered, and specific species. Compared to traditional detection methods, eDNA technology, combined with a variety of modern DNA analysis methods, enables non-destructive and real-time monitoring of species. Furthermore, within the development of specific PCR species identification technologies, multienzyme isothermal rapid amplification (MIRA) technology, with its advantages such as suitability for on-site testing, rapid speed, and high sensitivity, has demonstrated broad application potential in detection, making it an emerging technology in this field. In addition, MIRA is combined with lateral flow dipstick (LFD) technology to achieve rapid visual detection of amplified products. It has been used to authenticate the authenticity of beef, mutton, pork, chicken, and duck meat, and to detect various specific species or pathogens such as feline parvovirus and Bacillus cereus. This provides an important reference for real-time field monitoring of Zhenhai Acanthus. Summary of the Invention

[0004] The present invention aims to provide a primer-probe combination, kit, and detection method for monitoring Zhenhai Acanthus zhenhaiensis based on MIRA and eDNA. The present invention constructs a MIRA-LFD detection technology system for Zhenhai Acanthus zhenhaiensis based on eDNA from water samples. By designing specific primers and probes and combining the optimized MIRA amplification system with LFD, a MIRA-LFD detection method for Zhenhai Acanthus zhenhaiensis using eDNA from various materials, including water samples, has been developed. This method is simple to operate, highly sensitive, and specific, and results can be visualized within a short period of time. It does not require precision instruments and can obtain test results within 40 minutes at 37°C. This method is suitable for field surveys and monitoring of Zhenhai Acanthus zhenhaiensis, eliminating the reliance on survey time and direct observation of survey subjects, reducing field work risks, and improving monitoring accuracy and efficiency.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a primer-probe combination for monitoring Zhenhai Acanthus zhenhaiense based on MIRA and eDNA, wherein the primer-probe combination comprises a forward primer, a reverse primer and a probe;

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

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

[0009] The nucleotide sequence of the probe is shown in SEQ ID NO.3.

[0010] Preferably, the reverse primer is labeled with carboxyfluorescein, and the probe is labeled with biotin.

[0011] Preferably, the 3' end of the probe is modified with C3 spacer, the nucleotides G at positions 14, G at positions 16, and T at positions 22 are locked nucleic acids, and the nucleotide at position 24 is modified with THF.

[0012] The present invention also provides the use of the primer-probe combination in preparing a product for monitoring Zhenhai Acanthus.

[0013] Preferably, the product is a kit.

[0014] The present invention also provides a kit for monitoring Acanthus zhenhaiense, wherein the kit comprises the primer-probe combination.

[0015] The present invention also provides a method for monitoring Acanthus zhenhaiense using the primer-probe combination, comprising the following steps:

[0016] (1) extracting eDNA from the environment to be monitored, adding the primer probe combination and MIRA reaction reagent, performing MIRA amplification, and obtaining a MIRA amplification product;

[0017] (2) The MIRA amplified product was detected using LFD test strips labeled with anti-FAM and anti-Biotin.

[0018] Preferably, the objects for extracting eDNA include water samples, soil, humus or bottom sediments.

[0019] Preferably, the judgment criteria of the method are: when the control C line and the test T line appear on the test strip at the same time, it means that the sample to be tested is positive, that is, the sample contains Zhenhai Acanthus DNA; when the test strip only has the control C line, it means that the sample to be tested is negative, that is, the sample does not contain Zhenhai Acanthus DNA; when the test strip has no C line, it means that the detection process has failed and the sample needs to be retested.

[0020] The present invention provides a primer-probe combination, a kit and a detection method for monitoring Zhenhai Acanthus zhenhaiensis based on MIRA and eDNA. The primer-probe combination comprises a forward primer, a reverse primer and a probe; the nucleotide sequence of the forward primer is shown in SEQ ID NO.1; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; and the nucleotide sequence of the probe is shown in SEQ ID NO.3.

[0021] The primer probe set, kit and method can quickly and conveniently detect Zhenhai Acanthus zhenhaiense by eDNA with high sensitivity and strong specificity, thereby realizing convenient monitoring of Zhenhai Acanthus zhenhaiense in the wild.

[0022] The present invention designs specific MIRA primers and probes for Zhenhai Acanthus zhenhaiensis and optimizes their combination, adopts multi-enzyme constant temperature rapid amplification combined with lateral flow chromatography test strip (MIRA-LFD) detection technology, combines eDNA analysis with MIRA technology, and truly realizes a Zhenhai Acanthus zhenhaiensis field survey and monitoring method that is not limited by time and equipment, reduces the risk of investigators' field work, and improves accuracy.

[0023] This method requires minimal laboratory equipment and conditions, requiring no sophisticated instruments. Water, soil, humus, or bottom sediment samples can be collected in the field and brought back to the laboratory for testing. By testing the eDNA of the Zhenhai Acanthus zhenhaiensis, data on its distribution, activity, and habitat selection will be accumulated. This will be crucial for understanding the dynamics of the Zhenhai Acanthus zhenhaiensis population and provide scientific support for its conservation, reintroduction into the wild, and ecological restoration assessments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The amplification results of the 16S-F and 16S-R primers for Acanthus zhenhaiensis are shown in Figure 1. Lane M is 1200 DNA Marker; lanes 1 to 7 are Acanthus zhenhaiensis; and lane 8 is the negative control ddH2O.

[0025] Figure 2 The results of 12S rRNA primer amplification of the macro-DNA barcode of water sample eDNA are shown in Figure 1. Lane 1 represents sample S1; lane 2 represents sample S2; lane 3 represents S3; lane 4 represents S9141; lane 5 represents S9142; and NTC is the negative control.

[0026] Figure 3 Statistical donut chart of water sample eDNA species annotation results.

[0027] Figure 4 Taxonomic stacking diagram for species identification of eDNA from 10 water samples.

[0028] Figure 5The results of MIRA primer screening for Acanthus zhenhaiensis are shown in Figure 1, where M represents DNA Marker. Lane 1 is the amplified product of MIRA-F / R1; lane 2 is the amplified product of MIRA-F / R2; lanes 3 and 4 are negative controls for MIRA-F / R1 and MIRA-F / R2, respectively.

[0029] Figure 6 These are the screening results for Acanthus zhenhaiensis using MIRA-LFD probes MIRA-P1, P2, and P3. Test strips 1 and 2 used probe MIRA-P1; test strips 3 and 4 used probe MIRA-P2; and test strips 5 and 6 used probe MIRA-P3. Test strips 1, 3, and 5 contained Acanthus zhenhaiensis DNA; test strips 2, 4, and 6 contained negative control mixed DNA (carp, bullfrog, shrimp, cow, sheep, and pig); and test strip 7 was a ddH2O negative control.

[0030] Figure 7 This is an illustration of the design of MIRA-LFD primers and probes based on the CytB gene sequence of Acanthus zhenhaiense, where F1 is the forward primer, R1 is the reverse primer, and P1 is the probe.

[0031] Figure 8 The results are for optimizing the MIRA-LFD reaction temperature (27-47°C). Test strips 1-5 are 27°C, 32°C, 37°C, 42°C, and 47°C, respectively; test strip 6 represents the negative control (ddH2O).

[0032] Figure 9 To optimize the MIRA-LFD color development reaction at 37°C, test strips 1 to 5 represent color development times of 10 min, 15 min, 20 min, 25 min, and 30 min; test strip 6 represents the negative control (ddH2O).

[0033] Figure 10 These are the results of MIRA specificity tests, where (a) is the specificity result of PCR reaction, (b) is the specificity result of MIRA reaction, and (c) is the specificity result of MIRA-LFD reaction. Sample 1 represents Zhenhai Acanthus zhenhaiense; 2 represents crucian carp; 3 represents Jihu shrimp; 4 represents bullfrog; 5 represents dairy cow; 6 represents pig; 7 represents sheep; 8 is negative control (ddH2O is used as reaction template); and 9 is blank control.

[0034] Figure 11 is the detection limit of the MIRA-LFD assay, in which the initial DNA concentration in the sensitivity test was 5.1 ng / μL, all reactions consisted of 10-fold serial dilutions, and ddH2O was used as a negative control for NTC.

[0035] Figure 12Figure 5 shows the MIRA detection limit test results. (a) is the MIRA isothermal amplification product, and (b) is the MIRA conventional PCR amplification product. The initial DNA template concentration was 5.1 ng / μL. All reaction systems consisted of 10-fold serial dilutions. NTC used ddH2O as a negative control.

[0036] Figure 13 These are the PCR results of some water eDNA samples using 16S-F / R primers. Lanes 1 to 9 represent water eDNA samples, respectively; lane 10 represents the negative control (ddH2O).

[0037] Figure 14 These are the MIRA-LFD test results of some water eDNA samples. Test strips 1 to 9 are eDNA samples, and test strip 10 represents the negative control (ddH2O).

[0038] Figure 15 These are the MIRA-LFD detection and identification results of water sample eDNA. From left to right, 1 to 3 are positive water sample eDNA (including Zhenhai Acanthus zhenhaiensis), and 4 to 6 are negative water sample eDNA (excluding Zhenhai Acanthus zhenhaiensis). DETAILED DESCRIPTION

[0039] The present invention provides a primer-probe combination for monitoring Zhenhai Acanthus zhenhaiense based on MIRA and eDNA, wherein the primer-probe combination comprises a forward primer, a reverse primer and a probe;

[0040] The nucleotide sequence of the forward primer is shown in SEQ ID NO.1;

[0041] SEQ ID NO.1:

[0042] 5′-CCAGAGTGATACTTCCTATTTGC-3′;

[0043] The nucleotide sequence of the reverse primer is shown in SEQ ID NO.2;

[0044] SEQ ID NO.2:

[0045] 5′-FAM-GTATAGGATTGAGGCGATTTGTC-3′;

[0046] The nucleotide sequence of the probe is shown in SEQ ID NO.3;

[0047] SEQ ID NO.3:

[0048] 5′-CCAAACAAGTTAGGAGGAGTCTTGCTTTGCTTATATCC-3′.

[0049] In the present invention, the reverse primer is preferably labeled with carboxyfluorescein, and the probe is preferably labeled with biotin.

[0050] In the present invention, the 3' end of the probe is preferably modified with C3 spacer, the nucleotides G at positions 14, G at positions 16, and T at positions 22 are preferably locked nucleic acids, and the nucleotide at position 24 is preferably modified with THF.

[0051] The present invention also provides the use of the primer-probe combination in preparing a product for monitoring Zhenhai Acanthus.

[0052] In the present invention, the product is preferably a kit.

[0053] The present invention also provides a kit for detecting and monitoring Acanthus zhenhaiense, wherein the kit comprises the primer-probe combination.

[0054] The present invention also provides a method for monitoring Acanthus zhenhaiense using the primer-probe combination, comprising the following steps:

[0055] (1) extracting eDNA from the environment to be monitored, adding the primer probe combination and MIRA reaction reagent, performing MIRA amplification, and obtaining a MIRA amplification product;

[0056] (2) The MIRA amplified product was detected using LFD test strips labeled with anti-FAM and anti-Biotin.

[0057] In the present invention, the object for extracting eDNA preferably includes water samples, soil, humus or bottom sediment.

[0058] In the present invention, the judgment criteria of the method are: when the control C line and the test T line appear on the test strip at the same time, it means that the sample to be tested is positive, that is, the sample contains Zhenhai Acanthus DNA; when the test strip only has the control C line, it means that the sample to be tested is negative, that is, the sample does not contain Zhenhai Acanthus DNA; when the test strip has no C line, it means that the detection process has failed and the sample needs to be retested.

[0059] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] The reagents and equipment used in this invention are all commercially available. The MIRA system uses a DNA multi-enzyme constant temperature rapid amplification kit and a multi-enzyme polymerase amplification colloidal gold kit (Weifang Anpu Future Biotechnology Co., Ltd., China); the LFD test strips (Shanghai Sangon Biotechnology Co., Ltd.) are labeled with anti-FAM on the T line and anti-Biotin on the C line.

[0061] Example 1 Source and identification of Zhenhai Acanthus salamander samples

[0062] In April 2023, this research team collected 4 juveniles and 3 adults of Zhenhai Acanthus zhenhaiensis from the Zhenhai Acanthus zhenhaiensis Nature Reserve in Beilun District, Ningbo, Zhejiang Province during a field survey. The juveniles died mainly from cannibalism and were stored in anhydrous ethanol. 0.2 g of subcutaneous muscle tissue was taken from the Zhenhai Acanthus zhenhaiensis individual, and DNA was extracted using an animal tissue DNA kit (Hangzhou Xinjing Biological Company) according to the instructions. The extracted DNA was detected using NanoDrop 2000, and the integrity of the genomic DNA was detected by 1.5w% agarose electrophoresis, and stored at 4°C for use. The samples used as negative controls came from six species of bullfrogs, crucian carp, pigs, cows and sheep preserved in the laboratory, and fire shrimp caught in the waters of the nature reserve. Genomic DNA was extracted according to the instructions of the animal tissue DNA kit.

[0063] The concentration of DNA extracted from Zhenhai Acanthus tissue samples ranged from 50.2 ng / μL to 178.2 ng / μL, with an average concentration of 75.71±48.31 ng / μL. The purity of A260 / A280 ranged from 1.8 to 2.0 (average value was 1.94±0.11), indicating that it could be used for subsequent PCR analysis.

[0064] The mtDNA gene sequence of Echinops zhenhaiensis (EU880315.1) and the mtDNA gene sequences of bullfrog (NC_022696.1), crucian carp (NC_039257.1), prawn (NC_023823.1), pig (NC_012095.1), cow (NC_006853.1), and sheep (NC_001941.1) were downloaded from the NCBI database. Homology sequences were aligned using the MegAlign software package. The mitochondrial 16S rRNA gene sequence was selected as the identification target. Specific primers were designed and used to amplify the DNA of Echinops zhenhaiensis. The expected product size was 500 bp.

[0065] Upstream primer 16S-F:

[0066] 5'-AGGCGATAAACCTAACGAGCC-3' (SEQ ID NO.4),

[0067] Downstream primer 16S-R:

[0068] 5'-TCGTGATTGCCGAGTTCCTT-3' (SEQ ID NO. 5).

[0069] Positive products were sent to Hangzhou Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The sequencing results were compared and spliced ​​using Clustal X software (version 2.1) and further analyzed by Blast (blast.ncbi.nlm.nih.gov) in the GenBank database.

[0070] The products of the DNA samples of Zhenhai Acanthus zhenhaiensis amplified by primers 16S-F and 16S-R showed specific bands on the agarose gel ( Figure 1 ), the fragment length was about 500 bp, which was the same as the expected fragment. Blast comparison of the sequencing results showed that both were Acanthus zhenhaiensis (EU880315.1, similarity > 98%).

[0071] Example 2 Species Monitoring in Water Sample eDNA

[0072] In order to confirm other species in the water puddles of the Zhenhai Acanthus habitat and further verify the accuracy of the present invention, 5 positive water samples collected in the protected area (numbered S1, S2, S3, S5, S7) and 5 water samples outside the protected area where no Zhenhai Acanthus was found (numbered S9141, S9142, S91511, S91514, S91517) were selected. 200 ml of water sample was taken, filtered using a glass fiber microporous filter membrane (Shanghai Xingya Jinjing Company), and eDNA was extracted using the DNeasy Blood Tissue Kit (Shanghai Yanhui Biotechnology Co., Ltd.). The concentration of water sample eDNA ranged from 12.0 ng / μL to 300.0 ng / μL, and the purity range of A260 / A280 was 1.2 to 2.0, which basically met the requirements for subsequent PCR analysis. High-throughput sequencing and species analysis were performed using 12S rRNA macro DNA barcode primers.

[0073] Upstream primer sequence 12SV05F:

[0074] 5'-TTAGATACCCCACTATGC-3' (SEQ ID NO. 6);

[0075] Downstream primer 12SV05R:

[0076] 5'-TAGAACAGGCTCCTCTAG-3' (SEQ ID NO. 7).

[0077] The eDNA of 5 positive water samples from the Zhenhai Acanthus conservation area and 5 water samples from outside the conservation area where no Acanthus was found was amplified. The PCR products were detected by 2% agarose gel electrophoresis. It was found that the eDNA extracted from the 10 water samples was of good quality, and the PCR products were consistent with the expected fragment size, which met the requirements for library construction and subsequent sequencing analysis ( Figure 2High-throughput sequencing was performed by Shanghai Lingen Biotechnology Co., Ltd. The following databases were used for species identification:

[0078] Fish database: http: / / mitofish.aori.u-tokyo.ac.jp / download / ;

[0079] Plankton database: http: / / www.arb-silva.de;

[0080] Protist database: https: / / github.com / vaulot / pr2_database;

[0081] NT database: https: / / ftp.ncbi.nlm.nih.gov / blast / db / FASTA

[0082] The high-throughput sequencing results were analyzed and species annotations were performed. The number of annotated species for each sample was counted. The results showed that 54, 75, 49, 54 and 59 species were found in the eDNA of the five water samples positive for Acanthus zhenhaiense, respectively; and 96, 104, 84, 96 and 92 species were found in the eDNA of the five water samples without Acanthus zhenhaiense, respectively. The species annotation rate was 100% (see Figure 3 ). Stacking shock diagram ( Figure 4 ) showed that Zhenhai Acanthus was detected among the top 10 species in abundance, and the remaining species detected included cats, tree frogs, chickens, pigs, mice, marsh frogs, sheep, cattle, crucian carp and fruit bats.

[0083] Example 3 MIRA primer and probe design

[0084] By comparing the mtDNA gene sequences of Acanthus zhenhaiensis with those of a negative control species, the CytB gene (EU880315.1) sequence was selected as the target. Primers and probes were designed according to the MIRA kit instructions. Primer lengths generally ranged from 39 to 52 nt, probes from 45 to 52 bp, and product lengths from 150 to 300 bp. Primers and probes were designed using Clone Manager software: upstream primer MIRA-F, downstream primer MIRA-R1 (alternative primer MIRA-R2), and probe MIRAP1 (alternative probes MIRAP2 and MIRAP3).

[0085] The downstream primer was labeled with FAM at the 5' end, and the probe was biotin-labeled at the 5' end. A dSpacer (tetrahydrofuran, THF) was added approximately 23 nt from the 5' end, serving as the recognition site for the endonuclease nfo. Additionally, THF was added approximately 15 nt from the 3' end, and the 3' end was labeled with a C3 spacer modifier (Table 1). Primers and probes were synthesized by Anhui General Biotechnology Co., Ltd. and diluted to 10 μmol / L for later use.

[0086] Table 1 Sequences of primers and probes for MIRA of Acanthus zhenhaiensis

[0087]

[0088] Note: The letters in the boxes in the probe sequences represent locked nucleic acids. The sequences of probes MIRAP2 and MIRAP3 are identical. During the synthesis process, only the locked nucleic acids were modified, which changed the detection specificity, thereby selecting the probe with the best specificity.

[0089] Example 4 MIRA primer and probe combination optimization

[0090] The MIRA reaction used a recombinase polymerase amplification kit (Weifang Anpu Future Biotechnology Co., Ltd., China). The reaction system was prepared according to the instructions to be 50 μL, 2 μL of upstream and downstream primers (MIRAF / R1 and MIRAF / R2 combination), 29.4 μL A buffer, 2.5 μL B buffer, 5 μL DNA template and 9.1 μL ddH2O. The negative control used the mixed DNA of the 6 control species in Example 1. The reaction procedure was 30 minutes at 37°C. The amplified product was mixed with an equal amount of phenol: chloroform: isoamyl alcohol (25:24:1) to remove proteins. After the mixture was centrifuged at 12000 rpm for 5 minutes, the supernatant was analyzed by 2w% agarose gel electrophoresis to compare the specificity and amplification efficiency of the screening primer sets MIRAF / R1 and MIRAF / R2.

[0091] Two sets of MIRA primers were used to amplify the DNA of Acanthus zhenhaiensis. It can be seen that both the MIRA-F / R1 and MIRA-F / R2 primer sets specifically amplified the target gene and the size was consistent with the expected size ( Figure 5 ). With reference to the MIRA primer design principles and negative control results, the MIRA-F / R1 primer set was selected as the preferred primer.

[0092] The MIRA-LFD reaction was performed using a multi-enzyme polymerase amplification colloidal gold kit (Weifang Anpu Future Biotechnology Co., Ltd., China). The MIRA-LFD reaction system (50 μL) consisted of 29.4 μL of A buffer, 8.5 μL of deionized water, and 2.5 μL of B buffer. 2.0 μL of each of the preferred upstream and downstream primers MIRA-F and MIRA-R1 were used. DNA from Acanthus zhenhaiensis was used as a positive control, and deionized water was used as a blank control. The probes MIRA-P1, MIRA-P2, and MIRA-P3 were optimized at different concentrations (with a concentration gradient of 0.2, 0.6, 1.0, 1.2, and 1.5 μL). The results were directly interpreted using colloidal gold test strips to screen for the optimal probe and concentration.

[0093] The MIRA-F / R1 primer set was used to perform MIRA-LFD on DNA from Acanthus zhenhaiensis and a negative control, and the specificity of the three probes was compared. The results showed that the Acanthus zhenhaiensis DNA produced a clear signal band on the MIRA-P1 probe detection line on the lateral flow chromatography test strip, and there was no cross-reaction with the negative control. The repeated results were consistent, but the results of the MIRA-P2 and MIRA-P3 probes were false positive (see Figure 6 ). This indicates that the MIRA-F / R1 primer set and the MIRA-P1 probe set have good specificity and are suitable for the detection and identification of Echinopteryx zhenhaiensis. Therefore, the primer set MIRA-F / R1 and the probe MIRA-P1 (1.2 μmol / L) were selected to establish a detection system. The position of the primer set in the Cyt B gene sequence of Echinopteryx zhenhaiensis is shown in the figure. Figure 7 shown.

[0094] Example 5 MIRA primer-probe system optimization

[0095] For the selected upstream and downstream primers MIRA-F and MIRA-R1 and the probe MIRA-P1, five temperature gradients (27, 32, 37, 42, and 47°C) were set according to conventional MIRA-LFD parameters to optimize the MIRA reaction temperature. At the optimal temperature, reaction times of 10, 15, 20, 25, and 30 minutes were set to optimize the MIRA reaction time. The experiment was repeated three times.

[0096] The results showed that the MIRA-LFD reaction time was set to 20 min, and the color development results of the reaction products in the range of 27-47 ° C showed that the signal of the reaction product at 37 ° C was the most obvious ( Figure 8 Therefore, we chose to optimize the MIRA amplification time at 37°C ( Figure 9The results showed that the test line was observed within 10 to 30 minutes, but there was no significant difference in MIRA color development between 20 and 25 minutes, and the results were consistent with those of repeated experiments. Therefore, a 20-minute MIRA amplification incubation at 37°C was selected as the optimal incubation time for the MIRA-LFD method, and the LFD color development time was no less than 15 minutes, preferably 20 to 25 minutes.

[0097] Example 6 MIRA-LFD specificity test

[0098] In order to evaluate the specificity of the primers and probes selected for Zhenhai Acanthus zhenhaiense and their combinations in this study, specificity tests were carried out under the optimal reaction temperature and development time conditions, using DNA from Zhenhai Acanthus zhenhaiense as the positive sample and DNA from six species including bullfrog, crucian carp, shrimp, pig, cow and sheep as negative controls.

[0099] The specificity test of MIRA primers in this study included two levels: conventional PCR amplification and isothermal amplification using the MIRA primers designed in this study, and the amplified products were detected by agarose gel electrophoresis and LFD. The results showed that all amplified products could specifically identify Zhenhai Acanthus zhenhaiensis in agarose gel electrophoresis and LFD colorimetric detection. The results of conventional PCR using MIRA primers ( Figure 10 a) and isothermal amplification products ( Figure 10 b) The specificity of the agarose gel was consistent, but the amount of isothermal MIRA product was relatively small. The test strip test also had the same specificity, and the signal bands were clearer and easier to interpret than the electrophoresis test ( Figure 10 c) These results indicate that the designed MIRA primer and probe combination has good specificity and can be used to detect Echinops zhenhaiensis in water samples using eDNA.

[0100] Example 7 MIRA preferred primer-probe combination detection limit test

[0101] The minimum detection limit (LDL) was tested using a genomic DNA serial dilution method. Five DNA samples from Acanthus zhenhaiensis were mixed in equal proportions and diluted to a concentration of 5.1 ng / μL. The diluted mixture was used as a template for further 10-fold serial dilutions (5.1 ng / μL to 5.1 fg / μL). ddH2O was used as a blank control. The sensitivity of the MIRA-LFD assay was determined under optimal conditions. The MIRA reaction was performed for 30 minutes, the color development was performed for 20 minutes, and the results were observed three times independently. The results were then compared with those of conventional PCR amplification using the same MIRA primers and electrophoresis of isothermal amplification products.

[0102] The detection limit of MIRA-LFD was tested using 5 portions of mixed DNA from Acanthus zhenhaiensis. The results showed that as the DNA concentration decreased, the color of the test line gradually weakened. When the DNA concentration was diluted to 5.1×10 -4 ng / μL, a faint test line can be seen ( Figure 11 The detection limit of MIRA isothermal amplification was observed by agarose gel electrophoresis ( Figure 12 a), it can be seen that the lowest detectable DNA template concentration is 5.1×10 -3 ng / μL. The amplification results of the MIRA primers in the conventional PCR procedure are visible on the agarose gel electrophoresis results ( Figure 12 b) The minimum concentration of DNA template is 5.1×10 -2 ng / μL. In comparison, the detection sensitivity of MIRA-LFD was higher than that of isothermal amplification and conventional PCR amplification using MIRA primers, that is, MIRA-LFD > MIRA isothermal amplification-agarose detection > MIRA conventional PCR amplification-agarose detection.

[0103] Example 8 eDNA detection in water samples based on the MIRA-LFD detection system

[0104] From August to September 2024, in the Zhenhai Acanthus salamander protection area and the surrounding 5km 2 Search for water holes of the same type in the area (1-20m 2 ), and used a clean bottle to collect 2.0L of samples from 10cm below the water surface in the middle of the water area, and brought them back to the laboratory for use. A total of 197 samples were collected, including 5 water samples in which Zhenhai Acanthurus was found during the investigation of the Zhenhai Acanthurus Constrictor Reserve. 200mL of water sample was taken, filtered using a glass fiber microporous filter membrane (Shanghai Xingya Jinjing Company), and eDNA was extracted using the DNeasy Blood Tissue Kit (Shanghai Yanhui Biotechnology Co., Ltd.). Using NanoDrop 2000 detection, the concentration of eDNA in the water sample ranged from 2.0ng / μL to 375.9ng / μL, with an average concentration of 31.21±46.66ng / μL, and the purity range of A260 / A280 was 1.4 to 2.3 (average value of 1.88±2.18). The eDNA was measured using MIRA-LFD and amplified using the primer set 16S-F and 16S-R in Example 1, and some samples were extracted for sequencing analysis.

[0105] PCR amplification was performed on 197 water samples, and 51 specific products were observed on agarose gel ( Figure 13, only 9 samples are shown), the fragment length is about 500bp, which is consistent with the expected size. The PCR products were sequenced by Sanger sequencing and Blast alignment was performed at NCBI. All 51 positive samples were detected, and the positive rate was 25.88%. The results of MIRA-LFD test are shown in part. Figure 14 As shown, clear positive signals were observed for the eDNA in water samples from strips 1, 2, 3, 5, and 8, while weak detection line signals were observed for the eDNA in water samples from strips 6, 7, and 9. All positive MIRA-LFD results were consistent with the positive results amplified using primer sets 16S-F and 16S-R, demonstrating that MIRA-LFD can be used to detect Echinops zhenhaiensis based on water eDNA. Due to its higher sensitivity, MIRA-LFD detected 84 positive samples out of 197 water eDNA samples, for a detection rate of 48.7%.

[0106] As can be seen from the above examples, the present invention provides a primer-probe combination, a kit, and a detection method for monitoring Zhenhai Acanthus zhenhaiense based on MIRA and eDNA. The primer-probe combination includes a forward primer, a reverse primer, and a probe; the nucleotide sequence of the forward primer is shown in SEQ ID NO.1; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; and the nucleotide sequence of the probe is shown in SEQ ID NO.3. The present invention designs Zhenhai Acanthus zhenhaiense specific MIRA primers and probes and optimizes the combination, and uses MIRA reaction combined with LFD detection technology to achieve rapid and convenient detection and identification of Zhenhai Acanthus zhenhaiense from eDNA. The present invention has the advantages of high sensitivity and strong specificity, and has great advantages in field surveys of Zhenhai Acanthus zhenhaiense.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A primer-probe combination for monitoring Zhenhai Acanthus salamander based on MIRA and eDNA, characterized in that: The primer-probe combination includes a forward primer, a reverse primer and a probe; The nucleotide sequence of the forward primer is shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; The nucleotide sequence of the probe is shown in SEQ ID NO.

3.

2. The primer-probe combination according to claim 1, characterized in that The reverse primer was labeled with carboxyfluorescein, and the probe was labeled with biotin.

3. The primer-probe combination according to claim 1, characterized in that The 3' end of the probe is modified with C3 spacer, the 14th nucleotide G, the 16th nucleotide G, and the 22nd nucleotide T are locked nucleic acids, and the 24th nucleotide is modified with THF.

4. Use of the primer-probe combination according to any one of claims 1 to 3 in the preparation of a product for monitoring Acanthus zhenhaiensis.

5. The use according to claim 4, characterized in that The product is a test kit.

6. A kit for monitoring Acanthus zhenhaiensis, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 3.

7. A method for monitoring Acanthus zhenhaiensis using the primer-probe combination according to any one of claims 1 to 3, characterized in that: The steps include: (1) extracting eDNA from the environment to be monitored, adding the primer-probe combination according to any one of claims 1 to 3 and the MIRA reaction reagent, performing MIRA amplification, and obtaining a MIRA amplification product; (2) The MIRA amplified product was detected using LFD test strips labeled with anti-FAM and anti-Biotin.

8. The method according to claim 7, characterized in that The objects for extracting eDNA include water samples, soil, humus or bottom sediments.

9. The method according to claim 7, characterized in that The judgment criteria of the method are: when the control C line and the test T line appear on the test strip at the same time, it means that the sample to be tested is positive, that is, the sample contains Zhenhai Acanthus DNA; when the test strip only has the control C line, it means that the sample to be tested is negative, that is, the sample does not contain Zhenhai Acanthus DNA; when the test strip has no C line, it means that the detection process has failed and the sample needs to be retested.

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