Primer probe group and kit for rapidly detecting invasive species samarium red fallopian carp based on environmental DNA (Deoxyribonucleic Acid)

Abstract

The invention belongs to the technical field of biology, and particularly discloses a primer and probe combination for detecting an invasive species, namely Salvelinus pelitovii, in water by utilizing multiple PCR (Polymerase Chain Reaction). According to the invention, the RPA specific primer of the saw fat carp is innovatively designed, an eDNA technology and an RPA flow measurement chromatography detection technology are combined, and a technology for visually and timely detecting the saw fat carp is established. The method is simple to operate, does not need a precise instrument, can obtain a detection result within 50 minutes at 38 DEG C, and is suitable for field real-time monitoring research. The detection method can effectively save time and cost, is high in sensitivity, strong in specificity, high in efficiency and low in professional requirement, has a better detection rate when the concentration of a target species is low, and can obtain a detection result without the help of a special instrument.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a set of primers for detecting Pygocentrus nattereri and their applications. Background Art

[0002] Pygocentrus nattereri, also known as red-bellied piranha, Serrasalmus nattereri, red-bellied water tiger, etc., belongs to the phylum Chordata, class Osteichthyes, order Characiformes, family Characidae, genus Pygocentrus. It is native to the Amazon River Basin in South America. Currently, it is distributed in Brazil, Argentina, Bolivia, Colombia, Paraguay, Peru, the United States, and Bangladesh. In the early 1980s, Pygocentrus nattereri was developed as an ornamental fish and introduced into China. Currently, it has invaded the waters of Zhejiang, Jiangsu, Fujian, Guangdong, Guangxi and other places in China, and there is a tendency of further expansion. Pygocentrus nattereri has highly developed hearing, extremely sharp teeth, a strong lower jaw, and will not let go after biting its prey, and tears the meat off by twisting its body. Piranhas often appear in groups. During the dry season, when the water area becomes smaller, they will gather in groups to attack animals passing through this water area, seriously affecting the biodiversity of the invaded area. Due to the small number of individuals in the early stage of species invasion, it is difficult to detect invasive species in the early stage. Environmental DNA comes from the genetic material of species living in the environment and can more comprehensively represent the biodiversity information in the environment.

[0003] Currently, most of the most commonly used nucleic acid detection methods for environmental DNA are based on the PCR technology. However, since the PCR technology is temperature-variable amplification, it has very high requirements for technology, instruments, sites, personnel, etc., and is not suitable for on-site rapid detection. The isothermal nucleic acid amplification technology emerged to fill this technical gap. The recombinase polymerase technology, i.e., the RPA technology, is a commonly used nucleic acid isothermal amplification technology. The RPA reaction mainly relies on recombinase and single-stranded DNA binding protein. The enzyme binds to the primer to form a protein-DNA complex, and searches for the corresponding homologous sequence in the double-stranded nucleotide chain. Once the homologous sequence is located, the single-stranded binding protein will bind to the replaced nucleotide chain. At the same time, DNA synthesis begins. The RPA reaction is similar to the PCR reaction, but does not require high-temperature denaturation or amplification reaction. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a set of primer-probe groups for detecting Pygocentrus nattereri, and provide corresponding kits and applications. The detection method used in the present invention only requires a simple constant-temperature device to complete nucleic acid detection, without complex temperature-variable instruments and fluorescence detection devices. The operation is simple, the result reading is convenient, and it is convenient for on-site and grass-roots rapid detection of invasive species.

[0005] The technical solution of the present invention to solve the above problems is as follows:

[0006] In the first aspect, a set of primer-probe groups for detecting the DNA of Pygocentrus nattereri in the environment is provided. The primer-probe groups include 3 groups, and their sequences are shown in SEQ ID NO.1-9 respectively.

[0007] Furthermore, the sequences of 2 groups of primer groups and their corresponding amplification intervals are as follows:

[0008] The sequence of the first group of primer groups is shown in SEQ ID NO.1-2, and it is used to amplify the interval of 15121-15376 on the mitochondrion;

[0009] Its corresponding probe sequence is shown in SEQ ID NO.3;

[0010] The sequence of the second group of primer groups is shown in SEQ ID NO.4-5, and it is used to amplify the interval of 14488-14766 on the mitochondrion;

[0011] Its corresponding probe sequence is shown in SEQ ID NO.6.

[0012] The sequence of the second group of primer groups is shown in SEQ ID NO.7-8, and it is used to amplify the interval of 10350-10613 on the mitochondrion;

[0013] Its corresponding probe sequence is shown in SEQ ID NO.9.

[0014] Among them, SEQ ID NO.1, SEQ ID NO.4, and SEQ ID NO.7 are the forward primers (F) in each group of primer groups, and SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO.8 are the reverse primers (R) in each primer group.

[0015] Furthermore, preferably, the reverse primers SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO.8 and the probe sequences SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO.9 are modified by one or more of biotin, digoxin, and fluorescein.

[0016] Furthermore, preferably, the probe sequences SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO.9 are modified by fluorescein.

[0017] Furthermore, preferably, fluorescein includes, but is not limited to, fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives.

[0018] Further, preferably, the fluorescein is FAM dye.

[0019] Further, preferably, the reverse primer sequences SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8 are labeled with biotin.

[0020] In a second aspect, a kit for detecting the invasive species, red-bellied piranha (Pygocentrus nattereri) in the environment is provided, and the kit contains the above primer set and probe set.

[0021] Further, the kit contains DNA polymerase and its buffer. Preferably, the DNA polymerase is Bsu polymerase.

[0022] Further, the kit includes a nucleic acid recombinase and a single-stranded binding protein.

[0023] Further, the kit includes an exonuclease. Preferably, the exonuclease is exonuclease IV.

[0024] Further, the kit contains a negative control and a positive control. Preferably, the negative control is enzyme-free and sterile water; preferably, the DNA positive control is the amplification sequence corresponding to the primer set.

[0025] Further, the kit contains a test strip. Preferably, the test strip includes, but is not limited to, a sample loading part, a chromogenic substance loading part, a chromatography capture component, and a water absorption component.

[0026] Further, preferably, the colored nanoparticles in the sample loading part of the test strip are colloidal gold particles.

[0027] Further, preferably, the conjugate in the chromogenic substance loading part of the test strip is one or more of streptavidin or fluorescein monoclonal antibody.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] The primer-probe set and the kit for detecting red-bellied piranha provided by the present invention can complete on-site detection only with a simple constant temperature device. The operation is simple, the result reading is convenient, and it has the characteristics of strong specificity, high accuracy, high sensitivity, etc. Description of the Drawings

[0030] Figure 1: Electrophoresis result diagram of the availability test of 3 primer pairs in Example 2, which is a positive test result diagram. In the figure, Marker: DNA Marker; Blank (negative control): DNA is replaced with sterile and enzyme-free water, and the three primer sets are used; 1: The primer sets SEQ ID NO.1, SEQ ID NO.2; SEQ ID NO.4, SEQ ID NO.5; SEQ ID NO.7, SEQ ID NO.8 are used, and the DNA of Serrasalmus nattereri is used as shown in the figure. The 3 primer pairs successfully amplified the DNA of Serrasalmus nattereri and there is no band in the negative control. Figure 2 : Test result diagram of the amplification specificity of 3 primer pairs for related species and common species of the target species in Example 2. The specificity test includes the related species of Serrasalmus nattereri, Colossoma brachypomum, and 19 common freshwater fish species. In the figure, Marker: DNA Marker; Positive control: DNA of Serrasalmus nattereri, and the primer sets SEQ ID NO.1, SEQ ID NO.2; SEQ ID NO.4, SEQ ID NO.5; SEQ ID NO.7, SEQ ID NO.8 are used; The DNA of related species and 19 common freshwater fish species uses the primer sets SEQ ID NO.1, SEQ ID NO.2; SEQ ID NO.4, SEQ ID NO.5; SEQ ID NO.7, SEQ ID NO.8. The 3 primer pairs did not successfully amplify the DNA of related species and 19 common freshwater fish species and there is a band in the positive control.

[0031] Figure 3 : Test result diagram of the availability of the primer-probe combination for detecting the target species in Example 2. Blank (negative control): DNA is replaced with sterile and enzyme-free water, and the three primer sets are used; 1: The primer-probe sets SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3; SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6; SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 are used. The results are shown in the figure. All 3 primer-probe sets successfully amplified the DNA of Serrasalmus nattereri and there is no band in the negative control. Figure 4: Test result graph of the primer-probe combination for the target species *Serrasalmus rhombeus* in Example 2. Positive control: *Serrasalmus rhombeus* DNA, using primer-probe sets SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3; SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6; SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9; DNA of the related species *Colossoma brachypomum* and 19 common freshwater fish species using primer-probe sets SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3; SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6; SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9. The results are shown in the figure. Bands appeared when using the primer-probe sets SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 with the DNA of *Colossoma brachypomum* and 19 common freshwater fish species, while no bands appeared with the primer-probe set SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and the DNA of *Serrasalmus rhombeus* was successfully amplified.

[0032] Figure 5 : Test effect graph of the primer-probe combination for the target species *Serrasalmus rhombeus* on environmental DNA samples in Example 3. Blank (negative control): DNA was replaced with sterile and enzyme-free water. Using the primer-probe set SEQ ID NO 4-6 for testing, obvious bands appeared after adding the pre-prepared environmental DNA, and no bands appeared in the negative control.

[0033] Figure 6 : Test effect graph of the primer-probe combination for the target species *Serrasalmus rhombeus* on gradient-diluted environmental DNA samples in Example 4. Using the primer-probe set SEQ ID NO 4-6 for testing, the concentration of undiluted DNA was 22 ng / μl. From left to right, they were diluted 1×10 2 、5×10 2 、1×10 3 、1×10 5 times, and detection was still possible after dilution 1×10 5 times. Therefore, the sensitivity of this primer-probe pair was 2.2 ng / ml. Detailed implementation method

[0034] Example 1: Design of primers and probes

[0035] Using the mitochondrial genome sequences of Pygocentrus nattereri (NC_015840.1), Acipenser gueldenstaedtii (NC012576.1), African pike (Acipenser gueldenstaedtii, NC 012576.1), white sturgeon (Acipensergueldenstaedtii, NC 012576.1), bowfin (Acipenser gueldenstaedtii mitochondrion, NC 012576.1), Myloplus malabaricus (Acipenser gueldenstaedtii, NC 012576.1), paddlefish (Acipenser gueldenstaedtii, NC 012576.1), Pseudoscaphirhynchus fedtschenkoi (Acipensergueldenstaedtii, NC 012576.1), Bryconamericus stramineus (Acipenser gueldenstaedtii, NC012576.1), Sterlet (Acipenser gueldenstaedtii, NC 012576.1) and Megaleporinus macrocephalus (Acipenser gueldenstaedtii, NC 012576.1) publicly available in the NCBI Gene Bank of the National Center for Biotechnology Information, USA as reference sequences for target species.

[0036] The designed primer sets should meet the following requirements simultaneously:

[0037] 1) They cannot amplify other characidae fish species except the target species simultaneously.

[0038] 2) There are more than 3 bp differences between the target species and all background species in the primer binding region, and one of the following situations is satisfied:

[0039] The last 1 bp at the 3' end is different;

[0040] 2 out of the last 4 bases at the 3' end are different;

[0041] 3 out of the last 6 bases at the 3' end are different.

[0042] 3) The size of the amplified fragment is about 250 bp - 300 bp;

[0043] 4) The size of the primers is 30 bp - 35 bp;

[0044] 5) The GC content is 30% - 70%;

[0045] 6) Hairpin, < 40;

[0046] 7) polyX < 7.

[0047] 8) A modification group (commonly biotin) is labeled at the 5'-end of the downstream primer

[0048] The designed probe should meet the following requirements:

[0049] 1) Select a sequence with a length of 46 - 52 bp complementary to the target fragment between the upstream and downstream primers as the probe;

[0050] 2) The sequence should avoid palindromic sequences, internal secondary structures, and consecutive repeated bases;

[0051] 3) A antigen label (typically FAM) is modified at the 5'-end;

[0052] 4) A dSpacer (tetrahydrofuran, THF) is labeled at a sequence position approximately 30 nt from the 5'-end as the recognition site for nfo;

[0053] 5) THF is approximately 15 nt away from the 3'-end, and a modification group such as an amino group, a phosphate group, or a C3-spacer is labeled at the 3'-end.

[0054] Based on the above principles, the present invention has designed 3 groups of primer-probe sets as shown in the sequence listing. The present invention has no special restrictions on the sources of the primers in the designed primer sets, and the well-known primer sources in the art can be used. In the embodiments of the present invention, the primer sets were commissioned to be synthesized by Genewiz Suzhou Co., Ltd.

[0055] Example 2: Availability and Specificity Test of Primer and Probe Sets

[0056] To verify the amplification effect of the primer sets in the present invention on the target species, in this example, 3 pairs of primer sets were respectively used to amplify the DNA of the target species, and the results are shown in the appendix Figure 1 And in the appendix Figure 2 as shown.

[0057] Furthermore, to verify the specificity of the primer set of the present invention, 19 common freshwater fish species and closely related species of Colossoma brachypomum were selected as background species in this example. The 19 common freshwater fish species include: Channa argus, Paramisgurnus dabryanus, Hypophthalmichthys molitrix, Xenocypris argentea, Hypophthalmichthys nobilis, Culteralburnus, Rhodeus ocellatus, Cyprinus carpio, Paracanthobrama guichenoti, Tachysurus fulvidraco, Saurogobiodumerili, Pseudorasbora parva, Abbottina rivularis, Abbottina rivularis, Micropercops swinhonis, Carassius auratus, Ctenopharyngodon idella, (Hemiculter leucisculus) and Mylopharyngodon piceus.

[0058] The results are shown in Appendix Figure 3 and Appendix Figure 4 as shown.

[0059] Example 3: Detection of Pygocentrus nattereri Based on Environmental DNA

[0060] 1) Filter the water sample using a mixed cellulose ester membrane with a pore size of 0.45

[0061] 2) Cut the filter membrane into pieces, then add 500 μL of 10% chelex-100 solution and 20 μL of proteinase K solution into a centrifuge tube, incubate at 56 °C for 10 minutes using a portable metal bath, then adjust the temperature of the metal bath to 99 °C (maintain for 15 minutes during the process of heating up to 99 °C), and then centrifuge for 10 minutes using a portable small centrifuge. After centrifugation, take the supernatant for the next experiment.

[0062] 3) Configure the RPA system, where there are 29.4 μL of A buffer, 2 μL each of 10 μM upstream and downstream primers, 0.6 μL of 10 μM probe, and 2 μL of DNA template. After mixing each component by oscillation, add 2.5 μL of B buffer and 11.5 μL of sterilized deionized water. Vortex and centrifuge the mixed system. After incubating in a metal bath at 38°C for 4 min, take out the reaction system, vortex and centrifuge for 30 s, and then put it back into the 38°C metal bath for continued reaction for 8 min.

[0063] 4) After the reaction is completed, aspirate 10 μL of the reaction solution, add 190 μL of sterile deionized water, vortex and centrifuge. After mixing, aspirate 50 μL of the diluted solution and drop it on an LFD test strip labeled with anti-FAM and Biotin for detection.

[0064] Example 4: Sensitivity of the primer set

[0065] Furthermore, for RPA detection, in this example, the primer-probe pair will be used to test gradient-diluted DNA. The test results are shown in the appendix Figure 6 as follows. Using the primer-probe set of SEQ ID NO 4-6 for testing, the concentration of undiluted DNA is 22 ng / μl. From left to right, they are diluted 1×10 2 , 5×10 2 , 1×10 3 , 1×10 5 times, and the dilution of 1×10 5 times can still be detected. Therefore, the sensitivity of this primer-probe pair is 2.2 ng / ml.

Claims

1. A primer and probe set for detecting the invasive species red-bellied trout characin in water based on environmental DNA, characterized in that: The primer set and probe set include 3 sets, and the sequences of the 3 sets of primer probe sets are shown as SEQ ID NO.1-9.

2. The primer probe set according to claim 1, characterized in that: The 2 sets of primers and their corresponding regions are: The sequences of the first primer set are shown in SEQ ID NO.1 and SEQ ID NO.2, and are used to amplify the 15121-15376 interval in the mitochondria of the red-bellied characin; The probe sequence corresponding to the first primer set sequence is shown in SEQ ID NO.3; The sequences of the second primer set are shown in SEQ ID NO.4 and SEQ ID NO.5, which are used to amplify the 14488-14766 interval in the mitochondria of the red-bellied characin; The probe sequence corresponding to the sequence of the second primer set is shown as SEQ ID NO.

6. The sequences of the third primer set are shown in SEQ ID NO.7 and SEQ ID NO.8, which are used to amplify the 10350-10613 interval in the mitochondria of the red-bellied characin; The probe sequence corresponding to the sequence of the third primer set is shown in SEQ ID NO.

9.

3. The primer set as claimed in any of claim 1 is used in any of the following applications: (1) Identify or assist in the identification of red-bellied salmonids; (2) Prepare products for identification or auxiliary identification of red-bellied salmon characin.

4. A kit for detecting invasive red-bellied characin in water based on environmental DNA comprises the primer probe set described in claim 1.

5. The kit according to claim 4, characterized in that Including but not limited to a test strip consisting of a sample-carrying part, a color-developing substance-carrying part, a chromatographic capture part, and a water-absorbing part.

6. The kit according to claim 4, characterized in that The label of the primer or probe can be one or more of biotin, digoxigenin, and fluorescein.

7. The kit according to claim 4, characterized in that The fluorescein can be fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives.

8. The kit according to claim 4, characterized in that The colored nanoparticles in the sample-carrying portion of the test strip include but are not limited to colloidal gold particles.

9. The kit according to claim 4, characterized in that The colorimetric material-carrying part conjugate includes, but is not limited to, one or more of streptavidin, digoxigenin antibody or fluorescein monoclonal antibody.

10. The kit according to claim 4, characterized in that Test strips are not limited to having only a single test line.

11. The kit according to claim 4, characterized in that The kit components should also include positive quality control products and negative quality control products.

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