Method for accurately identifying spathiphyllum hylobatidaes by combining environmental DNA (deoxyribonucleic acid) and Taqman probe method

Abstract

The invention discloses a method for accurately identifying spathiphyllum hylobatidaes by combining environmental DNA and a Taqman probe method, and belongs to the technical field of biology.The method comprises the following steps that S1, eDNA to be detected is obtained, and real-time fluorescent quantitative PCR amplification reaction is conducted through spathiphyllum hylobatidaes primers and probes; wherein the sequence of the primer H.lar-F of the spathiphyllum hylobatidaes is as shown in SEQ ID NO.1, the sequence of the primer H.lar-R of the spathiphyllum hylobatidaes is as shown in SEQ ID NO.2, and the sequence of the probe H.lar-P of the spathiphyllum hylobatidaes is as shown in SEQ ID NO.3; and S2, if the detected CT value is less than 40, indicating that the to-be-detected eDNA contains the DNA of the spathiphyllum hylobatidaes. According to the invention, the TaqMan-MGB fluorescent probe with certain specificity and sensitivity is screened out, so that the probe can specifically detect the CT value of a target gene segment, thereby realizing rapid detection and identification on whether eDNA contains the genome DNA of the spathiphyllum hylobatidaes or not, and judging whether the spathiphyllum hylobatidaes inhabit in the environment or not.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, it relates to a method for accurately identifying the white-handed gibbon by combining environmental DNA and Taqman probe method. Background Art

[0002] Environmental DNA (eDNA) technology is a comprehensive detection method for species identification through the biological genetic information remaining in environmental media. Its detection objects cover environmental media such as soil, water, sediment, air, etc., and can also include biological traces carrying genetic material such as biological excrement and exfoliated cells. Based on the biological characteristic that organisms continuously release DNA during environmental activities, this technology realizes non-invasive biodiversity monitoring by capturing and analyzing trace genetic material in environmental samples.

[0003] Environmental DNA (eDNA) is the biological DNA directly obtained from environmental samples and is an important material for analyzing taxonomic information and gene function information related to biological traces in environmental samples. Extracting eDNA from environmental samples and analyzing these DNAs using molecular biology techniques can effectively identify the source species information of biological traces, thereby proving the existence of corresponding species in the sample collection environment. The purpose of eDNA is actually the same as that of traditional animal and plant taxonomic surveys. Compared with traditional morphological survey methods, this technology can break through the limitations of time and space, and reverse-deduce the evidence of the existence of organisms in the ecosystem through laboratory detection, significantly improving the monitoring efficiency of endangered species.

[0004] The white-handed gibbon inhabits tropical / subtropical dense forests and has a relatively small number of individuals. Traditional monitoring methods rely on field sighting records, vocalization monitoring, or infrared camera deployment, and there are three technical bottlenecks: high labor costs, large equipment losses, and long data acquisition cycles. Based on this, the present invention provides a method for accurately identifying the white-handed gibbon by combining environmental DNA and Taqman probe method. Summary of the Invention

[0005] In order to overcome the problems existing in the prior art, the present invention proposes a method for accurately identifying the white-handed gibbon by combining environmental DNA and Taqman probe method.

[0006] To achieve the above object, the present invention is realized by the following technical solutions: A method for accurately identifying the white-handed gibbon by combining environmental DNA and Taqman probe method, comprising the following steps: S1. Obtain the eDNA to be tested and perform a real-time fluorescence quantitative PCR amplification reaction using primers and probes specific to the white-handed gibbon; among them, the sequence of the white-handed gibbon primer H.lar-F is shown in SEQ ID NO.1, the sequence of the primer H.lar-R is shown in SEQ ID NO.2, and the sequence of the probe H.lar-P is shown in SEQ ID NO.3. S2. If the detected CT value is less than 40, it indicates that the eDNA to be tested contains the DNA of the white-handed gibbon.

[0007] Furthermore, the 5' end of the white-handed gibbon probe H.lar-P is modified with FAM, and the 3' end is modified with MGB.

[0008] Furthermore, the reaction system for the real-time fluorescence quantitative PCR amplification reaction in step S1 is as follows: 10 μL of 2xT5 FastqPCR Mix, 1 μL of 10 μM H.lar-F, 1 μL of 10 μM primer H.lar-R, 1 μL of 10 μM probe H.lar-P, 1 μL of DNA template, and ddH2O is used to make up the reaction system to 20 μL.

[0009] Furthermore, the reaction program for the real-time fluorescence quantitative PCR amplification reaction in step S1 is as follows: 95 °C for 2 min, one cycle; 95 °C for 15 s, 60 °C for 30 s, 40 cycles; fluorescence signals are collected after the end of the 60 °C stage in the cycle reaction.

[0010] Through the above technical solutions, the present invention can at least achieve the following beneficial effects: The present invention screens out TaqMan-MGB fluorescence probes with certain specificity and sensitivity, enabling the probes to specifically detect the CT value of the target gene fragment, thereby realizing the rapid detection and identification of whether the eDNA contains the genomic DNA of the white-handed gibbon, and determining whether there are white-handed gibbons inhabiting the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the standard curve of the plasmid standard in Example 2; Figure 2 is the amplification curve of the plasmid standard in Example 2; Figure 3 is the amplification curve for verifying the specificity of the primers and probes in Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0012] Unless otherwise specified, the materials and reagents used in the present invention are commercially available.

[0013] Example 1 Amplification and Identification of Target Samples Using the eDNA of the extract as a template, real-time fluorescence quantitative PCR detection was performed using the primer combination of H.lar-F, H.lar-R and the probe H.lar-P. Among them, the sequence of primer H.lar-F is shown in SEQ ID NO.1, the sequence of primer H.lar-R is shown in SEQ ID NO.2, and the sequence of probe H.lar-P is shown in SEQ ID NO.3; the 5' end of probe H.lar-P is modified with FAM, and the 3' end is modified with MGB. Among them, the primer and probe sequences are shown in Table 1, the reaction system of the amplification is shown in Table 2, the amplification program is shown in Table 3, and the PCR amplification product for obtaining the identification result is obtained after amplification.

[0014] Table 1 Primer and Probe Sequences Table 2 Reaction System Table 3 Reaction Program Example 2 Establishment of Standard Plasmid and Standard Curve Prepare a positive clone sample according to the PCR product, then perform sequencing, select the positive clone sample with completely correct sequence in the sequencing result, and use a plasmid extraction kit to complete the plasmid extraction.

[0015] Calculate the plasmid concentration copy number, and the calculation is as follows: C = A * B -1 x6.02x10 23 In the formula, A is the plasmid concentration (ng / μL), B is the molecular weight of plasmid DNA, and C is copies·μL -1 , and the concentration of the mother liquor is calculated: H.lar standard plasmid (2.75E+10 copies / μL) is used as the experimental plasmid standard Dilute the mother liquor concentration to 2.75E+8 copies / μL with a solvent, then dilute it to 5 gradients according to a concentration multiple of 10, and then use each diluted gradient plasmid standard as a template to perform real-time fluorescence quantitative PCR detection under the reaction conditions of Example 1. Each group of experiments is repeated three times to formulate a standard curve. The detection results of the plasmid standard are shown in Table 4 below, and the standard curve is shown in the appendix Figure 1 as shown, and the relevant amplification curves are shown in the appendix Figure 2 as shown.

[0016] Table 4 Detection Results of H.lar Plasmid Standard Example 3 Verification of Primer and Probe Specificity Each sample was amplified using primers and probes, with each sample repeated three times. The results are shown in Table 5 below, and the amplification curves are as Figure 3 shown.

[0017] Among the sample names: 1 represents the black crested gibbon (Nomascus concolor), 2 represents the eastern black crested gibbon (Nomascus nasutus), 3 represents the white-cheeked gibbon (Nomascus leucogenys), 4 represents the white-handed gibbon Yunnan subspecies (Hylobates lar yunnanensis), 5 represents the northern pig-tailed macaque (Macaca leonina), 6 represents the Assamese macaque (Macaca assamensis), 7 represents the stump-tailed macaque (Macaca arctoides), 8 represents the rhesus macaque (Macaca mulatta), 9 represents the Tibetan macaque (Macaca thibetana), 10 represents the slow loris (Nycticebus bengalensis), 11 represents the pygmy slow loris (Nycticebus pygmaeus), 12 represents the Indochinese gray langur (Trachypithecus crepusculus), 13 represents the Myanmar gray langur (Trachypithecus melamera), 14 represents the capped langur (Trachypithecus pileatus), and 15 represents Shortridge's langur (Trachypithecus shortridgei).

[0018] Table 5 Specificity detection results of primers and probes As shown in Table 5 above, the mixed primers and probes only had amplification reactions with specific species, had good specificity, and the CT values of the three repetitions were close, indicating good repeatability.

[0019] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for accurately identifying Hoolock leucogenys by combining environmental DNA and Taqman probe method, characterized in that: It includes the following steps: S1. Obtain the eDNA to be tested and perform a real-time fluorescence quantitative PCR amplification reaction using the primers and probes for the white-handed gibbon; among them, the sequence of the white-handed gibbon primer H.lar-F is as shown in SEQ ID NO.1, the sequence of the primer H.lar-R is as shown in SEQ ID NO.2, and the sequence of the probe H.lar-P is as shown in SEQ ID NO.3; S2. Detect that the CT value is less than 40, which indicates that the eDNA to be tested contains the DNA of the white-handed gibbon.

2. The method for accurately identifying Hoolock leucogenys by combining environmental DNA and Taqman probe method according to claim 1, wherein: The 5' end of the white-handed gibbon probe H.lar-P is modified with FAM, and the 3' end is modified with MGB.

3. The method for accurately identifying Hylobates lar using environmental DNA and Taqman probe method according to claim 1, characterized in that: The reaction system for the real-time fluorescence quantitative PCR amplification reaction in step S1 is: 2xT5 Fast qPCR Mix 10 μL, 10 μM H.lar-F 1 μL, 10 μM primer H.lar-R 1 μL, 10 μM probe H.lar-P 1 μL, DNA template 1 μL, and ddH2O is used to make up the reaction system to 20 μL.

4. The method for accurately identifying Hylobates lar using environmental DNA combined with Taqman probe method according to claim 1, characterized in that: The reaction program for the real-time fluorescence quantitative PCR amplification reaction in step S1 is: 95°C for 2 min, one cycle; 95°C for 15 s, 60°C for 30 s, 40 cycles; fluorescence signals are collected after the end of the 60°C cycle reaction stage.

Images