CN115992268A - Research method and detection kit for coral co-attached archaea diversity - Google Patents
Research method and detection kit for coral co-attached archaea diversity Download PDFInfo
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Abstract
The invention discloses a research method and a detection kit for coral co-attached archaea diversity. The invention provides a method for researching archaea diversity by NestedPCR or Semi-NestedPCR and a detection kit, which comprise 2 sets of primer groups, 2 rounds of PCR reaction systems and amplification flows. The invention is used for constructing, sequencing and analyzing the diversity of the archaea 16SrDNA V3, V4 and V5 variable region libraries; the method can detect archaea of all doors simultaneously, effectively shield the influence of coral, chrysophyta, various eukaryotes, viruses, most bacteria and the like, and remarkably increase the content of archaea, classification units and the like; can be used for environmental samples such as bottom mud, water body and the like and various biological samples, and is especially suitable for the study of biological co-attached archaea such as coral, sponge, sea anemone, calcareous algae and the like. The method can comprehensively, accurately and rapidly study the archaea diversity, and has the advantages of strong specificity, high sensitivity, good repeatability, simple operation, high sample fidelity and wide applicability.
Description
Technical field:
the invention belongs to the field of microbial molecular ecology detection, and relates to a method and a detection kit for researching diversity of co-attached archaea of coral, which are used for researching diversity of co-attached archaea of environment archaea and complex samples such as coral, sponge, sea anemone, seaweed and the like.
The background technology is as follows:
archaea, one of the earliest life forms, is widely distributed in various environments or co-produced in organisms. Archaea is a great variety of species, extremely abundant in diversity, and is of great importance in the bio-geochemistry process, and is an important driver of elements, substances and energy on the earth. Archaea is one of the characteristics of current bioelectrochemical research, and related research has important significance for clarifying the law of life activities, revealing the evolution of life origins and species, the interaction of organisms with organisms or habitats and the like. However, since the metabolic mechanism and growth conditions of archaea are still not completely understood, the species of archaea isolated and cultured in pure form are not so far many, and the related studies are limited. Pure cultured archaea can be comprehensively studied through genome, transcriptome, proteome, lipidome, metabolome and other histology technologies, and related researches on non-pure cultured archaea DNA are mainly focused on metagenome, gene library (high throughput sequencing library, clone library and the like), fingerprint spectrum technology such as Denaturing Gradient Gel Electrophoresis (DGGE), real-time PCR (Real-time PCR), functional genes, in-situ research, bioinformatics analysis based on databases and the like. Although advances in molecular biology technology and the creation of large databases provide much basic data for archaea research, archaea research still has many drawbacks, such as quality of extracted genomic DNA, limitations in designing primers, missing some key categories (even universal primers are unilateral), probe specificity, PCR product length and region, cloning efficiency, etc.
Coral reefs are one of the ecological systems with highest biodiversity and primary productivity on the earth, reef building corals are "frame" organisms in the coral reef ecological system, and play a decisive role in maintaining the health and stability of the coral reef ecological system and in material circulation and energy circulation. The archaea is in carbon of coral reef ecosystem,Has important ecological function in the biological geochemical process of nitrogen, sulfur and the like
et al 2015; vanwonterghem and Webster,2020; campos et al 2022). The archaea content of co-produced microorganisms in coral symbiota other than yellow algae varies from 1% up to about 50% depending on the coral species (Wegley et al 2007;Siboni et al, 2008;Blackall et al, 2015), an important functional community for coral symbiota. Currently, due to the technical limitations of archaea research, coral co-attachment archaea research is only small in archaea diversity (Siboni et al, 2008;Bourne et al, 2016), such as clone library, fingerprint, probe in-situ research, etc., the archaea diversity detected by high-throughput sequencing is relatively single and the content of archaea in the total obtained sequence is not high (Bourne et al, 2016; zhou et al, 2017;Huggett and Apprill,2019). Besides coral host, coral also comprises yellow worm algae, co-attached microorganisms such as bacteria, archaea, fungi, miniature eukaryotes, viruses and the like, and the components are complex and difficult to separate absolutely, so that the total DNA of coral contains the genomic DNA of coral and yellow worm algae occupying the main content and the genomic DNA mixture of other co-attached organisms, various interferences are more, and the study is relatively clear mainly of yellow worm algae and bacteria.
At present, archaea diversity studies are mainly performed on 16S rDNA high-throughput sequencing, and primers constructed by the reported library are not strong in universality, but can not amplify all the archaea in the current large-scale database (such as GenBank, silva, greengene, RDP (ribosomal RNA data base project) and the like). Archaea has special properties, and is similar to eukaryotes in terms of genetic information transfer and is similar to prokaryotes in terms of metabolic processes such as productivity and the like. Therefore, in complex samples, the archaea diversity study is easily interfered by other organisms, particularly eukaryote genomes, besides the lack of a universal strong primer for amplifying most archaea; the final archaea sequence of the complex sample of coral is far lower than that of bacteria, and the real archaea information can not be obtained by researching the conventional universal primer capable of amplifying bacteria and archaea. Based on the above problems, a method for detecting the archaea diversity in complex samples such as corals with strong specificity, sensitivity and high efficiency is needed to obtain higher archaea coverage and more real archaea diversity information.
The invention comprises the following steps:
the invention aims to solve the problems of the existing 16S rDNA high-throughput sequencing research on archaea diversity, and provides a method for researching archaea diversity and a rapid detection kit, wherein the method has strong primer universality and high archaea information coverage rate.
The invention provides the following technical scheme:
the first object of the present invention is to provide a primer set for rapidly detecting archaea diversity, wherein the primer set comprises Seq F1, seq F2, seq R1 and Seq R2, and the primer set is specifically as follows:
seq F1:5'-GCMCTAYGGKGYGCASCAGK-3'; as shown in SEQ ID NO. 1.
Seq F2：5’-XXXXXXGYMGCCRCGGKAAHAS-3’；
Seq R1:5'-TTCGGRSCRTRCNGACCTRCCG-3'; as shown in SEQ ID NO. 2.
Seq R2：5’-XXXXXXNYRTACTYCCCARGYRG-3’。
Wherein the degenerate primer codes are N (A, T, C, G), M (A, C), Y (C, T), K (G, T), S (G, C), R (A, G), W (A, T), H (A, T, C), B (G, T, C), respectively.
Preferably, the peripheral primer set comprises an upstream primer Seq F1 or Seq F2, a downstream primer Seq R1; the inner primer set comprises an upstream primer Seq F2 and a downstream primer Seq R2.
Further, the primer is a nucleotide fragment synthesized by chemistry. The Index sequence consisted of 6 bases, "XXXXXX", which was used to identify each sample 16S rDNA sequence; the following conditions are satisfied at the same time: the Index sequences of the primer sets of Seq F2 and Seq R2 of each set cannot be identical, the Index sequences of the primer sets of each set may be identical at one end, and the primer sets of each set cannot be identical for 2 indices and only are not identical in sequence.
A second object of the present invention is to provide a method for studying the diversity of co-archaea coral, which comprises extracting the total DNA of the genome of a sample, and performing a PCR reaction with the above-mentioned primer set, wherein the PCR reaction comprises a Nested PCR (Nested PCR) or a Semi-Nested PCR (Semi-Nested PCR) of 2 rounds of PCR reactions.
Preferably, the peripheral primer group is selected as the Seq F1 and the Seq R1 in the first round of PCR, and the inner peripheral primer group is selected as the Seq F2 and the Seq R2 in the second round of Nested PCR; or the inner peripheral primer set used in the second round of Semi-close PCR when the outer peripheral primer set of the first round of PCR is Seq F2 and Seq R1 is Seq F2 and Seq R2; since the inner primer sets were identical, the second round of Nest PCR was identical to the Semi-nested PCR amplification products.
Preferably, the method comprises the following steps:
step 1, extracting total DNA of a sample genome;
step 2, taking sample genome DNA as a template, selecting peripheral primer groups as Seq F1 and Seq R1 in the first round of PCR, and carrying out the second round of PCR by taking the first round of PCR products (diluted 10-20 times) as templates, wherein the second round of Nested PCR uses inner peripheral primer groups as Seq F2 and Seq R2; or taking the sample genome DNA as a template, taking the first round of PCR peripheral primer groups of Seq F2 and Seq R1 and taking the first round of PCR products (diluted 10-20 times) as the template to perform the second round of Semi-close PCR, and taking the inner peripheral primer groups of Seq F2 and Seq R2;
step 3, performing gel cutting purification on the PCR amplification products of the second round, constructing a library and sequencing;
and 4, bioinformatics and statistical analysis.
Further, the step 1 is to extract the total DNA of the genome of the sample by adopting a conventional CTAB method or a sediment DNA extraction kit.
Further, the second round of Nested PCR or Semi-close PCR amplification products can be purified and used directly for library construction and sequencing.
Further, the first round PCR reaction system comprises: 1 xEx Taq Buffer (Mg 2+ plus), 100-200 mu M dNTPs (2.5 mM each), 0.4-1.2 mu M final concentration of upstream and downstream primers, 0.025-0.04U/. Mu.L of Ex Taq Hs enzyme, and ddH 2 O quantifying each component to the final concentration, adding a sample genomic DNA template<10ng/μL。
Further, the first round of PCR program is 94-98 ℃ for 5min; (94-98 ℃ for 30s,65-68 ℃ for 15-30s,72 ℃ for 1 min) multiplied by 10-15cys; and at 72℃for 5min.
Further, the second round of Neted PCR or Semi-aged PCR reaction system: 1 xEx Taq Buffer (Mg 2+ plus), 100-200 mu M dNTPs (2.5 mM each), 0.4-1.0 mu M of final concentration of upstream and downstream primers, 0.025-0.04U/. Mu.L of Ex Taq Hs enzyme, and ddH 2 And O quantifying each component to the final concentration, taking a second round of PCR reaction system after the first round of PCR is finished, and adding the first round of PCR product diluted by 10-15 times as a template.
Further, the second round of Neted PCR or Semi-closed PCR reaction procedure: 94-98 deg.c for 5min; (94-98 ℃ for 30s,59-64 ℃ for 15-30s,72 ℃ for 30 s) x 20-25cys; and at 72℃for 5min.
When the number of the first PCR cycle is 10-15 and the number of the second PCR cycle is 20-25, the higher and more real archaea information coverage rate is obtained, the bioinformatics and statistical analysis of the sequencing result are facilitated, and the community structure and the abundance, alpha diversity and Beta diversity of the coral coanda archaea are determined.
The third object of the invention is to provide a kit for studying the diversity of coral co-attached archaea, which comprises the primer groups of Seq F1, seq F2, seq R1 and Seq R2, the 2-round PCR reaction system (except for a DNA template) and corresponding programs.
The invention provides application of the primer group and the research method or the kit to the diversity detection of archaea, and the primer group and the research method or the kit are used for the diversity research of the biological co-attached archaea of environmental archaea and complex samples such as coral, sponge, sea anemone, seaweed and the like.
Compared with the prior art, the invention has the following advantages: the invention can not only detect each archaea at the same time, but also effectively shield the influence of coral, yellow algae, various eukaryotes, viruses, most bacteria and the like, and the coral coanda archaea can be obviously increased; can be used for the environment such as bottom mud, water body and various biological samples, and is especially suitable for the study of biological co-attached archaea such as coral, sponge, sea anemone, calcareous algae and the like. The method can comprehensively, accurately and rapidly study the archaea diversity, and has the advantages of strong specificity, high sensitivity, good repeatability, simple operation, high sample fidelity and wide applicability.
Description of the drawings:
FIG. 1 shows the results of agarose gel electrophoresis of the second round of Nest-PCR amplification products of the 16S rDNA of three-bay 2 coral co-attached archaea and marine archaea. M is Marker DL2000, PD1, PD2, PD3 are the cup-shaped coral spring sample of cornu Cervi, PD4, PD5, PD6 are cup-shaped coral summer sample of cornu Cervi, GF7, GF8, GF9 are the cluster helmet shape is spring sample, GF10, GF11, GF12 are the cluster helmet shape summer sample, SW1, SW2, SW3 are the water environment spring sample, SW4, SW5, SW6 represent the water environment summer sample.
FIG. 2 is a bar graph showing the diversity of the three-bay 2 coral co-attached archaea and seawater archaea.
FIG. 3 is a bar graph of archaea community structure and abundance based on 16S rDNA for different sediment samples.
The specific embodiment is as follows:
the following examples are further illustrative of the invention and are not intended to be limiting thereof. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1:
a method for researching diversity of 2 coral coanda archaea in different seasons of three bays. The method comprises the following steps:
1.2 sets of primer sets for quick detection of archaea are synthesized by chemistry. The primers are respectively represented by the sequences of Seq F1, seq F2, seq R1 and Seq R2. Primer design covers the representative 16S rDNA sequence of each classification unit within the family of archaea in Silva and GenBank libraries; the design not only considers the universality of the primers, but also takes the influence of eukaryote genome and prokaryote genome such as coral, yellow worm algae and the like into consideration.
Seq F1：5’-GCMCTAYGGKGYGCASCAGK-3’；
Seq F2：5’-XXXXXXGYMGCCRCGGKAAHAS-3’；
Seq R1：5’-TTCGGRSCRTRCNGACCTRCCG-3’；
Seq R2：5’-XXXXXXNYRTACTYCCCARGYRG-3’。
2. The peripheral primer sets were selected for the first round of PCR as Seq F1 (5 '-GCMCTAYGGKGYGCASCAGK-3') and Seq R1 (5 '-TTCGGRSCRTRCNGACCTRCCG-3'), or Seq F2 (5 '-XXXXGYMGCCGGKAAHAS-3') and Seq R1 (5 '-TTCGGRSCRTRCNGACCTRCCG-3'). The second round of Nested PCR or Semi-close PCR selects the inner primer set Seq F2 and Seq R2 (5 '-XXXXXX NYRTACTYCCCARGYRG-3'); wherein the degenerate primer codes are N (A, T, C, G), M (A, C), Y (C, T), K (G, T), S (G, C), R (A, G), W (A, T), H (A, T, C), B (G, T, C), respectively.
3. The inventor respectively collects antler cup-shaped coral [ Pocillopora damicornis (Linnaeus, 1758) ] and clustered helmet-shaped coral [ Galaxea fascicularis (Linnaeus, 1758) ] samples and water body environment samples in 5 and 8 months of 2020, the water body environment samples are obtained by filtering seawater by using a 0.22 mu m filter membrane and collecting the filter membrane, and the samples are stored by alcohol. Sample numbers are respectively: PD1, PD2, PD3 are the cup-shaped coral spring samples, PD4, PD5, PD6 are the cup-shaped coral summer samples, GF7, GF8, GF9 are the cluster-helmet-shaped spring samples, GF10, GF11, GF12 are the cluster-helmet-shaped summer samples, SW1, SW2, SW3 are the water environment spring samples, and SW4, SW5, SW6 represent the water environment summer samples.
4. The conventional CTAB method extracts the total DNA of the sample genome and uses a Nanodrop 2000 spectrophotometer to determine the concentration.
5. First round PCR reaction System (50. Mu.L) 10 XEx Taq Buffer (Mg) 2+ plus) 5. Mu.L, dNTPs (2.5 mM each) 4. Mu.L, primer set of Seq F1 and Seq R1 each had a final concentration of 0.8. Mu.M, ex Taq Hs enzyme (5U/. Mu.L) 0.3. Mu.L, the above sample genomic DNA template was about 10 ng/. Mu.L, ddH was added 2 O quantifying the total volume to 50. Mu.L; the first round of PCR program is 94 ℃ for 5min; (95 ℃ C. 30s,68 ℃ C. 30s,72 ℃ C. 1 min). Times.12 cys; the first round PCR products were stored at-20℃for 5min at 72 ℃.
6. Second round Nested PCR reaction System (50. Mu.L): 10 xEx Taq Buffer (Mg 2+ plus) 5. Mu.L, dNTPs (2.5 mM each) 4. Mu.L, 0.8. Mu.M final concentration of Seq F2 and Seq R2, 0.3. Mu.L of Ex Taq Hs enzyme (5U/. Mu.L), 5. Mu.L of first round PCR product, ddH addition 2 The total volume was quantified to 50. Mu.L. The second round of Neted PCR (or Semi-closed PCR) reaction program: 94 ℃ for 5min; (95 ℃ C. 30s,59-64 ℃ C. 20s,72 ℃ C. 30 s). Times.23 cys; and at 72℃for 5min.
Nested PCR products (4. Mu.L) were detected by 7.1.2% agarose gel electrophoresis, as indicated by the single band of FIG. 1, at about 400bp. The remaining PCR product (-2.0. Mu.g) was used for target band purification recovery, library construction (TruSeq DNA PCR-Free Sample Preparation Kit, illumina, USA) and high throughput sequencing (Illumina NoveSeq platform), and after quality control of double-ended splice sequences, the Silva database was searched for archaea OTUs species annotation and bioinformatics analysis.
8. The result shows that the coral coanda archaea is detected to be rich in content and high in diversity. The total sequence contains archaea and bacterial 16S rDNA, and the invention effectively shields the influence of eukaryotes and viruses including coral, yellow worm algae, protozoa and the like, and obtains up to 2316 archaea OTUs. The average 44.21% (PD 4, PD5 and PD 6) of the sequence content of the aeolian sequences of the Corallium cervi Roxb is obviously increased compared with the average 31.91% (PD 1, PD2 and PD 3) of the sequence content of the aeolian sequences of the Corallium cervi Roxb in spring; the cluster helmet-shaped coral coanda archaea has stable structure and similar abundance (36.53% in spring and 37.85% in summer); archaea abundance in the water environment varied significantly between spring (27.08%) and summer (49.07%) (table 1). The diversity aspect compares to the conventional method results (2-3 archaea phylum), the invention achieves a significant increase in coral co-epiphyte phylum (up to 76 genera except 13 phylum of primary archaea) as shown in figure 2; the co-attached archaea community of the platycerus cervi coral and the clustered helmet-shaped coral has similar composition, the dominant archaea is nitrosobacteria (Nitrososphaeria) (more than 63%) of the ammoxidation archaea, but the platycerus cervi coral has stronger structural variability and is more active in horizontal transfer with the environment compared with the clustered helmet-shaped coral co-attached archaea community.
TABLE 1 coral co-attached archaea and method for obtaining archaea sequence ratio by using 16S rDNA library of archaea in water environment
Example 2:
a method for researching the diversity of archaea. The method comprises the following specific steps:
1. and collecting sediment samples in different environments. The bottom mud samples were as follows: ZJ1, ZJ2, ZJ3: collecting a bottom mud sample of the mouth of the pearl river (stored for 3 months at normal temperature) at 1 month of 2022; ZJ4, ZJ5, ZJ6: the fresh bottom mud sample of the mouth of the pearl river; d2 D3, D4: a base mud sample of the three-bay coral reef area (-20 ℃ for 2 years); d7 D8, D9: a base mud sample of the three-bay coral reef area (-4 ℃ for 3 months); HF1, HF2, HF3, KRR, DZ: south seafloor mud samples (8 months of normal temperature storage); l23: gantry mountain fault mud (18 months of preservation at normal temperature).
2. Using MP
Spin Kit for Soil the kit is used for extracting the total DNA of the substrate sludge genome, and the concentration is measured by a Nanodrop 2000 spectrophotometer.
3. A first round of PCR amplification was performed on the 16S rDNA V3-V5 region with the primer sets Seq F2 and Seq R1. The reaction system and the procedure were described in example 1 "method for studying diversity of 2 coral co-archaea in different seasons of three bays" using the total DNA of the substrate sludge genome as a template.
4. The first round of PCR products are used as templates, the primer group Seq F2 and the primer group Seq R2 are used for carrying out the second round of Semi-close PCR amplification on the 16S rDNA of the archaea, and the OTUs archaea species annotation and bioinformatics analysis of the obtained product purification and recovery, library construction, high-throughput sequencing and splicing sequences are all referred to the method for researching the diversity of 2 coral coadditive archaea in different seasons of Sanwan in example 1.
5. The results are shown in Table 2, and the archaea diversity information is truly and accurately reflected. The content of the archaea 16S rDNA sequences detected in samples with different preservation modes and preservation time is different (18.20% -95.31%), the sample fidelity is high, and the total number of OTUs obtained from all sediment samples is up to 3012. As shown in FIG. 3, the primer has strong universality and high archaea information coverage rate, different substrate sludge samples contain abundant archaea and have high sensitivity (containing 3 sequences of genus represented by OTUs) in total of 13 gate 76 genus horizontal classification units; archaea in the large Silva database currently contains 14 gates; undetected archaea (Korarchaeota) were mainly present in the extreme hydrothermal zone.
TABLE 2 archaea 16S rDNA V3-V5 region library sequencing archaea ratio conditions of substrate sludge sample archaea
| Sample name | HF1 | HF2 | HF3 | ZJ1 | ZJ2 | ZJ3 | ZJ4 | ZJ5 | ZJ6 |
| Archaea sequence | 14187 | 13100 | 9636 | 17925 | 21037 | 24777 | 29835 | 31066 | 36683 |
| Total sequence | 38487 | 38487 | 38487 | 38487 | 38487 | 38487 | 38487 | 38487 | 38487 |
| Archaea ratio | 36.86％ | 34.04％ | 25.04％ | 46.57％ | 54.66％ | 64.38％ | 77.52％ | 80.72％ | 95.31％ |
| Sample name | KRR | DZ | L23 | D2 | D3 | D4 | D7 | D8 | D9 |
| Archaea sequence | 12641 | 10536 | 13355 | 11838 | 7004 | 7953 | 22192 | 20990 | 25267 |
| Total sequence | 38487 | 38487 | 38487 | 38487 | 38487 | 38487 | 38487 | 38487 | 38487 |
| Archaea ratio | 32.84％ | 27.38％ | 34.70％ | 30.76％ | 18.20％ | 20.66％ | 57.66％ | 54.54％ | 65.65％ |
The above is a further detailed description of the present invention in connection with the preferred embodiments, and it should not be construed that the invention is limited to the specific embodiments described above. All other embodiments, or modifications and improvements in the invention will be apparent to those skilled in the art without departing from the principles and concepts of the invention.
Claims (10)
1. A primer group for rapidly detecting archaea diversity, which is characterized by comprising Seq F1, seq F2, seq R1 and Seq R2, and specifically comprises the following components:
Seq F1：5’-GCMCTAYGGKGYGCASCAGK-3’；
Seq F2：5’-XXXXXXGYMGCCRCGGKAAHAS-3’；
Seq R1：5’-TTCGGRSCRTRCNGACCTRCCG-3’；
Seq R2：5’-XXXXXXNYRTACTYCCCARGYRG-3’。
wherein the degenerate primer codes are N (A, T, C, G), M (A, C), Y (C, T), K (G, T), S (G, C), R (A, G), W (A, T), H (A, T, C), B (G, T, C), respectively.
2. The primer set of claim 1, wherein the peripheral primer set comprises an upstream primer set of Seq F1 or Seq F2, a downstream primer set of Seq R1; the inner primer set comprises an upstream primer Seq F2 and a downstream primer Seq R2.
3. A method for studying diversity of co-archaea coral, which comprises extracting total DNA of a sample genome, and performing a PCR reaction using the primer set of claim 1, wherein the PCR reaction comprises a Nested PCR or a Semi-close PCR of 2 rounds of PCR reactions.
4. The method of claim 3, wherein the first round of PCR uses the set of peripheral primers Seq F1 and Seq R1, and the second round of Nested PCR uses the set of peripheral primers Seq F2 and Seq R2; or the second round of Semi-nestedPCR uses the inner set of primers Seq F2 and Seq R2 when the outer set of primers of the first round of PCR is Seq F2 and Seq R1.
5. A method of investigation according to claim 3, comprising the steps of:
step 1, extracting total DNA of a sample genome;
step 2, taking sample genome DNA as a template, selecting a peripheral primer group as a Seq F1 and a Seq R1 in the first round of PCR, and diluting a 10-20 times of the first round of PCR product as the template to perform a second round of PCR, wherein the second round of Nested PCR uses an inner peripheral primer group as a Seq F2 and a Seq R2; or taking the sample genome DNA as a template, taking the first round of PCR peripheral primer groups of Seq F2 and Seq R1 and taking the first round of PCR products diluted 10-20 times as the template to carry out the second round of Semi-close PCR, and taking the inner peripheral primer groups of Seq F2 and Seq R2;
step 3, performing gel cutting purification on the PCR amplification products of the second round, constructing a library and sequencing;
and 4, bioinformatics and statistical analysis.
6. The method according to claim 5, wherein the step 1 of extracting the total genomic DNA of the sample uses a conventional CTAB method or a substrate sludge DNA extraction kit.
7. The method of claim 4 or 5, wherein the first round of PCR reaction system comprises: 1 xEx Taq Buffer (Mg 2+ plus), 100-200 mu M dNTPs (2.5 mM each), 0.4-1.2 mu M final concentration of upstream and downstream primers, 0.025-0.04U/. Mu.L of Ex Taq Hs enzyme, and ddH 2 O quantifying each component to the final concentration, adding a sample genomic DNA template<10 ng/. Mu.L; the first round of PCR program is 94-98 ℃ for 5min; (94-98 ℃ for 30s,65-68 ℃ for 15-30s,72 ℃ for 1 min) multiplied by 10-15cys; and at 72℃for 5min.
8. The method according to claim 4 or 5, wherein the second round of Nested PCR or Semi-Nested PCR reaction system: 1 xEx Taq Buffer (Mg 2+ plus), 100-200 mu M dNTPs (2.5 mM each), 0.4-1.0 mu M of final concentration of upstream and downstream primers, 0.025-0.04U/. Mu.L of Ex Taq Hs enzyme, and ddH 2 O quantifying each component to the final concentration, taking a second round of PCR reaction system after the first round of PCR is finished, and adding a first round of PCR product diluted by 10-15 times as a template; the second round of Neted PCR or Semi-closed PCR reaction procedure: 94-98 deg.c for 5min; (94-98 ℃ for 30s,59-64 ℃ for 15-30s,72 ℃ for 30 s) x 20-25cys; and at 72℃for 5min.
9. A kit for studying diversity of coral co-attached archaea, which is characterized by comprising the Seq F1, the Seq F2, the Seq R1, the Seq R2, a PCR reaction reagent and a program in the primer set of claim 1.
10. Use of the primer set of claim 1, the research method of claim 3 or the kit of claim 9 for archaea diversity detection.
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