Microorganism absolute quantification kit using Cas9 targeted enzyme digestion method

Abstract

The invention discloses a microbe absolute quantification kit using a Cas9 targeted enzyme digestion method, the kit comprises sgRNA and Cas9 molecules, the kit tries to use a Cas9 enzyme digestion technology, and the kit is capable of specifically recognizing sgRNA for indicating an internal reference, and the Cas9 molecules can specifically recognize the sgRNA for indicating the internal reference. The kit is used for replacing gel cutting and recycling operation in a traditional bacterial flora detection method for designing a specific internal reference standard substance, and experiments find that the kit can effectively perform enzyme digestion on an internal reference sequence, increase the proportion of effective sequencing data, avoid influence on quantitative internal reference and ensure the accuracy of absolute quantitative results of microorganisms; compared with a gel cutting recovery method, the Cas9 enzyme digestion method has the advantages that the internal reference data proportion is lower, and the effective data proportion is higher; the correlation between the Cas9 enzyme digestion method and direct sequencing is higher, and the accuracy of a quantitative result is higher, so that the Cas9 enzyme digestion method has the advantages of the direct sequencing method and the gel cutting recovery method.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a microorganism absolute quantification kit, and specifically relates to a microorganism absolute quantification kit using a Cas9 targeted enzyme cleavage method. Background Art

[0002] As a powerful tool for studying differences in microbial diversity and community structure, 16S rDNA amplicon sequencing technology has been one of the important strategies for metagenomic research since its introduction. However, traditional 16S rDNA amplicon sequencing technology can only detect the relative abundance of each taxonomic unit. Due to the technology itself and the analysis method, it is impossible to detect the absolute abundance that is closer to the essence of the truth. This not only limits the application of 16S rDNA amplicon sequencing technology, but also causes erroneous conclusions in certain research scenarios. In response to this situation, researchers have successively developed a series of 16S rDNA amplicon sequencing absolute quantification technologies, mainly including qPCR methods and internal reference sequence methods.

[0003] Based on the qPCR method. After using traditional qPCR to absolutely quantify the total copy number of 16S, the method of calculating the absolute abundance by combining the relative abundance of each taxonomic unit obtained by amplicon sequencing is the current mainstream method and can easily achieve absolute quantification of microorganisms. This method completely separates "amplicon sequencing" from "quantification". The advantage is that the experiment is flexible and the samples can be selectively analyzed for absolute quantification. However, the qPCR absolute quantification technology has high requirements for sample quality and is especially sensitive to PCR inhibitors, which is an unavoidable problem for the main objects of microbial research such as soil / intestinal tract. In addition, this method inevitably prolongs the detection time while ensuring flexibility. At the same time, because sequencing technology and qPCR technology rely on completely different experimental platforms, the hardware requirements for researchers are also high.

[0004] Based on the internal reference sequence method. This type of method adds a certain proportion of internal reference sequence fragments to the sample DNA, and constructs an amplicon sequencing library simultaneously with the microbial 16S rDNA. During the data analysis process, the absolute abundance of each taxonomic unit is calculated based on the absolute copy number of the internal reference sequence fragment. The existing methods based on the internal reference sequence have quantitative inaccuracies, cannot give the upper / lower limit of detection, or have no effective solution to control the addition ratio of the internal reference sequence, which will result in high sequencing costs and failure to meet the expected experimental requirements.

[0005] Therefore, in the patent "Method for detecting bacterial flora composition and absolute content based on internal reference sequences" (patent number ZL201910295389.X), several "quantitative internal references" mixed in a 10-fold gradient ratio are used to achieve a single library single sequencing to obtain the composition and absolute quantitative data of microorganisms in the sample. At the same time, an "indicator internal reference" is introduced to estimate the proportion of the "quantitative internal reference". A method based on capillary electrophoresis is invented to estimate the proportion of the "quantitative internal reference" by the peak area ratio of the fluorescence peak.

[0006] When applying the above patented method to absolute quantitative sequencing of microorganisms, the presence of "indicator internal reference" will occupy part of the sequencing data, resulting in a waste of valid data. When using traditional agarose gel electrophoresis to remove the "indicator internal reference" by gel excision and recovery, due to the polymorphism of the 16S length of the sample microorganism, it is inevitable that there will be 16S amplification products close to the length of the "indicator internal reference" to be excised, which has the risk of introducing deviations; and the gel excision and recovery step has the disadvantages of easy cross-contamination and incompatibility with workstation automation operations.

[0007] The CRISPR-Cas9 system is a natural immune mechanism in bacteria. After being invaded by a virus, bacteria can store fragments of viral genes in their own DNA repeat sequences, namely CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). When they encounter a virus invasion again, bacteria can recognize specific DNA sequences and cut the viral DNA through the Cas9 nuclease, rendering it ineffective. Scientists have developed a common gene editing technology based on the CRISPR-Cas9 system. According to the target gene sequence, a specific single-stranded guide RNA (sgRNA) is designed and delivered to the target cell together with the Cas9 nuclease, which can achieve precise gene editing operations such as gene knockout, insertion or replacement. CRISPR-Cas9 technology has been widely used in the study of specific gene functions, genetic diseases and tumors, and species improvement in the fields of agriculture and microbiology. In the high-throughput sequencing process, CRISPR-Cas9 has also begun to be used to remove nucleic acid sequences other than the research target in the sequencing library, such as removing rRNA sequences in transcriptome sequencing (RNA-seq) and removing mitochondrial sequences in chromatin accessibility sequencing (ATAC-seq). Compared with the gel-cutting recovery method, CRISPR-Cas9 targeted enzyme cutting technology is based on the sequence characteristics of nucleic acids rather than the difference in the length of the target and interference fragments, so it has the advantages of high specificity, good flexibility, and simple operation.

[0008] Although CRISPR-Cas9 has the above advantages, in practical applications, there are still problems such as the efficiency of enzyme cutting being greatly affected by the sgRNA sequence. In the "Method for detecting bacterial flora composition and absolute content based on internal reference sequences", the "indicator internal reference" is used to indicate the content of microorganisms in the sample during the amplification of the 16S region, but at the same time it will occupy additional data in the sequencing stage and needs to be removed during the library preparation process. Conventional gel cutting and recovery operations are cumbersome and greatly affected by factors such as the operator and the diversity of the 16S regions of different microorganisms.

[0009] In response to the above technical problems, the inventors attempted to use Cas9 targeted enzyme cleavage technology for absolute quantitative detection of bacterial community composition (microbial absolute quantification), thereby providing a microbial absolute quantification kit and detection method for specifically removing indicator internal references. By screening out sgRNA with high enzyme cleavage efficiency, the "indicator internal reference" used to estimate the proportion of the "quantitative internal reference" is effectively removed to avoid the problems and risks introduced by gel cutting and recovery, thereby improving the accuracy and stability of the results. Summary of the invention

[0010] The purpose of the present invention is to provide a microbial absolute quantification kit using a Cas9 targeted enzyme cleavage method, which can specifically enzymatically remove the "indicator internal reference" used to estimate the proportion of the "quantitative internal reference", thereby increasing the proportion of effective sequencing data while avoiding affecting the "quantitative internal reference" and ensuring the accuracy of the microbial absolute quantification results.

[0011] In order to achieve the above object, a technical solution provided by a specific embodiment of the present invention is as follows:

[0012] In one aspect, the present invention provides an sgRNA, comprising a target sequence without a PAM sequence and a backbone sequence, wherein the backbone sequence is located at the 3' end of the sgRNA, and the nucleotide sequence of the backbone sequence is shown in SEQ ID NO: 1:

[0013] GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGC UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUUU.

[0014] On the other hand, the present invention also provides a DNA sequence encoding the above-mentioned sgRNA, further comprising a T7 promoter, wherein the T7 promoter is located at the 5' end of the DNA sequence, and the nucleotide sequence of the T7 promoter is shown in SEQ ID NO: 2: TAATACGACTCACTATAGG.

[0015] In one or more embodiments of the present invention, the nucleotide sequence of the DNA sequence is shown in SEQ ID NO: 3:

[0016] AGTCTAATACGACTCACTATAGGCACTTTTCTCCTCCAGAGGTGTTTAA GAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATC AACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.

[0017] In yet another aspect, the present invention also provides a vector comprising the above-mentioned DNA sequence.

[0018] In another aspect, the present invention further provides a CRISPR / Cas9 system for indicating an internal reference in the absolute quantification of Cas9 targeted enzyme cleavage of microorganisms, comprising the above-mentioned sgRNA and a Cas9 molecule, wherein the nucleotide sequence of the indicating internal reference is shown in SEQ ID NO:13.

[0019] On the other hand, the present invention also provides a kit for indicating an internal reference in the absolute quantification of Cas9 targeted enzyme cleavage of microorganisms, comprising the above-mentioned sgRNA, or the above-mentioned DNA sequence, or the above-mentioned vector, or the above-mentioned CRISPR / Cas9 system.

[0020] On the other hand, the present invention also provides the use of the above-mentioned sgRNA, or the above-mentioned DNA sequence, or the above-mentioned vector, or the above-mentioned CRISPR / Cas9 system in absolute quantitative detection of microorganisms.

[0021] In another aspect, the present invention also provides a method for absolute quantitative detection of microorganisms with a Cas9 targeted enzyme cleavage indicator internal reference, comprising the following steps:

[0022] 1) Taking a sample to be tested, extracting total DNA, and obtaining a total DNA sample;

[0023] 2) mixing the total DNA sample with a quantitative internal reference standard, wherein the quantitative internal reference standard includes a quantitative internal reference and an indicator internal reference, to obtain a first mixture, and then subjecting the first mixture to a 16S rDNA PCR amplification reaction to obtain a 16S rDNA PCR amplification product;

[0024] 3) Then, the 16S rDNA PCR amplification product is used as a template to perform subsequencing library amplification to obtain a subsequencing library PCR product, and then the subsequencing library PCR product is standardized to obtain a standardized subsequencing library PCR product, wherein the concentration of the standardized subsequencing library PCR product is preferably 140 ng / 5 μL;

[0025] Preferably, the subsequencing library PCR product is standardized by a method comprising the following steps: adding the subsequencing library PCR product to a LIZ 500-HiDi premix, denaturing the product by a PCR instrument, and then analyzing the product by 3730xl DNA analysis software to obtain the sum of the peak areas of the 16S rDNA and the quantitative internal reference of the sample to be tested, and the peak area of ​​the indicator internal reference, and adjusting the amount of the quantitative internal reference added to the sample according to the peak area of ​​the indicator internal reference;

[0026] 4) adding the standardized subsequencing library PCR product to the CRISPR / Cas9 system to perform enzyme digestion to remove the indicator internal reference, thereby obtaining the subsequencing library PCR product after enzyme digestion, wherein the CRISPR / Cas9 system includes sgRNA and Cas9 molecules; then performing secondary PCR amplification on the subsequencing library PCR product after enzyme digestion, and then performing second-generation sequencing to obtain the 16S rDNA read sequence of the sample to be tested and the read sequence of the quantitative internal reference;

[0027] 5) Then the 16S rDNA read sequence of the sample to be tested and the read sequence of the quantitative internal reference are analyzed to obtain the classification composition and relative abundance information of the bacterial community structure, as well as the number of read sequences of 16S rDNA of each species in the sample to be tested and each quantitative internal reference; and then the total amount of bacteria in the sample to be tested and the content of each type of bacteria are calculated according to the quantitative standard.

[0028] In one or more embodiments of the present invention, the sgRNA includes a target sequence without a PAM sequence and a backbone sequence, wherein the backbone sequence is located at the 3' end of the sgRNA, and the nucleotide sequence of the backbone sequence is shown in SEQ ID NO: 1:

[0029] GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGC UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUUU; the nucleotide sequence of the indicator internal reference is shown in SEQ ID NO: 13;

[0030] Preferably, the DNA sequence encoding sgRNA is as shown in SEQ ID NO: 3:

[0031] AGTCTAATACGACTCACTATAGGCACTTTTCTCCTCCAGAGGTGTTTAA GAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATC AACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT;

[0032] Further preferably, in step 4), the molar ratio of sgRNA:Cas9:subsequencing library PCR product is 10:10:1.

[0033] In one or more embodiments of the present invention, the CRISPR / Cas9 system is assembled by incubating Cas9 and sgRNA at 25° C. for 10 minutes.

[0034] In one or more embodiments of the present invention, in step 4), after obtaining the PCR product of the subsequencing library after enzyme cleavage, the following step is further included: the area of ​​the indicator internal reference removed in the product after enzyme cleavage is calculated by using the ratio of the target peak area before and after enzyme cleavage to the peak area of ​​the indicator internal reference before and after enzyme cleavage, thereby calculating the enzyme cleavage efficiency, specifically calculated according to the following formula I:

[0035] Enzyme digestion efficiency = [1-(A(Af,I)*A(Bf,T)) / (A(Af,T)*A(Bf,I))]*100% Formula Ⅰ

[0036] Among them, the target peak area before enzyme cleavage is A(Bf,T), the indicating internal reference peak area before enzyme cleavage is A(Bf,I), the target peak area after enzyme cleavage is A(Af,T), and the indicating internal reference peak area after enzyme cleavage is A(Af,I).

[0037] In one or more embodiments of the present invention, the quantitative internal reference standard comprises at least three selected from the following group of sequences:

[0038] 1) a first quantitative internal reference sequence R1, wherein the nucleotide sequence of the first quantitative internal reference sequence is shown in SEQ ID NO: 4;

[0039] 2) a second quantitative internal reference sequence R2, the nucleotide sequence of which is shown in SEQ ID NO: 5;

[0040] 3) a third quantitative internal reference sequence R3, the nucleotide sequence of which is shown in SEQ ID NO: 6;

[0041] 4) a fourth quantitative internal reference sequence R4, the nucleotide sequence of which is shown in SEQ ID NO: 7;

[0042] 5) a fifth quantitative internal reference sequence R5, the nucleotide sequence of which is shown in SEQ ID NO: 8;

[0043] 6) a sixth quantitative internal reference sequence R6, the nucleotide sequence of which is shown in SEQ ID NO: 9;

[0044] 7) a seventh quantitative internal reference sequence R7, the nucleotide sequence of which is shown in SEQ ID NO: 10;

[0045] 8) an eighth quantitative internal reference sequence R8, the nucleotide sequence of which is shown in SEQ ID NO: 11;

[0046] 9) A ninth quantitative internal reference sequence R9, the nucleotide sequence of which is shown in SEQ ID NO:12.

[0047] Compared with the prior art, the microbial absolute quantification kit using the Cas9 targeted enzyme cleavage method of the present invention adopts a method different from the traditional detection method to design internal reference standards, attempts to use Cas9 enzyme cleavage technology, designs a sgRNA that can specifically identify the indicator internal reference, and uses it to replace the gel excision recovery operation in the traditional bacterial flora detection method for designing specific internal reference standards, and experiments have found that it can effectively enzyme-cleave the indicator internal reference, increase the proportion of effective sequencing data, and avoid affecting the "quantitative internal reference", thereby ensuring the accuracy of the absolute quantitative results of microorganisms; and compared with the gel excision recovery method, the Cas9 enzyme cleavage method of the present invention has a lower proportion of indicator internal reference data and a higher proportion of effective data; the Cas9 enzyme cleavage method has a higher correlation with direct sequencing, and the accuracy of the quantitative results is higher, which shows that it has the advantages of both direct sequencing and gel excision recovery methods, and the Cas9 enzyme cleavage method of the present invention can control the proportion of indicator internal references in the sample to less than 30%, and the method is stable, reliable and compatible. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 This is a result diagram of checking the absolute quantitative library length distribution and calculating the enzyme cutting efficiency using Peakscan software in Example 1 of the present invention;

[0050] Figure 2 The same absolute quantitative library in Example 3 of the present invention is respectively subjected to direct sequencing, Cas9 enzyme digestion to remove the indicator internal reference, and gel excision recovery to remove the indicator internal reference, and the proportion of the indicator internal reference in the sequencing data;

[0051] Figure 3This is a schematic diagram of the correlation between the proportion of the indicated internal reference in the sequencing data in which the indicated internal reference is removed by the Cas9 enzyme cleavage method and the gel excision recovery method in Example 3 of the present invention, and the proportion of the indicated internal reference in the direct sequencing library data. DETAILED DESCRIPTION

[0052] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0053] Unless otherwise specified, the reagents and materials herein can be purchased from conventional channels.

[0054] Unless otherwise specified, the "enzyme cleavage method" and "Cas9 enzymatic cleavage method" in this article refer to the "absolute quantitative detection method of microorganisms using the Cas9 targeted enzymatic cleavage internal reference sequence of the present invention".

[0055] Unless otherwise specified, the “indicator peak” herein refers to the “peak area of ​​the indicator internal reference”.

[0056] Unless otherwise specified, the "target peak" or "target peak area" in this article refers to "the sum of the peak areas of 16S rDNA of the sample to be tested and the quantitative internal reference", which is separated into "abundance (read sequence) of 16S rDNA of the sample to be tested" and "abundance (read sequence) of the quantitative internal reference" after second-generation sequencing.

[0057] Example 1 Design and synthesis of sequences and preliminary detection of enzyme cleavage efficiency

[0058] 1. According to the prediction of the design software, based on the unique sequence of the "indicator internal reference", the target targeting sequence containing the PAM sequence (NGG) was predicted by the software (such as CHOPCHOP: https: / / chopchop.cbu.uib.no), and based on the indicator internal reference in the patent "Method for detecting bacterial flora composition and absolute content based on internal reference sequence" (patent number ZL201910295389.X), a series of sgRNAs were designed, and through multiple experimental screening, one of the sgRNAs with the highest enzyme cleavage efficiency was selected, which includes a target sequence without a PAM sequence, and a sequence (skeleton sequence) that can form a hairpin structure recognized by Cas9 at the 3' end;

[0059] The nucleotide sequence of the backbone sequence is shown in SEQ ID NO: 1: GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

[0060] 2. Chemically synthesize a plasmid vector containing a sgRNA sequence, which contains a T7 promoter, a target sequence, and a sgRNA backbone structure. The DNA sequence of the sgRNA is:

[0061] AGTCTAATACGACTCACTATAGGCACTTTTCTCCTCCAGAGGTGTTTAAGAGCTATG CTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCCGTTATCAACTTGAAAAAGTGGC ACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 3).

[0062] 3. Then transcribe the vector in vitro to obtain sgRNA.

[0063] 4. According to the method described in the example of the patent "Method for detecting bacterial flora composition and absolute content based on internal reference sequences" (patent number ZL201910295389.X), a microbial absolute quantitative library (standardized subsequencing library PCR product) is prepared, which is an absolute quantitative library obtained by PCR reaction containing the following quantitative internal reference standards. The specific quantitative internal reference standards include quantitative internal references and indicator internal references:

[0064] 1) A first quantitative internal reference sequence R1, the nucleotide sequence of which is shown in SEQ ID NO: 4:

[0065] ACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATTACTAGCTTCGTTTCCCACCAGGATAGTTAGGAGTGCCGACCCGTTATAGAAGTGCAGTGTCCTTTCTCTGCACTCGAGTTAAGTCGACAAGTCCTCTTACGCTAGGACTCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTCATCGCGAGGCTTTATACGAGGCACCAAATAAGCACCGTAATAAGTGAGTCCCGCGGGCTTATTGTGCTGCAGTATAGCTACTATAGCGTAGGGATCGATATCAGCTATACCTAGATGAGAGCCCATTTCCGCTCGATATACCTAGGGACACGTAGATGTACTATTTCGGCGACTTGGATGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATCTACCACATCAGGCACTTGGCTATGAAGACTGCGTAAGCCATTTAGAGTTCGGGCTCCTTCTAAGGCTTAGCAGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCT;

[0066] 2) The second quantitative internal reference sequence R2, the nucleotide sequence of the second quantitative internal reference sequence is shown in SEQ ID NO: 5:

[0067] ACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATAATGCGACGCACGTTAGCAGGCCCTAGTTATTAGCCCGTAGCTTGAAGCACTAGATTCTACGCGGGTTCATCAGCCCAGACCCAACAATGAGGGTCCAATCCATGGCTAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTGTAAGGCGACTTCTCTTATGACCAAAGTGGGCGTCCATGGCTTAGACTCGTGTGGCTCGAACCGAAGTCTTGACGTGATCTCGGGAGGGATGGTCGAGCTACTACCACACTCTCGGCTCAATTACCGTGTGACATCGGATACTCCAACATGGCACGGCGACTGTATTACACGATCCTGGTGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGCGTTCATGATAGGTTCTCGGCAGCTAAAGGACTGCTATCTACTGGGAATAGCTGCCTTGTGACACTGTTCCTTGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCT;

[0068] 3) The third quantitative internal reference sequence R3, and the nucleotide sequence of the third quantitative internal reference sequence is shown in SEQ ID NO: 6:

[0069] ACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATCATCTGGTCAGGTCTCCTCGACCTACCTACATGTGTGGCACGTTCGAGAGCGTGCTTTCGACCAGTATTGGCGCTCGTCCATAGCTAAGCGCCTAACTGCATAGCTACCTCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGGTTAGGCACACATGGCACCGGTCTGCGTTGTGAGCGTACCTTTCACCTCCTATCGCCGTCGTCGGCGTTATAGACAGCACTCCTTCGTTGCTACTACCTGGGTAGGTTAGCACCACGAATCATCCCGACGAAATGACACCGGATCGGGCTGATAGGTGACTCGGAAAGCTGAATCGAGGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGCTTTCCTATGGACTGGACAAGTGCCTCTCCAGCTAACTGCAACAGTCGTGGATCAGGTAATGGTAGAGCTCAGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCT;

[0070] 4) The fourth quantitative internal reference sequence R4, the nucleotide sequence of the fourth quantitative internal reference sequence is shown in SEQ ID NO: 7:

[0071] ACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATTGCGCTCCGAGTATCGACTCAAGCCTCACTAGGAACAGCGGGCGTTTGCACGAACCCTGTTCCGCCGGCTTTGAGTTACTGTCGTGCTGACGCCGTTGAGGCGAGGGTTGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTGAAGGCGTTGCGTTCTTCTCGCTGGACCAGACTCTAATGGCATGTCTCATTCTGTCGTGGCCTGTATTCGCGGAGTGTGTGAGTCTTTGGGCAATGTCAGCAGTGAGCTACCCTGGGAGCAGCCGGACCTCTCTTGAGCGAATGCAGACGGGTTACCGAATCACACCCACCCTCACAGCGGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATAGGCGTTCTGGTGCCCACAATCCTGATCCTGGCATGGGACTACTGTTGGGCGTTATCGAACACACGGAGAGACGTCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCT;

[0072] 5) The fifth quantitative internal reference sequence R5, the nucleotide sequence of the fifth quantitative internal reference sequence is shown in SEQ ID NO:8:

[0073] ACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATTTCGGACCTGTGAGCTATGCTGCGCTATGTGCTTAGGGTCGCCTGGACGTTCACAGGATTCCGTCGTGATGCCCATCTTCGAGGTGTGGTAGCGGACGGACGCCATCCGTCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTGCCGGGTATTGTAGAGACGTCGCACTTACTTGCTCCACCCGACTCGACCCTGTTGGGTTACCGCGGAAAGTTTGGTCGTCCTCTCTGCCATCAGGCGGGATATAGGGAGCTCCGGCAAACGTGGTGCATCCGCAGAGCGGGATGCTGTGGTCAGCTTGGACTTGACGACTGTACCTCAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATACACGGGCTAATCTGTAATCTCCGGTTCCTTGACGCTGCCATGGCGTTTACGACGTTACGGACCACTTGCCAGAACCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCT;

[0074] 6) The sixth quantitative internal reference sequence R6, the nucleotide sequence of the sixth quantitative internal reference sequence is shown in SEQ ID NO: 9:

[0075] ACTGAGACACGGTCCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAAGCTATGCCACAGGTTCGGACGGCTGTTAGTGCGGACTGCGGTCCTTAAAGCCGTCAGCCATCCGTACCGTTAGCTCAGCGTCCCTAGCCTTCGCATCGAACGCGACACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGCTGGCTTGCTATGGAGTTGGATCTCACAGTGTGTGTAACATGGCAGCGCTCCCGATTTCAACAGAGGCCGTTGACGCGCCGATTGAGCCTCCCATGGCATTGGTCGACCATCAACACGAGCTCGTTGGCTGTGCTGAATAGGGTGCGAGCAGCATCTAGGCGGAATTTCCATCGTGGTGTGGGTATCAAACAGGATTAGATACCCTGGTAGTCCACACGGTAAACGATGCTTAGATGACGGGTGCAGATCCTCTAGATTCCACCGCGAATAGGTCCCGTGAACTCTGCTCCCGGTTGGTACGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCGGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCT;

[0076] 7) The seventh quantitative internal reference sequence R7, and the nucleotide sequence of the seventh quantitative internal reference sequence is shown in SEQ ID NO: 10:

[0077] ACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATATTCCGTCAGAGCCGCACATAAGGCCAGCAGGGATGACTAGATATTCCCGCACGCCGACACTGACACTGTCAACGGGTGCAGGTACCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTACTGGGCGTAAAGGGAGCGTAGGTGGATTGCCTAGCCTAGCGGTTGGAGCGGGAATACTAAGGTGCAGTGTTTCCCATGGCCGGATTTAGGCGCTTCACGGGATCGTCGAGTTCGTCTGGGCATAGGTCGCGTCACGCTTTCATTCGAAGCCGATCTCGGACCGCCACCGATAAGGCAGTAGTCGCCTGTGGCATGTGCCTCGCTGAGGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATTGTAAGGTAGCCCGTGTAAGGGTACCTCGCAGATTCGCCAAGAACGAGCAAGGGCTAGTGTGTTGACGGTTACTCTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAGACT;

[0078] 8) The eighth quantitative internal reference sequence R8, the nucleotide sequence of the eighth quantitative internal reference sequence is shown in SEQ ID NO: 11:

[0079] ACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGCCGAAAGGCTGACCCTGAACTCTTGGGATGCGACGTTGAGGGCTGCTGGCATTAGCGAACCGCAATCCCGTACTTGGTAGACAACGAACCCAACACTCCGGAGAGACTGCCTACGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTGTCCGGAATCACTGGGCGTAAAGGGCGCGTAGGCGGTCCGATACGAAACTTCTTGCACAGGCATGAGGCACGCGTGCGTACCAGACGGCCTCGGAATACACCGGAAACCTTTGAGGCCGCTCCCAGGTGTACGCGAGGCACCAAAGCGGTATTCCATGGTAGAGAACACTGCTGCGGATTACCGACATTTAGCCTCGCGTATAGCACCCTGCCGTTGCGTGGGGAGCAAACAGGATTAGATCCCTGGTAGTCCACGCCGTAAACGATGGACAACCACGCGGTTACACGCGGAGATCCCGCCAGATGAGAGCCCAGGCATCACAGCGATCAGGCACTTGACATACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCCAGGCT;

[0080] 9) The ninth quantitative internal reference sequence R9, the nucleotide sequence of the ninth quantitative internal reference sequence is shown in SEQ ID NO: 12:

[0081] TGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGCTAAGAATATTCCGCAATGGACGAAAGTCTGACGTAAATCGCCACCTGGAATCAGGGTGCGCTGTCGTGTGCGGATCGCATGACCGCCAATTCCGTGTAGCAGGGATAGCCTCCCACCTTCGATGATGGCTCGGCATCTGCTCCAACGGCTAATTACGTGCCAGCAGCCGCGGTAACACGTAAGTTGCGAGCGTTGTTCGGAATTATTGAGCGTAAAGGGCATGTAGGCGGTTGTTTGCTCGACATGGTTCGAGCTGGTAGAGATGCGGCCGTCCTAAGAGGAGAGTAGTGCGTCGCAAAGCACCCGGGTCAAGAGCCGGAGTTGACAGCACACCTTGACTCACTCGTATGGCAATAGCAGGACCACATTCGGGTTCGCGCATTACGATTACCACGTGGTCCTTGCTCGACCCGCGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCGCACAGTCAACTATAGGGTGGCTGATGGAGGTAGAGACGACGGACTGCGAGGTGTGGTAGTTTCCTTCCGAGGGTGCACGGTTAGCCCGCAAGGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGGTACGCGAGGAACCTTACCTGGGTT;

[0082] The quantitative internal reference standard also contains the indicator internal reference R0, and the nucleotide sequence of the indicator internal reference (R0) is as shown in SEQ ID NO: 13:

[0083] TGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGCTAAGAATATTCCGCAATGGACGAAAGTCTGACGTAAATCGCCACCTGGAATCAGGGTGCGCTGTCGTGTGCGGATCGCATGACCGCCAATTCCGTGTAGCAGGGATAGCCTCCCACCTTCGATGATGGCTCGGCATCTGCTCCAACGGCTAATTACGTGCCAGCAGCCGCGGTAACACGTAAGTTGCGAGCGTTGTTCGGAATTATTGAGCGTAAAGGGCATGTAGGCGGTTGTTTGCTCGACATGGTTCGAGCTGGTAGAGATGCGGCCGTCCTAAGAGGAGAGTAGTGCGTCGCAAAGCACCCGGGTCAAGAGCCGGAGTTGACAGCACACCTTGACTCACTCGTATGGCAATAGCAGGACCACATTCGGGTTCGCGCATTACGATTACCACGTGGTCCTTGCTCGACCCTCCCTTGTCTCCCTACCTCTGGAGGAGAAAAGTGTTGACATGGGCGCTCCCGGCGCAAGGGCCAAAGGAGTCTCCGATTTCTTATTCCCGAATGACATGCGCGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCGCACAGTCAACTATAGGGTGGCTGATGGAGGTAGAGACGACGGACTGCGAGGTGTGGTAGTTTCCTTCCGAGGGTGCACGGTTAGCCCGCAAGGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGGTACGCGAGGAACCTTACCTGGGTT;

[0084] 5. The standardized absolute quantitative libraries of different samples from the same source were mixed according to equal mass and standardized to 140 ng / 5 μL to obtain a standardized absolute quantitative library (standardized subsequencing library PCR product), and the subsequencing library PCR product was standardized by a method comprising the following steps: adding the subsequencing library PCR product to a LIZ500-HiDi premix, performing a denaturation step on a PCR instrument, and then analyzing with 3730xl DNA analysis software to obtain the sum of the peak areas of 16S rDNA and the quantitative internal reference of the sample to be tested, and the peak area of ​​the indicator internal reference, and adjusting the amount of the quantitative internal reference added to the sample according to the peak area of ​​the indicator internal reference;

[0085] 6. The mixed and standardized absolute quantitative library was subjected to targeted enzyme digestion using a Cas9 kit. The reaction system was prepared according to the table below, incubated at 25°C for 10 minutes, and Cas9 assembly was performed;

[0086] Table 1

[0087]

[0088] 7. After the reaction is completed, add the standardized absolute quantitative library to the system, mix well by pipetting, and incubate at 37°C for 2 hours for enzyme digestion;

[0089] 8. (Optional) After the digestion, take 1 μL of the product before and after the digestion to a new 96-well plate, add 9 μL of LIZ500-HiDi premix and mix well, denature at 95°C for 5 min on a PCR instrument, ice bath for 2 min, and load on a 3730xl DNA analyzer;

[0090] 9. (Optional) Use Peakscan software to check the length distribution of the absolute quantitative library and calculate the enzyme cutting efficiency. Taking the 16S-V3V4 absolute quantitative library as an example, the ratio of the fluorescence peak area of ​​the 581-607bp segment of the product before enzyme cutting (target peak) to the fluorescence peak area of ​​the 706bp internal reference long fragment position (indicator internal reference peak area) is used to calculate the area of ​​the long fragment internal reference removed in the product after enzyme cutting, thereby calculating the enzyme cutting efficiency.

[0091] The enzyme cutting efficiency calculation formula is:

[0092] Enzyme digestion efficiency = [1-(A(Af,I)*A(Bf,T)) / (A(Af,T)*A(Bf,I))]*100% Formula Ⅰ

[0093] Among them, the target peak area before enzyme cleavage is A(Bf,T), and the indicator internal reference peak area before enzyme cleavage is A(Bf,I).

[0094] The target peak area after enzyme cleavage is A(Af,T), and the indicator internal reference peak area after enzyme cleavage is A(Af,I).

[0095] The result is shown in Figure 1. Figure 1 It can be seen that the target peak contains the amplification products of the sample DNA and the "quantitative internal reference", which cannot be distinguished; the indicator peak only contains the amplification products of the "indicator internal reference". After enzyme digestion, the area of ​​the indicator peak is significantly reduced.

[0096] 10. Obtain the PCR product of the subsequencing library after restriction digestion, purify it using 24 μL (0.8x) of DNA purification magnetic beads, and then perform secondary PCR amplification on the PCR product of the subsequencing library after restriction digestion according to the following table;

[0097] Table 2 Amplification reaction system

[0098]

[0099] Table 3 PCR amplification reaction conditions

[0100]

[0101] 11. The PCR product after secondary amplification was purified using 18 μL (0.9×) of DNA purification magnetic beads; the purified product was sequenced by the second generation sequencing (high throughput sequencing) of the Illumina sequencing method to obtain the read sequence of 16S rDNA of the sample to be tested and the read sequence of the quantitative internal reference;

[0102] 12. Then analyze the 16S rDNA read sequence of the sample to be tested and the read sequence of the quantitative internal reference to obtain the bacterial community structure classification composition and relative abundance information, as well as the number of read sequences of 16S rDNA of each species in the sample to be tested and each quantitative internal reference; then calculate the total amount of bacteria in the sample to be tested and the content of each type of bacteria based on the quantitative standard.

[0103] Example 2. Calculation of the efficiency of the internal reference enzyme digestion

[0104] 1. Add the quantitative internal reference and the indicating internal reference prepared by the method in the patent "Method for detecting bacterial flora composition and absolute content based on internal reference sequence" (patent number ZL201910295389.X) to each real sample in Table 7 or 8 below (see Example 1 for details), and PCR amplify the microbial 16S-V3V4 or 16S-V4 region;

[0105] 2. The amplified product was diluted 5 times and subsequencing library amplification (Index PCR) was performed to obtain the subsequencing library PCR product, which was then purified by magnetic beads to obtain a microbial absolute quantitative library containing a quantitative internal reference standard;

[0106] 3. Determine the library concentration using the Qubit fluorescence quantification instrument and standardize it to 140 ng / 5 μL to obtain a standardized absolute quantitative library;

[0107] 4. The sgRNA prepared in Example 1 was then used to prepare the reaction system according to Table 4 below, incubated at 25°C for 10 minutes, and the CRISPR / Cas9 system was assembled:

[0108] Table 4

[0109]

[0110] 5. After the CRISPR / Cas9 system reaction is completed, the standardized absolute quantitative library is added to the reaction system, mixed by pipetting, and incubated at 37°C for 2 hours for enzyme digestion. The enzyme digestion product is then purified using 24 μL (0.8x) of DNA purification magnetic beads;

[0111] 6. Then, the absolute quantitative library before restriction digestion (step 2) and after restriction digestion (step 5) was amplified by PCR reaction. The PCR amplification system and conditions were as follows. The forward primer used in PCR was 5'FAM fluorescently labeled. The absolute quantitative library PCR amplification reaction system was prepared according to the system in Table 5 below, and PCR amplification was performed according to the reaction conditions in Table 6 below.

[0112] Table 5

[0113]

[0114] Table 6

[0115]

[0116] 7. Prepare the LIZ500-HiDi mixture in advance according to the ratio of 10 μL molecular weight internal standard GeneScan-500 LIZ and 890 μL HiDi;

[0117] 8. Take 1 μL of the products before and after the enzyme digestion after PCR amplification in step 6 above to a new 96-well plate, add 9 μL of LIZ500-HiDi premix and mix well, denature at 95°C for 5 min on a PCR instrument, ice bath for 2 min, and put on a 3730xl DNA analyzer;

[0118] 9. Use Peakscan software to view the denatured PCR products in step 8 above, analyze the library length distribution and peak area before and after enzyme digestion, and calculate the digestion efficiency. Taking the 16S-V3V4 absolute quantitative library as an example, the ratio of the fluorescence peak area of ​​the 581-607bp segment of the product before digestion (target peak) to the fluorescence peak area of ​​the 700-702bp internal reference long fragment position (indicator internal reference peak) is used to calculate the area of ​​the excised long fragment indicator internal reference in the digested product, thereby calculating the digestion efficiency. The specific formula is as follows:

[0119] Enzyme digestion efficiency = [1-(A(Af,I)*A(Bf,T)) / (A(Af,T)*A(Bf,I))]*100% Formula Ⅰ

[0120] Among them, the target peak area before enzyme cleavage is A(Bf,T), the indicating internal reference peak area before enzyme cleavage is A(Bf,I), the target peak area after enzyme cleavage is A(Af,T), and the indicating internal reference peak area after enzyme cleavage is A(Af,I).

[0121] The results are shown in Tables 7-8 below.

[0122] Table 7 Calculation of enzyme digestion efficiency of real sample 16S-V3V4 library

[0123]

[0124]

[0125] Table 8 Calculation of enzyme digestion efficiency of real sample 16S-V4 library

[0126]

[0127] It can be seen from Tables 7-8 above that the effect of enzyme digestion to remove the indicator internal reference is extremely good, and the enzyme digestion efficiency basically reaches more than 90%.

[0128] Example 3. Comparison of library construction effects between enzyme digestion and gel excision

[0129] 1. Add the quantitative internal reference and the indicator internal reference prepared by the method in the patent "Method for detecting bacterial flora composition and absolute content based on internal reference sequence" (patent number ZL 201910295389.X) to each real sample in Table 9 below (see Example 1 for details), and PCR amplify the microbial 16S-V3V4 region;

[0130] 2. The amplified product was diluted 5 times, and subsequencing library amplification (Index PCR) was performed to obtain the subsequencing library PCR product, which was then purified by magnetic beads to obtain a microbial absolute quantitative library containing a quantitative internal reference standard, and then standardized to 140 ng / 5 μL to obtain a standardized absolute quantitative library;

[0131] 3. The standardized absolute quantitative library was divided into three parts: one part was directly sequenced; one part was purified by magnetic beads and amplified by PCR after removing the indicator internal reference using the enzyme digestion kit of the present invention (refer to the sgRNA prepared in Example 1, and carried out according to the methods and steps therein) and sequenced; and one part was sequenced after removing the indicator internal reference by agarose gel electrophoresis gel excision recovery and amplification by PCR;

[0132] 4. According to the steps and methods in Example 1, the data volume proportions of quantitative internal references and indicator internal references in the sequencing data of the three methods were counted and compared.

[0133] The results are as follows Figure 2-3 As shown, from Figure 2 It can be seen that both Cas9 enzyme cleavage and gel recovery methods can significantly reduce the proportion of indicator internal references in sequencing data. After Cas9 targeted enzyme cleavage, the proportion of indicator internal references is even lower. Figure 3 It can be seen that compared with the gel excision recovery method, the Cas9 enzyme cutting method has a lower proportion of internal reference data and a higher proportion of valid data. In addition, the Cas9 enzyme cutting method has a higher correlation with direct sequencing, and the quantitative results are more accurate.

[0134] Table 9 Type information of real samples

[0135]

[0136] Example 4. Absolute quantitative library ratio of real sample Cas9 digestion method

[0137] 1. Add the quantitative internal reference and the indicator internal reference prepared by the method in the patent "Method for detecting bacterial flora composition and absolute content based on internal reference sequence" (patent number ZL201910295389.X) (see Example 1 for details) to each real sample in Table 10 below, and PCR amplify the microbial 16S-V3V4, 16S-V4V5 or 16S-V4 region;

[0138] 2. The amplified product was diluted 5 times and subsequencing library amplification (Index PCR) was performed to obtain the subsequencing library PCR product, which was then purified by magnetic beads to obtain a microbial absolute quantitative library containing an internal reference standard;

[0139] 3. The standardized absolute quantitative libraries (subsequencing libraries, Index PCR products) of different samples from the same source were mixed at equal mass, and then standardized to 140 ng / 5 μL according to the method of Example 1 or 2 to obtain a standardized absolute quantitative library mixture;

[0140] 4. The absolute quantitative library mixture is subjected to the Cas9 enzyme digestion method described in the present invention (with reference to the sgRNA prepared in Example 1, and carried out according to the methods and steps therein) to remove the quantitative internal reference, and the reaction product is purified by magnetic beads;

[0141] 5. The purified product is amplified by low cycle PCR and the amplified product is purified by magnetic beads;

[0142] 6. Perform the second generation sequencing, count according to the steps and methods in the embodiment, and compare the data volume ratio of the quantitative internal reference and the indicator internal reference in the sequencing data. The results are shown in Table 10 below.

[0143] Table 10 Types of real samples and test results

[0144]

[0145] As can be seen from Table 10, the real sample test results show that the Cas9 targeted enzyme cleavage technology described in the present invention can be applied to the absolute quantification of microorganisms in various environmental samples such as soil, feces, sludge, water, etc. The library type is compatible with 16S-V3V4, 16S-V4, and 16S-V4V5 regions, and the proportion of internal references can be controlled below 30%. The method is stable, reliable, and has good compatibility.

[0146] The microbial absolute quantification kit using the Cas9 targeted enzyme cleavage method of the present invention attempts to use the Cas9 enzyme cleavage technology to replace gel excision recovery for the absolute quantification method of bacterial flora composition based on the internal reference sequence, designs a sgRNA that can specifically identify the indicator internal reference, and uses it to replace the gel excision recovery operation in the traditional bacterial flora detection method for designing specific internal reference standards, and experiments show that it can effectively enzyme-cleave the indicator internal reference, increase the proportion of effective sequencing data, and avoid affecting the quantitative internal reference, thereby ensuring the accuracy of the microbial absolute quantification result; and compared with the gel excision recovery method, the Cas9 enzyme cleavage method of the present invention has a lower proportion of indicator internal reference data and a higher proportion of effective data; the Cas9 enzyme cleavage method has a higher correlation with direct sequencing, and the quantitative result has a higher accuracy, which shows that it has the advantages of both the direct sequencing method and the gel excision recovery method, and the Cas9 enzyme cleavage method of the present invention can control the proportion of the indicator internal reference in the sample to less than 30%, and the method is stable, reliable and compatible.

[0147] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0148] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A sgRNA, comprising a target sequence without a PAM sequence and a backbone sequence, wherein the backbone sequence is located at the 3' end of the sgRNA, and the nucleotide sequence of the backbone sequence is shown in SEQ ID NO: 1: GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGC UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUUUUU.

2. A DNA sequence encoding the sgRNA according to claim 1, characterized in that: It also includes a T7 promoter, which is located at the 5' end of the DNA sequence. The nucleotide sequence of the T7 promoter is shown in SEQ ID NO: 2: TAATACGACTCACTATAGG.

3. The DNA sequence according to claim 2, characterized in that The nucleotide sequence of the DNA sequence is shown in SEQ ID NO: 3: AGTCTAATACGACTCACTATAGGCACTTTTCTCCTCCAGAGGTGTTTAA GAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATC AACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.

4. A vector comprising the DNA sequence according to claim 2 or 3.

5. A CRISPR / Cas9 system for indicating an internal reference in the absolute quantification of Cas9 targeted enzyme cleavage of microorganisms, comprising the sgRNA according to claim 1 and a Cas9 molecule, wherein the nucleotide sequence of the indicating internal reference is shown in SEQ ID NO:

13.

6. A kit for indicating an internal reference in the absolute quantification of Cas9 targeted enzyme digestion of microorganisms, comprising the sgRNA according to claim 1, or the DNA sequence according to claim 2 or 3, or the vector according to claim 4, or the CRISPR / Cas9 system according to claim 5.

7. Use of the sgRNA according to claim 1, or the DNA sequence according to claim 2 or 3, or the vector according to claim 4, or the CRISPR / Cas9 system according to claim 5 in absolute quantitative detection of microorganisms.

8. A method for absolute quantitative detection of microorganisms with Cas9 targeted enzyme cleavage as an indicator internal reference, comprising the following steps: 1) Taking a sample to be tested, extracting total DNA, and obtaining a total DNA sample; 2) mixing the total DNA sample with a quantitative internal reference standard, wherein the quantitative internal reference standard includes a quantitative internal reference and an indicator internal reference, to obtain a first mixture, and then subjecting the first mixture to a 16S rDNA PCR amplification reaction to obtain a 16S rDNA PCR amplification product; 3) Then, the 16S rDNA PCR amplification product is used as a template to perform subsequencing library amplification to obtain a subsequencing library PCR product, and then the subsequencing library PCR product is standardized to obtain a standardized subsequencing library PCR product, wherein the concentration of the standardized subsequencing library PCR product is preferably 140 ng / 5 μL; Preferably, the subsequencing library PCR product is standardized by a method comprising the following steps: adding the subsequencing library PCR product to a LIZ 500-HiDi premix, denaturing the product by a PCR instrument, and then analyzing the product by 3730xl DNA analysis software to obtain the sum of the peak areas of the 16S rDNA and the quantitative internal reference of the sample to be tested, and the peak area of ​​the indicator internal reference, and adjusting the amount of the quantitative internal reference added to the sample according to the peak area of ​​the indicator internal reference; 4) adding the standardized subsequencing library PCR product to the CRISPR / Cas9 system, performing enzyme digestion to remove the indicator internal reference, and obtaining the subsequencing library PCR product after enzyme digestion, wherein the CRISPR / Cas9 system includes sgRNA and Cas9 molecules; then performing secondary PCR amplification on the subsequencing library PCR product after enzyme digestion, and then performing second-generation sequencing, thereby obtaining the 16S rDNA read sequence of the sample to be tested and the read sequence of the quantitative internal reference; 5) Then the 16S rDNA read sequence of the sample to be tested and the read sequence of the quantitative internal reference are analyzed to obtain the bacterial community structure classification composition and relative abundance information, as well as the number of read sequences of 16S rDNA of each species in the sample to be tested and each quantitative standard, and then the total amount of bacteria in the sample to be tested and the content of each type of bacteria are calculated according to the quantitative internal reference.

9. The method for absolute quantitative detection of microorganisms with Cas9 targeted enzyme cutting as an internal reference according to claim 8, characterized in that: The sgRNA includes a target sequence without a PAM sequence and a backbone sequence, wherein the backbone sequence is located at the 3' end of the sgRNA, and the nucleotide sequence of the backbone sequence is shown in SEQ ID NO: 1: GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGC UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUUU; the nucleotide sequence of the indicator internal reference is shown in SEQ ID NO: 13; Preferably, the DNA sequence encoding sgRNA is as shown in SEQ ID NO: 3: AGTCTAATACGACTCACTATAGGCACTTTTCTCCTCCAGAGGTGTTTAA GAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATC AACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT; Further preferably, in step 4), the molar ratio of sgRNA:Cas9:subsequencing library PCR product is 10:10:1; More preferably, the CRISPR / Cas9 system is assembled by incubating Cas9 and sgRNA at 25° C. for 10 min.

10. The method for absolute quantitative detection of microorganisms with Cas9 targeted enzyme cutting indicating internal reference according to claim 8 or 9, characterized in that: In step 4), after obtaining the PCR product of the subsequencing library after enzyme cleavage, the method further includes the step of calculating the enzyme cleavage efficiency by the following method: using the ratio of the target peak area before and after enzyme cleavage to the peak area of ​​the indicating internal reference before and after enzyme cleavage, the area of ​​the indicating internal reference removed in the product after enzyme cleavage is calculated, thereby calculating the enzyme cleavage efficiency, specifically according to the following formula I: Enzyme digestion efficiency = [1-(A(Af,I)*A(Bf,T)) / (A(Af,T)*A(Bf,I))]*100%, Among them, the target peak area before enzyme cleavage is A(Bf,T), the indicating internal reference peak area before enzyme cleavage is A(Bf,I), the target peak area after enzyme cleavage is A(Af,T), and the indicating internal reference peak area after enzyme cleavage is A(Af,I).

11. The method for absolute quantitative detection of microorganisms with Cas9 targeted enzyme cutting indicator internal reference according to any one of claims 8 to 10, characterized in that: The quantitative internal reference comprises at least three selected from the following groups of sequences: 1) a first quantitative internal reference sequence R1, wherein the nucleotide sequence of the first quantitative internal reference sequence is shown in SEQ ID NO: 4; 2) a second quantitative internal reference sequence R2, the nucleotide sequence of which is shown in SEQ ID NO: 5; 3) a third quantitative internal reference sequence R3, the nucleotide sequence of which is shown in SEQ ID NO: 6; 4) a fourth quantitative internal reference sequence R4, the nucleotide sequence of which is shown in SEQ ID NO: 7; 5) a fifth quantitative internal reference sequence R5, the nucleotide sequence of which is shown in SEQ ID NO: 8; 6) a sixth quantitative internal reference sequence R6, the nucleotide sequence of which is shown in SEQ ID NO: 9; 7) a seventh quantitative internal reference sequence R7, the nucleotide sequence of which is shown in SEQ ID NO: 10; 8) an eighth quantitative internal reference sequence R8, the nucleotide sequence of which is shown in SEQ ID NO: 11; 9) A ninth quantitative internal reference sequence R9, the nucleotide sequence of which is shown in SEQ ID NO:12.

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