CN115725608A

CN115725608A - Pathogen specific nucleic acid gene and acquisition and detection method

Info

Publication number: CN115725608A
Application number: CN202211392578.7A
Authority: CN
Inventors: 王强; 梁笑辉
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2022-11-08
Filing date: 2022-11-08
Publication date: 2023-03-03

Abstract

The invention discloses a pathogen specific nucleic acid gene and an acquisition and detection method, wherein a whole genome sequence is obtained: establishing a database for the obtained whole genome sequence and carrying out multi-sequence comparison analysis to obtain an overlapping sequence which is the intraspecific common sequence of the bacteria; obtaining interspecific specific sequences: carrying out BLAST comparison on the obtained intra-species common sequence of each bacterium with the intra-species common sequences of all bacteria, the genome sequences of all bacteria and the genome sequences of human beings for three times in sequence, and then taking the non-overlapped sequences as inter-species specific sequences, wherein the inter-species specific sequences are pathogen specific nucleic acid genes; removing repeated sequences: overlapping repeats in the pathogen-specific nucleic acid gene. The invention can be used for quickly identifying pathogens, and has high positive detection rate and short detection period in clinical application.

Description

Pathogen specific nucleic acid gene and acquisition and detection method

Technical Field

The invention relates to a pathogen specific nucleic acid gene and an acquisition and detection method, belonging to the technical field of pathogen detection.

Background

The identification of pathogenic bacteria from a patient body fluid sample based on colony culture belongs to a traditional pathogen detection method and is also the gold standard of clinical microbiological examination at present. The method comprises the steps of collecting a body fluid sample of a patient through aseptic operation, transferring the body fluid sample into an aerobic or anaerobic aseptic culture bottle, firstly carrying out enrichment culture on pathogenic bacteria, then inoculating the body fluid sample into a flat culture dish, finally picking a monoclonal to carry out dyeing microscopy observation, carrying out differential growth experiments on a selective culture medium or carrying out various biochemical tests, and realizing identification on the pathogenic bacteria in the patient sample according to the phenotypic characteristics and the physiological characteristics of the pathogenic bacteria. The traditional culture method is not only time-consuming (usually 2-5 days are needed to obtain etiological information), but also cannot culture target pathogenic bacteria because of the severe growth conditions required by some anaerobic pathogenic bacteria, so that the test result is false negative. In addition, contamination may be introduced during the bacterial culture and isolation process due to handling problems, resulting in false positive results, leading to misdiagnosis and misuse of antibacterial agents.

Of the major pathogenic enterobacteriaceae, serratia marcescens is one of the least studied pathogenic bacteria to date. Researchers have long thought that serratia marcescens belongs to nonpathogenic bacteria or opportunistic pathogens, and mainly affects newborns, children and the elderly with immature immune functions. Serratia marcescens is known to cause respiratory, urinary, blood and eye infections, with the trachea, bronchi and urinary tracts being the most accessible sites for storing the bacteria. This bacteria is also commonly detected in the digestive tract of children. When the immune function of the organism is low, various infections such as pulmonary infection, intracranial infection, sepsis and the like can be caused. In the last 20 years, serratia marcescens has developed continuous outbreaks of infection in neonatal wards of developed countries, as well as in adult patient populations of ICU. The studies by italian researchers have found that pneumonia and sepsis are the most serious complications of infant infection with this bacterium, with mortality rates up to 7% following infection, and that blood and sputum are the major clinical isolates of this pathogen, suggesting that clinicians need to focus on the complications of this pathogen infection. For departments such as ICU and neurology department, the pathogeny information needs to be rapidly and accurately acquired so as to timely and reasonably carry out antibacterial treatment, so that the clinical characteristics of patients are improved, and the cure rate is increased. However, the current clinical pathogen detection technology, traditional culture method, is far from meeting the clinical diagnosis and treatment requirements.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides a pathogen specific nucleic acid gene and an acquisition and detection method thereof.

The technical scheme is as follows: in order to realize the purpose, the invention adopts the technical scheme that:

a method for obtaining a pathogen-specific nucleic acid gene comprises the following steps:

step 1, obtaining a whole genome sequence: obtaining the whole genome sequence of common microorganism.

Step 2, obtaining the common sequences in the same kind of bacteria: establishing a database for the obtained whole genome sequence and carrying out multi-sequence comparison analysis, firstly using a sequence with high sequencing quality in the whole genome sequence of a certain bacterium as a target sequence, respectively carrying out double-sequence comparison with the whole genome sequences of other strains belonging to the same bacterium to obtain two-by-two common sequences, and then carrying out multi-sequence comparison comprehensive analysis on the two-by-two common sequences to obtain an overlapped sequence which is the intraspecific common sequence of the bacterium.

And 3, obtaining an interspecific specific sequence: and carrying out BLAST comparison on the obtained intra-species common sequence of each bacterium with the intra-species common sequences of all bacteria, the genome sequences of all bacteria and the genome sequences of human beings for three times, and then taking the non-overlapped sequences as inter-species specific sequences, wherein the inter-species specific sequences are pathogen specific nucleic acid genes.

And 4, removing repeated sequences: overlapping repeats in the pathogen-specific nucleic acid gene.

Preferably: comprises removing repeated sequences: overlapping repeats in the pathogen-specific nucleic acid gene.

A serratia viscous pathogen specific gene is obtained by a pathogen specific nucleic acid gene obtaining method, and the nucleotide sequences of the serratia viscous pathogen specific gene are respectively shown in a sequence table SEQ ID NO:4-40.

A recombinant expression vector comprises a pathogen-specific gene of Serratia viscosa.

A transformant comprising a host cell of a recombinant expression vector.

An application of a serratia marcescens pathogen specific gene is used for detecting the serratia marcescens pathogen.

A method for identifying a pathogen-specific gene of Serratia viscosa comprises the following steps:

s101, obtaining a sample with the pathogen of the serratia viscosus.

S102, detecting whether the sample has the pathogen specific gene of the serratia viscous pathogen or detecting the content of the pathogen specific gene of the serratia viscous in the sample. The corresponding nucleotide sequences of the pathogen specific genes of the serratia viscosus are respectively shown in a sequence table SEQ ID NO:4-40, or a complement thereof, wherein the presence or amount of said serratia marcescens pathogen-specific gene reflects the presence or amount of a pathogen in said sample corresponding to said serratia marcescens pathogen-specific gene, respectively. The nucleic acid molecule is not less than 50 nucleotides in length.

Preferably, the following components: the method for detecting whether the sample has the pathogen specific gene of the serratia viscous pathogen or detecting the content of the pathogen specific gene of the serratia viscous pathogen in the sample comprises the following steps: and carrying out amplification reaction on the pathogen specific gene of the serratia viscous, and determining whether the pathogen specific gene of the serratia viscous exists or not or the content of the pathogen specific gene of the serratia viscous by detecting whether the amplification product exists or not or the quantity of the amplification product. The amplification reaction is a PCR amplification reaction.

Preferably: in the amplification reaction, the primer for amplifying the pathogen specific gene of the serratia viscous is shown as a sequence table SEQ ID NO:1 and SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

Preferably: and (3) detecting an amplification product by using the trans-cleavage activity of Crispr/Cas family nuclease, wherein the target sequence of the crRNA used together with the Crispr/Cas family nuclease is shown in a sequence table SEQ ID NO:3, and (b) is the sequence shown in the specification. The Crispr/Cas family nuclease is Cas12 or the Crispr/Cas family nuclease is LbCas12.

A kit for detecting a pathogen in a sample comprising

1) Primers for amplifying pathogen-specific nucleic acid fragments in the sample to produce amplification products; and

2) A Crispr/Cas family nuclease with trans-cleavage activity, a crRNA with at least partial sequence of the amplification product as a target sequence, and a single-stranded DNA reporter molecule with a fluorescent group and a quenching group at the 5 'end and the 3' end respectively, wherein

Pathogen-specific nucleic acid fragments corresponding to serratia marcescens are selected from SEQ ID NO:4-40, or a complement thereof

In some embodiments, the kit further comprises the above-described nucleic acid fragments for use as a positive standard.

In some embodiments, the criprpr/Cas family protein is LbCas12.

In some embodiments, the primers used to amplify the pathogen-specific nucleic acid fragment of serratia marcescens include SEQ ID NO:1 and SEQ ID NO:2 in sequence

In some embodiments, the primers for amplification of a pathogen-specific nucleic acid fragment of serratia marcescens include SEQ ID NO:1 and SEQ ID NO:2, and the target sequence of the crRNA comprises SEQ ID NO:3, and (b) a sequence shown in (3).

In some embodiments, the sample is sputum or alveolar lavage fluid from a patient with severe pneumonia.

In some embodiments, the kit is for use against serratia marcescens in the sample.

Compared with the prior art, the invention has the following beneficial effects:

the invention can be used for rapidly identifying pathogens, and has high positive detection rate and short detection period (for example, less than 3 hours) in clinical application.

Drawings

FIG. 1 is a flow chart of a method of obtaining pathogen-specific nucleic acid fragments as described herein.

Fig. 2 is a schematic diagram showing the trans-cleavage activity of Cas12 a.

Fig. 3 is a primer set against serratia viscosa with the primer set SEQ ID NO:1 and SEQ ID NO:2, testing the electrophoresis result chart of the amplified product after the conventional PCR amplification of the DNA templates of various pathogens which are common in clinic.

FIG. 4 shows the results of fluorescence signals detected for different amplification products using LbCas12a and crRNA.

Detailed Description

The present invention is further illustrated by the following description in conjunction with the accompanying drawings and the specific embodiments, it is to be understood that these examples are given solely for the purpose of illustration and are not intended as a definition of the limits of the invention, since various equivalent modifications will occur to those skilled in the art upon reading the present invention and fall within the limits of the appended claims.

"pathogen-specific nucleic acid fragment", also referred to as pathogen or pathogen-specific nucleic acid fragment (usually referred to as DNA sequence, but also including RNA sequence), refers to: DNA sequences widely present in genomes of strains that belong to the same species of pathogenic bacteria in biological taxonomy, but are not present in genomes of other species of pathogenic bacteria, i.e.have DNA sequences specific between species within a species. The species of the bacteria can be accurately detected and identified according to the specific DNA sequence of the pathogenic bacteria.

In some embodiments, a DNA sequence specific for a pathogen is obtained herein by:

1) Obtaining a whole genome sequence: acquiring whole genome sequences of hundreds of common microorganisms from public databases such as NCBI;

2) Obtaining the intraspecific common sequences of the same bacteria: establishing a database for the obtained whole genome sequence and carrying out multi-sequence comparison analysis, firstly, using a sequence with higher accuracy (high sequencing quality) in the whole genome sequence of a certain bacterium as a target sequence, respectively carrying out double-sequence comparison with the whole genome sequences of other strains belonging to the same bacterium to obtain two-by-two common sequences, and then carrying out multi-sequence comparison comprehensive analysis on the two-by-two common sequences to obtain an overlapped (overlap) sequence which is the intraspecific common sequence of the bacterium;

3) Obtaining interspecific specific sequences: carrying out BLAST comparison on the obtained intra-species common sequence of each bacterium, the intra-species common sequence of all bacteria, the genome sequence of all bacteria (excluding self comparison during comparison) and the human genome (hg 19) sequence for three times in sequence, and then taking the non-overlapped sequence as an interspecific specific sequence which is a specific DNA sequence of the pathogenic bacteria;

4) Removing repeated sequences: the repetitive sequence is overlaid using a tool such as RepeatMasker.

In this way, we obtained a plurality of specific nucleic acid fragments of various bacteria in which the nucleotide sequences of Serratia viscosa are shown in SEQ ID NO:4-40.

After obtaining the sequences of these pathogen-specific nucleic acid fragments, they can be conveniently used to detect various pathogens in a patient or patient sample. For example, the presence of a particular pathogen-specific nucleic acid fragment can be used to indicate that the particular pathogen is included in the patient or patient sample; the amount of a particular pathogen-specific nucleic acid fragment can be used to indicate the amount of that particular pathogen in a patient or patient sample. The sequences of these pathogen-specific nucleic acid fragments may themselves be included in some test kits, for example, as positive controls. It is understood that, in the detection process, the full length of the specific nucleic acid fragment against Serratia marcescens provided herein (SEQ ID NO: 4-40) can be detected; alternatively, only a partial fragment thereof, e.g., a fragment of 50, 60, 80, 100 nucleotides or more in length, is detected. Similarly, the pathogen-specific nucleic acid fragments included in the kit can also be full-length fragments or partial fragments (e.g., fragments of 50, 60, 80, 100 nucleotides or more in length). In some preferred embodiments, the pathogen-specific nucleic acid fragment is at least 60 nucleotides in length.

Nucleic acid amplification

The application of nucleic acid amplification technology changes the diagnosis mode of microbial pathogens and is also a molecular biology technology commonly used in the detection and identification means of pathogenic microorganisms at present. In the 80's of the 20 th century, a variety of techniques for DNA amplification appeared in succession, including mainly the Polymerase Chain Reaction (PCR) technique, the Ligase Chain Reaction (LCR) technique, and the isothermal amplification technique.

The discovery and application of DNA polymerase with high temperature resistance makes PCR technology the most common DNA amplification technology. The principle of PCR technology for detecting pathogenic microorganisms is to utilize specific oligonucleotide chains as primers, target nucleic acids containing sequences to be amplified as templates, and to realize exponential amplification of double-stranded DNA by continuously converting temperature, so as to obtain a large amount of target DNA fragments (i.e., amplification products) for subsequent identification. The technology has the advantages of high sensitivity and easy operability, can complete detection on trace pathogenic bacteria, and is very important for detection of pathogenic microorganisms with long growth period, harsh culture conditions or atypical biochemical reaction characteristics.

LCR is another in vitro amplification technology developed after the advent of PCR technology. The principle of the technology is as follows: using thermostable DNA ligase and four primers, two adjacent forward primers and two reverse primers paired with their reverse complements, there are typically 1 gap between two adjacent primers, which serves as a template for DNA ligase ligation. DNA ligase has high specificity and is not tolerant to base mismatches, so the technique is often used for SNP detection.

Isothermal amplification of nucleic acids is a simple technique that can rapidly and efficiently accumulate nucleic acid sequences at a constant temperature, and since the early 90 s of the 20 th century, various isothermal amplification techniques have been developed as alternatives to PCR. In contrast to PCR, isothermal amplification techniques do not require complex thermal cycling processes (and thus can reduce amplification costs) and only achieve amplification reactions at one specific temperature. Isothermal amplification techniques commonly used in the art include: nucleic Acid Sequence-Based Amplification (NASBA), strand Displacement Amplification (SDA), recombinase Polymerase Amplification (RPA), helicase-dependent Isothermal DNA Amplification (HDA), loop-mediated Isothermal Amplification (LAMP), rolling Circle Amplification (RCA), and the like. These isothermal amplification techniques are well known to those skilled in the art and will not be described further herein.

In some embodiments herein, the nucleic acid in the sample can be amplified prior to detection of the pathogen-specific nucleic acid fragment to increase the sensitivity of the detection.

CRISPR/Cas Gene editing System and Trans-cleavage Activity of Cas12a

CRISPR/Cas systems have the potential to target cleavage of nucleic acid molecules, and gene editing tools based on this system are increasingly being developed and exploited in large quantities. At present, gene editing tools based on CRISPR/Cas9 and CRISPR/Cas12a systems are most commonly used. Their working principle is briefly described as follows: firstly, by means of the targeting function of an RNA molecule, a Cas protein is guided to directionally cut double-stranded DNA of a target gene, so that the integrity of a DNA chain is damaged; then, the corresponding DNA repair system in the cell is activated, mainly comprising NHEJ repair mechanism and HDR repair mechanism, thereby completing the destruction or targeted modification of the target gene.

Compared with the prior gene editing tools, the CRISPR/Cas-based gene editing technology has considerable advantages: the composition is simple, consisting of only one Cas protein and sgRNA (only crRNA for Cas12 a), so editing at different sites requires only the replacement of different sgrnas (or only seed region sequences in which the target nucleic acid binds). The simultaneous editing of multiple gene loci can be realized by designing multiple pairs of sgRNAs, and the possibility is provided for the functional research of multiple copies of genes.

In recent two years, cas12a was found to have not only cis (cis) cleavage activity for targeted cleavage of DNA but also trans (trans) cleavage activity for non-specific cleavage of any single-stranded DNA (see fig. 2). Binding of the Cas12a-crRNA complex to the crRNA reverse complementary paired target DNA stimulates trans-cleavage activity of Cas12a, resulting in indiscriminate cleavage of any single-stranded DNA molecules in the vicinity of Cas12a (i.e., no sequence specificity). Therefore, in the presence of a single-stranded reporter DNA molecule, the target DNA molecule can be detected using the trans-cleavage activity of Cas12 a. The detection process includes, for example: firstly, performing exponential amplification on target DNA in a sample to be detected by utilizing technologies such as PCR (polymerase chain reaction) or RPA (reverse transcription amplification) and the like to improve the detection sensitivity; the amplification product is then detected by using Cas12a, crRNA and single-stranded reporter DNA, and if a DNA sequence corresponding to a seed region of the crRNA (a PAM sequence, such as TTTA, is also present upstream of the 5') is present in the amplification product, the trans-cleavage activity of Cas12a is excited to cleave the reporter DNA, thereby generating a fluorescent signal.

In some embodiments, the crRNA used in conjunction with Cas12crRNA consists of a 21nt backbone region (backbone) and 20-24 nt seed regions (seed region/spacer) for recognition of the target sequence of interest. In a more specific embodiment, the sequence of the synthesized crRNA transcribed in vitro is: 5'-AAUUUCUACUAAGUGUAGAUCAGGUAAGGCGCCUCGGGUG-3' (SEQ ID NO: 41). In some embodiments, the corresponding crRNA can be obtained by in vitro transcription with T7 RNA polymerase using DNA as a template, and purified crRNA can be obtained by purification.

The single-stranded reporter DNA molecule has a fluorescent group modification at one end and a quencher group modification at the other end. Due to the presence of the quencher, the complete single-stranded reporter DNA molecule does not generate a fluorescent signal, and when the trans-cleavage activity of Cas12a is excited, the single-stranded reporter DNA molecule is cleaved, such that the quencher is separated from the fluorescent group, generating a fluorescent signal. The presence or intensity of the fluorescent signal indicates the presence or amount of amplification product.

In this study, we first obtained species-specific DNA sequences against serratia marcescens using bioinformatics, and designed and synthesized specific crRNA for targeted recognition of the pathogen from these sequences. Next, we expressed and purified LbCas12a protein derived from helicobacter pili (Lachnospiraceae bacteria) using E.coli pronucleus, and verified that the protein has cis-and trans-cleavage activity. Subsequently, single-stranded reporter DNA, lbCas12a protein and specific crRNA are utilized to assist in a PCR amplification technology, a CRISPR/Cas12 a-based pathogenic bacterium detection method is developed, and the accuracy and the specificity are verified. Finally, the rapid detection of clinical samples of severe pneumonia patients is realized within 3 hours by using a CRISPR/Cas12 a-based pathogen detection tool. The pathogen detection method based on CRISPR/Cas12a provided by the invention is expected to become a novel rapid clinical pathogen detection means, and provides important technical support for improving diagnosis and treatment level of severe pneumonia.

In some embodiments herein, the pathogen-specific nucleic acid fragment or amplification product thereof is detected using the trans-cleavage activity of Cas12a, which further increases the specificity of detection due to the need for the crRNA to bind to a complementary pair of target DNA.

The invention is further illustrated by the following specific examples.

Example 1 specificity verification of CRISPR/Cas-based pathogen detection method

Pathogenic bacteria for detection are all sourced from intensive care units of drugstore hospitals, are separated and cultured from clinical samples of patients with severe pneumonia, and the strain types are determined by microbiological inspection departments of the drugstore hospitals (each pathogenic bacteria has two groups, and is respectively sourced from different patients).

1. Specificity test

In order to prove the specificity and reliability of the pathogen detection method based on the CRISPR/Cas system, cross detection experiments are respectively carried out on amplification primers and corresponding crRNAs.

1.1 primer specificity test

In order to determine the specificity of the amplification primers, the amplification primers aiming at the serratia viscosus respectively use genome DNAs extracted from the serratia viscosus and other 11 clinically common pathogenic bacteria as templates to carry out cross-type PCR reaction.

The forward and reverse primers of the amplification primers used were:

SEQ ID NO:1 and 2, amplifying the genome DNA of the serratia viscosus;

the target sequence (or seed region) of crRNA used in conjunction with the above primer pair is shown in SEQ ID NO:3, respectively.

Examples of the use of Serratia viscosa pathogen-specific nucleic acid fragments to design amplification primers and crRNA sequences are shown below, in which the sequences corresponding to the upstream and downstream primers (SEQ ID NOS: 1 and 2) are underlined, the target sequence of the crRNA (SEQ ID NO: 3) is underlined, and the PAM sequence is boxed.

Use of I-5 ^TM Master Mix (TsingKe) for PCR

TIANeq HiFi Amplification Mix PCR reaction System:

and (3) PCR reaction conditions:

and setting corresponding positive control and negative control for PCR amplification, and respectively taking the genomic DNA and water of the target pathogenic bacteria as amplification templates.

After the PCR reaction was completed, 6 Xgel Loading Dye (NEB) was added in a corresponding volume, mixed well and detected by agarose electrophoresis. And judging the specificity of the primer according to the existence of an obvious bright band of the size of the target DNA in the result of the gel chart.

1.2 crRNA specificity test

PCR products amplified by corresponding primers are respectively detected by using LbCas12a and the crRNA designed aiming at the specific DNA sequence of pathogenic bacteria, and each sample is provided with 3 repeats.

Reaction system:

wherein the structure and sequence of ssDNA-reporter are: 5'-FAM-TTATT-BHQ1-3'.

Adding the reaction solution into a 384-well plate, and reacting for 30-45 min at 37 ℃. After the reaction is finished, a fluorescence value of each hole is detected by using a microplate reader (Infinite M200 Pro multifunctional microplate reader, austria Tecan), and detection parameters are set as follows:

2. results of the experiment

2.1 primer specificity test results

As shown in FIG. 3, although a small amount of non-specific bands are present only in individual non-target pathogenic bacteria, a large amount of target DNA products can be amplified using genomic DNA corresponding to the pathogenic bacteria as a template, indicating that the primer specificity for amplification by Serratia viscocola is good.

2.2 Results of crRNA specificity test

And detecting the PCR reaction solution after the corresponding primers are amplified by utilizing LbCas12a and crRNA aiming at the serratia marcescens. The fluorescence results of fig. 4 show: the detection system of the pathogenic bacteria of the serratia viscosus can generate obvious fluorescent signals only when the amplification product obtained after the PCR amplification is carried out by taking the genome DNA of the pathogenic bacteria of the serratia viscosus as a template, and the intensity of the fluorescent signals of the other non-target pathogenic bacteria is consistent with the negative control, which shows that the detection method has excellent specificity by combining with crRNA.

Example 2 detection of Severe pneumonia patient clinical specimens Using CRISPR/Cas System

1. Experimental procedures

All 12 clinical samples (sputum or alveolar lavage fluid) were obtained from the intensive care unit of the drumbeat hospital and were compared with pathogen types determined by the microbiological laboratory of the drumbeat hospital using the conventional culture isolation assay.

1.1 kit method for extracting clinical sample DNA

Clinical sample DNA extraction reference Quick-DNA/RNA ^TM The method is carried out by a Patholon Miniprep Kit (ZYMO RESEARCH) product specification, and the operation steps are briefly as follows: (the centrifugal rotation speeds are all 16,000 Xg)

a) Aspirate 50-200. Mu.L of sample, add 800. Mu.L of DNA/RNA Shield reagent, and vortex for 60s.

b) Centrifuge at 16,000 Xg for 1min and aspirate 200. Mu.L of supernatant.

c) Add 2. Mu.l of Proteinase K reagent to 200. Mu.l of supernatant and mix well.

d) 1ml Pathogen DNA/RNA buffer reagent was added, mixed well and allowed to stand at room temperature for 5min.

e) The solution was pipetted onto a DNA binding column (which had been placed in a recovery tube), centrifuged for 30 seconds, and the column-through solution was discarded.

f) Add 500. Mu.l of Pathologen DNA/RNA Wash buffer to the column, centrifuge for 30s, and discard the column. This step is repeated once.

g) Add 500. Mu.l ethanol (95-100%) to the column, centrifuge for 1min at 16,000 Xg to ensure the ethanol is cleared, discard the recovery tube, place the DNA binding column in a DNase-free 1.5ml centrifuge tube.

h) 50 mul of 65 ℃ double distilled water is absorbed into the matrix of the column, the column is kept still for 2 to 5min, and the column is centrifuged for 1min at 16,000 Xg, and the eluent is collected.

i) The DNA concentration was determined and stored in a freezer at-20 ℃.

1.2PCR amplification of target sequences

PCR amplification reactions were performed using 1 pair of primers described in example 1, respectively, using the extracted total DNA of the clinical specimen as a template. The positive control group uses genome DNA of Serratia viscosa as template, and the negative control group uses water as template.

The PCR reaction system and reaction conditions were the same as in example 1.

1.3 Cas12a detection

The unpurified PCR reaction stock was detected using LbCas12a, crRNA and ssDNA-reporter. 3 repetitions are set for the same sample to be detected;

the reaction system and reaction conditions were the same as in example 1.

2. Results of the experiment

And respectively carrying out PCR amplification on the total DNA of 12 clinical samples of the severe pneumonia patients by using 1 pair of amplification primers, and then detecting corresponding PCR reaction liquid by using the specific crRNA of the single-stranded reporter DNA, the LbCas12a and the serratia viscosus. The results of detection based on fluorescent signal determination are shown in table 1 and compared with the conventional culture method.

TABLE 1 comparison of detection results of Serratia viscosus in 12 clinical samples of alveolar lavage fluid

As can be seen from the table, serratia viscosus was detected in clinical samples No. 1, no. 5, no. 8 and No. 10 based on CRISPR/Cas12a detection, and the remaining samples showed negativity. The clinical culture results showed the presence of serratia viscosus in samples No. 1, no. 5 and No. 8. The second generation sequencing technology was examined for 5 out of 12 samples, among which Serratia viscosa was detected in clinical samples No. 5, no. 8 and No. 10, and samples No. 2 and No. 4 showed negativity. It is noteworthy that the second-generation sequencing technology in sample No. 10 showed the presence of a small amount of serratia marcescens pathogen, while the clinical culture method was not detected, and the PCR-CRISPR/Cas12a combined detection technology developed by us showed positive.

In conclusion, compared with the traditional detection method relying on pathogen isolation and culture, the pathogen detection tool based on CRISPR/Cas12a developed by the research and development shows good detection effect, particularly for a sample with low bacterial load, such as a No. 10 sample, and the traditional detection period is shortened from several days to be within 4 hours, so that the detection method can be used as a potential detection tool for rapidly diagnosing the existence condition of the serratia tenacicola in a clinical sample. In addition to Serratia viscosa, a similar approach can be used to identify pathogenic microorganisms for a variety of other infectious diseases based on the specific nucleic acid fragments for the numerous pathogens provided herein.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims

1. A method for obtaining a pathogen-specific nucleic acid gene, comprising the steps of:

step 1, obtaining a whole genome sequence: obtaining a whole genome sequence of a common microorganism;

step 2, obtaining the common sequences in the same kind of bacteria: establishing a database for the obtained whole genome sequence and carrying out multi-sequence comparison analysis, firstly, using a sequence with high sequencing quality in the whole genome sequence of a certain bacterium as a target sequence, respectively carrying out double-sequence comparison with the whole genome sequences of other strains belonging to the same bacterium to obtain two-by-two common sequences, and then carrying out multi-sequence comparison comprehensive analysis on the two-by-two common sequences to obtain an overlapped sequence which is the intraspecific common sequence of the bacterium;

and 3, obtaining an interspecific specific sequence: carrying out BLAST comparison on the obtained intra-species common sequence of each bacterium with the intra-species common sequences of all bacteria, the genome sequences of all bacteria and the genome sequences of human beings for three times in sequence, and then taking the non-overlapped sequences as inter-species specific sequences, wherein the inter-species specific sequences are pathogen specific nucleic acid genes;

2. The method for obtaining a pathogen-specific nucleic acid gene according to claim 1, wherein: comprises removing repeated sequences: overlapping repeats in the pathogen-specific nucleic acid gene.

3. A serratia marcescens pathogen specific gene is characterized in that: the pathogen-specific gene of serratia viscosus obtained by the method for obtaining a pathogen-specific nucleic acid gene according to claim 1, wherein the nucleotide sequences of the pathogen-specific gene of serratia viscosus are respectively as shown in SEQ ID NO:4-40.

4. A recombinant expression vector characterized by: comprising the pathogen-specific gene of Serratia viscosa according to claim 4.

5. Use of the pathogen-specific gene of Serratia viscosa according to claim 4, wherein: the kit is used for detecting the pathogen of the serratia viscosa.

6. A method for identifying a gene specific to a pathogen of Serratia viscosa according to claim 4, comprising the steps of:

s101, obtaining a sample with a serratia viscous pathogen;

s102, detecting whether the sample has the pathogen specific gene of the serratia viscous pathogen or detecting the content of the pathogen specific gene of the serratia viscous in the sample.

7. The identification method according to claim 7, wherein: the method for detecting whether the sample has the pathogen specific gene of the serratia viscous pathogen or detecting the content of the pathogen specific gene of the serratia viscous pathogen in the sample comprises the following steps: and carrying out amplification reaction on the pathogen specific gene of the serratia marcescens, and determining whether the pathogen specific gene of the serratia marcescens exists or not or the content of the pathogen specific gene of the serratia marcescens by detecting whether the amplification product exists or the quantity of the amplification product.

8. The identification method according to claim 8, wherein: in the amplification reaction, the primer for amplifying the pathogen specific gene of the serratia viscous is shown as a sequence table SEQ ID NO:1 and SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

9. The method of identifying a pathogen of serratia viscosus according to claim 9, wherein: and the detection of amplification products is carried out by using the trans-cleavage activity of Crispr/Cas family nuclease, and the target sequence of the crRNA used together with the Crispr/Cas family nuclease is shown in a sequence table SEQ ID NO:3, and (b) is the sequence shown in the specification.

10. A kit for detecting a Serratia viscosa pathogen in a sample, comprising the gene specific to the Serratia viscosa pathogen of claim 3.