CN112226488A - Method and kit for constructing pathogenic microorganism sequencing library based on total nucleic acid and metagenome - Google Patents

Abstract

The invention provides a method and a kit for constructing a pathogenic microorganism sequencing library based on total nucleic acid and metagenome. The method comprises the steps of extracting total nucleic acid of pathogenic microorganisms, simplifying the process of respectively and independently detecting DNA and RNA, enriching the RNA in the obtained total nucleic acid, and then performing the steps of one-chain cDNA synthesis, two-chain cDNA synthesis, end repair, tailing reaction, joint connection and the like to obtain a sequencing library of the pathogenic microorganisms in an infected sample. The method realizes the simultaneous detection of DNA and RNA pathogens in a single-tube reaction, can cover up to 13396 pathogens, and obviously improves the detection rate of the pathogens of complex infection such as bacteria, fungi, viruses and the like on the basis of reducing the detection cost and detection steps.

Description

Method and kit for constructing pathogenic microorganism sequencing library based on total nucleic acid and metagenome

Technical Field

The invention belongs to the technical field of microorganism detection, relates to a method for constructing a sequencing library of pathogenic microorganisms, and particularly relates to a method for constructing a sequencing library of pathogenic microorganisms based on total nucleic acid and metagenome.

Background

Infectious diseases are one of the common clinical diseases, are local or systemic inflammations or organ dysfunction caused by pathogens such as bacteria, viruses, fungi and the like and products thereof, and have larger harmfulness and higher fatality rate. Infection is one of the leading causes of death in critically ill patients. Severe infection is acute in onset, rapid in progress and complex in pathogen, and whether pathogenic microorganisms are clearly caused in a short time is crucial.

There are various ways to detect pathogenic microorganisms in the field of infectious diseases. Wherein, (1) the living body for obtaining the pathogen is a gold standard for infectious disease diagnosis, and is mainly positive for bacteria, fungi and viruses in culture, but the in vitro culture of the pathogen is generally long in time consumption, the operation steps are complicated, and most of the pathogens can not be cultured; (2) the method is characterized in that an immunological method such as a complement fixation test, a neutralization test, an enzyme-linked immunosorbent assay, an immunofluorescence method, an enzyme-labeled spot immunoassay and the like is utilized to detect pathogenic microorganisms, although the operation is simpler, the number of researched and developed antigens and antibodies can not meet the market demand greatly due to the variety of pathogens; (3) the PCR detection of the pathogenic microorganisms has extremely high sensitivity and specificity, but high-throughput screening cannot be completed, and the detection rate is low; (4) the gene chip technology can only carry out intention screening on the genome of known pathogens, but cannot detect new unknown pathogens.

However, the conventional detection methods still have the problems of being unable to quickly and accurately determine pathogen information, and the like, which easily causes that patients with infectious diseases cannot be timely and effectively cured, thereby deteriorating the disease conditions. Therefore, the rapid, specific and high-flux pathogen detection method has important significance for effectively diagnosing and timely preventing and treating infectious diseases.

The etiology diagnosis technology based on the metagenomic sequencing (mNGS) is a non-targeted broad-spectrum etiology screening technology, and more researches in recent years show that the pathogeny diagnosis technology plays a key role in the field of severe infection, particularly severe infection caused by difficult and rare diseases. Some challenges still remain, such as:

(1) the influence of background bacteria, in the mNGS report result, it is determined whether the bacteria are customized, background bacteria or pathogenic bacteria by combining the sample collection type, microbial background, clinical characteristics of patients, detection report and auxiliary examination of traditional pathogens, etc.

(2) The detection rate of intracellular bacteria/fungi is low, and the detection sensitivity is low mainly due to the fact that the content of intracellular infectious bacteria released into body fluid is low, such as mycobacterium tuberculosis, legionella, brucella and the like;

(3) drug resistance detection has certain difficulty, and the coverage of drug resistance related genes is low mainly due to some differences in the degree of association between the currently reported drug resistance genotype and the drug resistance phenotype and consideration of detection methods and detection cost;

(4) the detection of RNA pathogens has certain difficulties in clinical detection of RNA viruses because human transcripts have higher abundance and complexity than genomes, and RNA is easy to degrade and has higher requirements for transportation and storage.

At present, most of the DNA-level-based mNGS detection developed in the market is less RNA-based macrotranscriptome detection; the method can be used for carrying out the sequencing application of the metagenome and the macrotranscriptome, and also can be used for carrying out the independent detection of the DNA and the RNA and the data merging analysis; or, RNA is first reverse transcribed and double-stranded synthesized, and then mixed with DNA, which results in high detection cost and is not favorable for popularization of detection application.

Therefore, there is a need to provide a kit, which can be used in a single-tube reaction for simultaneously performing DNA and RNA library construction, and can save both the usage amount of detection samples and the detection cost and period.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method and a kit for constructing a pathogenic microorganism sequencing library based on total nucleic acid and metagenome. The method simultaneously builds the DNA and RNA libraries in a single tube reaction, reduces the construction steps, and can better keep the original nucleic acid of the sample in the construction process, so the method has higher application value under the condition of less sample amount.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for constructing a sequencing library of a pathogenic microorganism based on total nucleic acids and metagenomics, the method comprising the steps of:

(1) respectively extracting DNA and RNA in a sample infected by pathogenic microorganisms, enriching the RNA, and then mixing the obtained DNA and RNA to obtain Total Nucleic Acid (TNA) to obtain a sample to be detected;

or directly extracting TNA in the sample infected by the microorganism as a sample to be detected;

(2) the sample to be detected contains DNA and RNA of pathogenic microorganisms, then the RNA in the sample to be detected is fragmented, and simultaneously, one-chain cDNA synthesis is carried out;

(3) after the first-strand cDNA synthesis is finished, synthesizing second-strand cDNA to obtain a cDNA/DNA compound;

(4) and (3) breaking the cDNA/DNA complex by using a fragmentation enzyme, and performing end repair and tailing reaction, joint connection, PCR enrichment and purification to obtain a sequencing library constructed based on TNA.

The method is a sequencing method based on total nucleic acid, can be abbreviated as TNA-seq, and specifically comprises the following steps: extracting total nucleic acid of the microbial pathogens, wherein when the sample amount is large, DNA and RNA are extracted independently, and the sample amount is small, so that the total nucleic acid TNA of the microbial pathogens is extracted; adding RNA fragment enzyme to carry out RNA fragmentation and one-strand synthesis buffer and enzyme; performing double-strand synthesis to form a cDNA/double-stranded DNA complex; performing enzyme digestion fragmentation and end repair on a cDNA/double-stranded DNA compound and adding an A tail; and connecting and purifying the joint, enriching by PCR, and purifying to obtain the single-tube co-sequencing library with the DNA/RNA as the template.

The method simplifies the process of respectively and independently detecting DNA and RNA, greatly reduces the detection cost, and can obviously improve the detection rate of pathogenic bacteria of composite infection of bacteria, fungi, viruses and the like. The simultaneous detection of DNA and RNA pathogen infection in a single-tube reaction is realized; meanwhile, the coverage area is wide, and the comprehensive coverage of various clinical pathogens of different types can be realized by one-time detection.

Compared with the method for simultaneously detecting the pathogenic microorganisms of DNA and RNA by constructing the library by using Tn5 transposase, the method has the advantages of no selection preference, good compatibility and high detection rate after co-construction of the library. In the construction method using the Tn5 transposase, the Tn5 transposase has the preference of nucleic acid fragment selection, is not suitable and is widely applied and popularized, and the possibility of missing detection of a detection result is high; meanwhile, the construction method using the Tn5 transposase can only ensure that the quality of the library construction is qualified, and has no obvious library co-construction effect on the detection rate of DNA and RNA pathogens.

As a preferred technical scheme of the invention, the pathogenic microorganism in the step (1) comprises any one or the combination of at least two of bacteria, fungi, DNA viruses, RNA viruses, chlamydia, mycoplasma or parasites.

The sequencing library of the pathogenic microorganism constructed in the invention can be 13396 in types.

Preferably, the microorganism-infected sample of step (1) comprises any one of alveolar lavage fluid, sputum, cerebrospinal fluid, pleural effusion or punctured tissue or a combination of at least two thereof.

In the present invention, the biological sample to be detected can be carried out in two different ways. For samples with large sample size, such as alveolar lavage fluid, sputum and the like, DNA and RNA in the samples can be respectively extracted; for samples with small sample size, such as cerebrospinal fluid and the like, the sample nucleic acid can be obtained by extracting the total nucleic acid in the sample, so that the complete nucleic acid in the sample can be obtained under any condition, and the missing of the microbial pathogens caused by the missing in the process of extracting the sequencing library is prevented. The method can be used for establishing a library of all DNA and RNA virus nucleic acids in a sample, so that the detection rate of pathogens is improved, and the sensitivity and specificity are ensured.

Preferably, the enrichment reaction system comprises Turbo deoxyribonuclease (Turbo DNase) and/or Baseline deoxyribonuclease (Baseline DNase).

Preferably, the Kit used for enriching RNA is ZYMO RNA Clean & Concentrator Kit, and the enrichment step is carried out according to the instruction of the Kit, so as to enrich RNA in the sample. In the invention, when the sample amount is large, after the DNA and the RNA of the sample are respectively extracted, the RNA is purified and enriched, and then the DNA and the RNA are combined and the next step is carried out.

Preferably, the reaction system of step (2) comprises DNA, RNA, reverse transcriptase, RNA fragmenting enzyme and dNTPs in the sample.

In the invention, when the single-Strand cDNA is synthesized, the reaction system comprises RNA Fragmentation Mix, 1st Strand Buffer and 1st Strand Enzyme Mix; wherein the 1st Strand Enzyme Mix contains reverse transcriptase. In this step, DNA and RNA are present in the reaction solution, and the presence of DNA does not affect the fragmentation of RNA and the occurrence of reverse transcription step, and the synthesis of single-stranded cDNA is normally performed.

Preferably, the volume ratio of the reverse transcriptase to the RNA fragmentation enzyme is 1 (5-8).

Preferably, the reaction conditions for the single strand cDNA synthesis are: the reaction is carried out for 5-10 min at 20-30 ℃, then for 10-15 min at 40-42 ℃, and then for 10-20 min at 65-70 ℃.

As a preferred embodiment of the present invention, the reaction system in the step (3) comprises DNA, single strand cDNA, DNA polymerase and RNaseH in the sample.

In the present invention, the double-stranded cDNA synthesis is performed immediately after the completion of the single-stranded cDNA synthesis step. The reaction system used included the products of one-Strand cDNA synthesis, 2nd Strand Buffer and 2nd Strand Enzyme Mix.

Preferably, the reaction conditions for the two-strand cDNA synthesis are: the reaction is carried out for 20-40 min at 20-30 ℃ and then for 10-15 min at 60-65 ℃.

After the synthesis of the double-stranded cDNA is finished, the magnetic beads can be used for extracting and purifying nucleic acid in the reaction solution to obtain a product cDNA/DNA compound.

Preferably, the fragmenting Enzyme of step (4) comprises DNA Frag Enzyme.

Preferably, the reaction system used in the end repairing and tailing reaction of step (4) comprises: end Repair-adding tailase and corresponding premix buffer (End Repair-A Tailing Enzyme Mix), DNA fragmentation Enzyme and corresponding premix buffer (DNA Frag Enzyme Mix) and DNA fragmentation-adding tailase Enhancer and enhancing premix buffer (DNA Frag-A Tailing Enzyme).

In the invention, after a cDNA/DNA compound is synthesized, a fragmentation enzyme is used for breaking the compound, and a system of end repair and A-tail addition reaction is adopted for modifying the fragmented product.

Preferably, the reaction temperature of the joint connection is 22-26 ℃, and the reaction time is 10-15 min.

Preferably, the Adaptor connection reaction system in the step (4) comprises an Adaptor (Adaptor), and the using amount ratio of the fragmented DNA to the Adaptor is (15-20) ng:1 muL.

In the present invention, the amount of Adaptor used and the DNA content in the reaction solution are calculated in the following ratio, and the good connection effect of the linker can be ensured according to the amounts of Adaptor used shown in the following table.

DNA content	Joint dosage (mu L)	Dilution factor of joint
			100ng	5	Is not diluted
50ng	5	1:2
			10ng	5	1:10
1ng	5	1:100

Preferably, the concentration of the sequencing library in the step (4) is more than or equal to 2.0 ng/. mu.L.

Preferably, the length of the DNA fragments in the sequencing library in the step (4) is 220 bp-400 bp.

Preferably, the method for purifying nucleic acid in the method is a magnetic bead purification method.

Preferably, the method further comprises the step of subjecting the sequencing library to high throughput sequencing and bioinformatic analysis.

As a preferred technical scheme of the invention, the method comprises the following steps:

(1) respectively extracting DNA and RNA in alveolar lavage fluid, sputum or pleural effusion of an infected patient, enriching the RNA, and mixing the obtained DNA and RNA to obtain a sample to be detected;

or, directly extracting TNA in cerebrospinal fluid or punctured tissues of the infected patient to be used as a sample to be detected;

(2) mixing the sample to be detected with reverse transcriptase, RNA fragmentation enzyme and dNTPs, fragmenting and reverse transcribing RNA in the sample to be detected, and simultaneously performing one-strand cDNA synthesis;

the reaction conditions for the single-strand cDNA synthesis are as follows: firstly reacting for 5-10 min at 20-30 ℃, then reacting for 10-15 min at 40-42 ℃, and then reacting for 10-20 min at 65-70 ℃;

(3) mixing the product obtained by synthesizing the first-strand cDNA with DNA polymerase, and synthesizing second-strand cDNA to obtain a cDNA/DNA compound;

the reaction conditions for the two-strand cDNA synthesis are as follows: firstly reacting for 20-40 min at 20-30 ℃, and then reacting for 10-15 min at 60-65 ℃;

(4) fragmenting the cDNA/DNA compound by using a fragmenting enzyme, and performing end repair, tailing reaction and joint connection, wherein the usage amount ratio of the fragmented DNA to the joint in a joint connection reaction system is (15-20) ng:1 muL;

after the joint connection is finished, PCR enrichment and purification are carried out to obtain a sequencing library constructed based on TNA, the concentration of the obtained sequencing library is more than or equal to 2.0 ng/mu L, and the length of the DNA fragment is 220 bp-400 bp.

In a second aspect, the present invention may also provide a kit for constructing a sequencing library of a pathogenic microorganism using the method of the first aspect, the kit comprising:

DNA and RNA extraction fractions;

RNA enrichment components including Turbo DNase, Baseline DNase and RNA adsorption columns;

RNA reverse transcription component, including reverse transcriptase, reverse transcription reaction buffer solution and primer;

a fragmentation component comprising an RNA fragmentation enzyme and a DNA fragmentation enzyme;

the cDNA/DNA compound library building component comprises DNA fragmenting enzyme and corresponding premixed buffer solution, terminal repair-tailing enzyme A and corresponding premixed buffer solution, DNA fragmentation-tailing enzyme A enhancer, reinforced premixed buffer solution and a joint.

Illustratively, the kit comprises the following components:

in the co-database building process, the kit increases the template enrichment process of an RNA detection link, improves the detection rate of RNA viruses in the detection process of infected samples, and has the characteristic of higher detection sensitivity. Therefore, the kit can be used for constructing a library of pathogenic microorganisms, and after the library is constructed, the type of the infected pathogenic microorganisms can be analyzed by performing on-machine detection.

The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between non-recited numerical ranges, and is not intended to be exhaustive or to limit the invention to the precise numerical values encompassed within the range for brevity and clarity.

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

the construction method of the sequencing library provided by the invention realizes the simultaneous construction of the DNA and RNA pathogen sequencing library in a single-tube reaction, can comprehensively cover 13396 pathogens in clinic, lays a foundation for a comprehensive detection method of pathogenic microorganisms based on TNA (DNA and RNA co-construction library) sequencing, and can be suitable for mixed infection of pathogens (bacteria, fungi, viruses and mycoplasma) with staggered symptoms and difficult judgment, infection of rare pathogens (adenovirus, plasmodium and the like) and the condition that the pathogens are negative after being cultured for many times in clinic but seriously suspected to be infected; meanwhile, the method has the advantages of small sample initial amount, quick detection period, low detection cost, high sensitivity and the like, and the detection rate of pathogens is obviously improved.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

In the following examples, the room temperature was 25 ℃;

in the following examples, Turbo DNAse was purchased from Thermo fisher; baseline DNAse was purchased from Epicentre, Illumina; RNA Clean & Concentrator Kit was purchased from Zymo Research; 1st Strand Buffer, 1st Strand Enzyme Mix, 2nd Strand Buffer and 2nd Strand Enzyme Mix were purchased from Dynasty Gene; RNA Fragmentation Enzyme and corresponding pre-Mix Buffer (RNA Fragmentation Mix), DNA Fragmentation Enzyme and corresponding pre-Mix Buffer (DNA fragment Enzyme), End Repair-A labeling Enzyme Mix, DNA fragment-A labeling Buffer, DNA Fragmentation-A Tailing Enhancer and enhancing pre-Mix Buffer (DNA fragment-A labeling Enhancer), Ligation Buffer (Ligation Buffer), Ligase (Ligase), PCR reaction solution (PCR Mix) and linker adapter (IL) are all purchased from KAPA Biosystems; the remaining reagents not mentioned are all commercially available from the reagent manufacturers customary in the art.

Example 1

This example provides a method for metagenomics and TNA to construct a sequencing library of pathogenic microorganisms while using clinical samples to validate the reliability of the detection method.

In this example, 60 clinical samples were tested and the types of pathogens tested included bacteria, fungi, viruses, parasites, mycobacteria, mycoplasma/chlamydia. Comparing each pathogen type sample by adopting a clinical standard detection technology, wherein bacteria, fungi and mycoplasma adopt a separation culture method; the virus and the tubercle bacillus are detected by a PCR method to compare results, and meanwhile, a standard strain and healthy human nucleic acid are respectively used as a positive control and a negative control.

The specific detection steps are as follows:

1. sample preparation

(1) Sample pretreatment: adding 1mL of sputum DTT and PBS buffer solution to blow and suck uniformly aiming at the condition that the sample is viscous and cannot be sampled by using a gun head, if the sample is viscous, continuously adding 1mL of sputum, blowing and sucking uniformly by using a pipette head, and placing the sample in a metal bath at 37 ℃ for incubation for 15min until the gun head is not blocked;

(2) and (3) cracking treatment: centrifuging 8000g of 2mL sample for 5min, collecting precipitate, adding 600 mu L of lysis solution RLplus, vortexing for 30s, transferring all the solutions into a DNA adsorption column at 12000rpm (13 to 400 Xg), centrifuging for 1min, collecting filtrate (the filtrate is used for RNA extraction), placing the DNA adsorption column in a collection tube at room temperature or 4 ℃ until DNA is subsequently extracted;

(3) RNA extraction: adding 1 time volume of 70% ethanol into the filtrate, mixing, transferring into an RNA adsorption column, centrifuging at 12000rpm for 1min to remove waste liquid, adding 700 μ L deproteinization solution RW1 into the RNA adsorption column, centrifuging at 12000rpm for 1min to remove waste liquid, adding 500 μ L rinsing solution RW (ethanol is added before use) into the RNA adsorption column, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, and rinsing twice; placing the RNA adsorption column at room temperature for 5 minutes, adding 30 microliter of eluent, placing at room temperature for 2 minutes, and centrifuging at 12000rpm for 1 minute to obtain RNA solution;

(4) DNA extraction: adding 500 μ L buffer GD (ethanol is added before use) into the DNA adsorption column, centrifuging at 12000rpm for 1min to remove waste liquid, adding 500 μ L rinsing liquid PW into the DNA adsorption column, standing at room temperature for 2min, centrifuging at 12000rpm for 1min to remove waste liquid, and rinsing twice; the DNA adsorption column was left at room temperature for 5 minutes, 50. mu.L of the eluent was added thereto, left at room temperature for 2 minutes, and centrifuged at 12000rpm for 1 minute to obtain a DNA solution.

2. RNA enrichment (Kit ZYMO RNA Clean & concentrate Kit)

(1) Pretreatment of an RNA adsorption column: in order to remove trace DNA, adding 80 muL of DNase I reaction solution, then uniformly mixing 5 muL of DNase I and 75 muL of DNA digest Buffer, adding the mixture into an adsorption column, and incubating for 15min at room temperature;

(2) sample lysis: sucking 500mL of sample, directly adding 500mL of Trizol lysate into the sample, uniformly mixing, standing at room temperature for 10min, centrifuging at 4 ℃ and 12000g for 1min after the sample is cracked, taking out supernatant, transferring the supernatant into a new centrifuge tube, adding absolute ethyl alcohol with the same volume into the centrifuge tube, and discarding tissues and precipitates which cannot be cracked;

(3) RNA adsorption: the mixture was added to a pretreated adsorption column and centrifuged for 1 min. Removing the filtrate; adding 400 μ L RNA Prep Buffer to the adsorption column, centrifuging at 12000g for 1min, and removing the filtrate;

(4) adding 700 μ L of RNA Wash Buffer to the adsorption column, centrifuging at 12000g for 1min, removing the filtrate, and repeating once;

(5) taking out the adsorption column, placing into a centrifuge tube without RNase, adding 50 μ L RNase-free water into the middle part of the adsorption membrane, standing at room temperature for 2min, centrifuging for 1min, and collecting RNA.

And mixing the enriched RNA with the extracted DNA, and continuing the experiment.

3. Single-stranded cDNA Synthesis

(1) The RNA Fragmentation Mix, 1st Strand Buffer was thawed from the freezer, equilibrated to room temperature, and 1st Strand enzymeMix was placed on ice for thawing.

A one-strand cDNA synthesis reaction system was set up on ice as shown in the following table:

components	Volume (μ L)
		DNA/RNA	8.5
RNA Fragmentation Mix	8.5
		1st Strand Buffer	7
1st Strand Enzyme Mix	1
		Total volume	25

Gently pipetting for 10 times by using a pipette, fully mixing, and performing short separation.

(2) First strand cDNA synthesis was performed in a PCR instrument running the following program:

immediately after completion, the second strand cDNA synthesis reaction was carried out.

4. Double-stranded cDNA Synthesis

(1) The 2nd Strand Buffer was thawed from the freezer, equilibrated to room temperature, and the 2nd Strand Enzyme Mix was thawed on ice.

The two-strand cDNA synthesis reaction system was configured on ice as shown in the following table:

components	Volume (μ L)
		Products of the above step	25
2nd Strand Buffer	3.6
		2nd Strand Enzyme Mix	6.5
Nuclease-free Water	14.9
		Total amount of	50

Gently pipetting for 10 times by using a pipette, fully mixing, and performing short separation.

(2) Second strand cDNA synthesis was performed in a PCR instrument running the following program:

step (ii) of	Temperature (. degree.C.)	Time of day
			1	16	30min
2	65	15min
			3	4	Holding

(3) Adding 90 μ L of magnetic Beads DNAQUARAcces Hyper Pure Beads which are pre-balanced for 40min at room temperature after the reaction is finished, uniformly mixing, and standing for 5min at room temperature;

(4) placing on a magnetic frame, standing for clarification, and removing supernatant;

(5) adding 200 μ L of 80% ethanol, standing for 30s, removing supernatant, and repeating the steps once;

(6) quickly centrifuging, removing residual ethanol with 10 μ L pipette, standing at room temperature, and air drying;

(7) taking down the magnetic frame, adding 32 mu L of nucleic-free Water resuspension magnetic beads, fully sucking and uniformly mixing, standing at room temperature for 2min, placing on the magnetic frame, carefully sucking 31.3 mu L of supernatant after the solution is clarified, transferring to a new Nuclease-free tube, and placing on ice for later use.

5. cDNA/DNA Complex cleavage fragmentation

(1) Placing the DNA Frag Enzyme and the End Repair-A labeling Enzyme Mix on ice to melt, flicking and uniformly mixing with fingers, placing the DNA Frag-A labeling Buffer and the DNA Frag-A labeling Enzyme on ice to melt, and carrying out vortex oscillation for 2 s;

(2) the PCR instrument was set up according to the following table, the hot lid temperature was set to 70 ℃, the program was started, and the time was suspended when the temperature reached 4 ℃;

step (ii) of	Temperature (. degree.C.)	Time of day
			1	4	1min
2	32	22min
			3	65	30min
4	4	Holding

(3) Adding 5 mu L of DNA Frag-A labeling Buffer and 2.5 mu L of DNA Frag-A labeling Enhancer, gently blowing, beating and uniformly mixing for 6 times;

(4) 10 mu.L of DNA Frag Enzyme and 1.2 mu.L of End Repair-A Tailing Enzyme Mix were added to each sample, gently beaten and mixed 6 times;

(5) and (4) centrifuging the mixed sample to the bottom of the tube briefly, immediately transferring the mixed sample to a pre-cooled PCR instrument, and continuing to run the program to the end.

6. Joint connection

(1) Taking out the Ligation Buffer, adapter (IL) and Ligase, and configuring a reaction system connected by a joint according to the following table:

components	Volume (μ L)
		Products of the above step	50
Adaptor(IL)	5
		Ligation Buffer	16
Ligase	6
		Nuclease-free Water	3
Total	80

Blowing, beating, mixing uniformly, separating for a short time, and placing on a PCR instrument to execute the following procedures:

step (ii) of	Temperature (. degree.C.)	Time of day
			1	25	10min
2	4	Holding

After completion, 64. mu.L of magnetic beads were added for purification.

7、Pre-PCR

(1) Taking down the reaction tube from the magnetic frame, adding 15 μ L of nucleic-free Water resuspension magnetic beads, mixing well with a vortex oscillator, and standing at room temperature for 2 min;

then 25. mu.L of PCR Mix, 8. mu.L of Pre-Primer Mix were added, mixed well, centrifuged rapidly for 5s for 1min on a magnetic stand, the supernatant was transferred to a new PCR tube, and the following PCR reaction was performed:

wherein N is determined according to the DNA content, and the specific numerical values are as follows:

DNA	number of cycles (N)
		100ng	6-9
50ng	9-11
		10ng	11-13
1ng	14-16

After the reaction is finished, 40 mu L of magnetic beads are added for purification; and (3) taking the tube off the magnetic frame, adding 21 mu L of nucleic-free Water resuspension magnetic beads, fully sucking, uniformly mixing, standing at room temperature for 2min, placing on the magnetic frame, carefully sucking 20 mu L of supernatant after the solution is clarified, and transferring to a new Nuclease-free tube.

8. Library quality inspection

Quantifying by using the Qubit, wherein the concentration of the library is more than or equal to 2.0 ng/mu L and reaches the qualified standard of the on-machine;

and (3) performing quality control by adopting an Agilent 2100DNA1000 chip, wherein the size of a quality control fragment is 220 bp-400 bp, and the quality inspection is qualified.

9. And performing on-machine sequencing NextCN500 and PE75 mode double-ended Index sequencing on the library qualified by quality control, and performing bio-signaling flow analysis.

10. The experimental results are as follows: the results obtained are shown in table 1, where mNGS is the test result obtained using the method described in this example:

TABLE 1

As can be seen from the above table, the consistency of the types of pathogenic bacteria of 60 samples is 53.3%;

in 60 experimental samples, 21 virus samples were detected by TNA-mNGS seq, and only 3 virus samples were detected clinically.

At present, the clinical microorganism detection method has limitations in the aspects of sensitivity, specificity, timeliness, information content and the like, and the sensitivity of separation and culture in clinical detection is about 34.2%, so that the detection result obtained by the TNA-mNGS seq provided by the invention is in an acceptable range.

Example 2

This example simulates the sequencing of TNA by mixing a clinically positive DNA pathogen sample with a clinically positive RNA pathogen sample, and the results are shown in Table 2 below:

TABLE 2

As can be seen from the above table, the sequences are consistent with the reads with higher abundance detected from the DNA sequencing result alone and the RNA sequencing result alone. Therefore, the kit and the detection method can simultaneously detect DNA virus and RNA virus infected samples.

In conclusion, the method for constructing the pathogenic microorganism sequencing library based on the metagenomics and the TNA has high sensitivity and accuracy and high coverage rate, so that the condition of missed detection is not easy to occur in the process of detecting the infection sample, and an effective and reliable basis can be provided for the diagnosis of the disease state of the infected patient.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Claims (10)

1. A method for constructing a sequencing library of a pathogenic microorganism based on total nucleic acids and metagenomics, the method comprising the steps of:

(1) respectively extracting DNA and RNA in a sample infected by pathogenic microorganisms, enriching the RNA, and mixing the obtained DNA and RNA to obtain total nucleic acid serving as a sample to be detected;

or directly extracting total nucleic acid in a microorganism-infected sample as a sample to be detected;

(2) the sample to be detected contains DNA and RNA of pathogenic microorganisms, then the RNA in the sample to be detected is fragmented, and simultaneously, one-chain cDNA synthesis is carried out;

(3) after the first-strand cDNA synthesis is finished, synthesizing second-strand cDNA to obtain a cDNA/DNA compound;

(4) the cDNA/DNA complex is broken by using a fragmentation enzyme, and terminal repair and tailing reaction, linker ligation, PCR enrichment and purification are carried out to obtain a sequencing library constructed based on total nucleic acid.

2. The method according to claim 1, wherein the pathogenic microorganisms of step (1) comprise any one or a combination of at least two of bacteria, fungi, DNA viruses, RNA viruses, chlamydia, mycoplasma or parasites;

the sample infected by the pathogenic microorganism in the step (1) comprises any one or the combination of at least two of alveolar lavage fluid, sputum, cerebrospinal fluid, pleural effusion or punctured tissue;

directly extracting total nucleic acid in cerebrospinal fluid and/or punctured tissues in the step (1) to be used as a sample to be detected.

3. The method according to claim 1, wherein the enrichment reaction system of step (1) comprises Turbo dnase and/or Baseline dnase.

4. The method according to claim 1, wherein the reaction system of step (2) comprises DNA, RNA, reverse transcriptase, RNA fragmenting enzyme and dNTPs in the sample;

the volume ratio of the reverse transcriptase to the RNA fragmentation enzyme is 1 (5-8);

the reaction conditions for the single-strand cDNA synthesis are as follows:

the reaction is carried out for 5-10 min at 20-30 ℃, then for 10-15 min at 40-42 ℃, and then for 10-20 min at 65-70 ℃.

5. The method according to claim 1, wherein the reaction system of step (3) comprises DNA, single-strand cDNA, DNA polymerase and ribozyme-free water in the sample;

the reaction conditions for the two-strand cDNA synthesis are as follows:

the reaction is carried out for 20-40 min at 20-30 ℃ and then for 10-15 min at 60-65 ℃.

6. The method according to claim 1, wherein the reaction system of the end repairing and tailing reaction of step (4) comprises: DNA fragmentation enzyme and corresponding premixed buffer, tail end repair-tailing enzyme A and corresponding premixed buffer, DNA fragmentation-tailing enzyme A enhancer and reinforced premixed buffer;

the reaction temperature of the joint connection in the step (4) is 22-26 ℃, and the reaction time is 10-15 min.

7. The method according to claim 6, wherein in the adaptor-ligated reaction system in step (4), the ratio of the amount of fragmented DNA to adaptor used is (15-20) ng:1 μ L;

the concentration of the sequencing library in the step (4) is more than or equal to 2.0 ng/mu L;

the length of the DNA fragment in the sequencing library in the step (4) is 220 bp-400 bp.

8. The method according to claim 1, wherein the method for purifying nucleic acid in the method is a magnetic bead purification method;

the method further comprises the step of subjecting the sequencing library to high throughput sequencing and bioinformatic analysis.

9. Method according to claim 1, characterized in that it comprises the following steps:

(1) extracting DNA and RNA in alveolar lavage fluid, sputum or pleural effusion of an infected patient respectively, enriching the RNA, and mixing the obtained DNA and RNA to obtain total nucleic acid serving as a sample to be detected;

or directly extracting total nucleic acid in cerebrospinal fluid or punctured tissue of the infected patient to be used as a sample to be detected;

(2) mixing the sample to be detected with reverse transcriptase, RNA fragmentation enzyme and dNTPs, fragmenting and reverse transcribing RNA in the sample to be detected, and simultaneously performing one-strand cDNA synthesis;

the reaction conditions for the single-strand cDNA synthesis are as follows: firstly reacting for 5-10 min at 20-30 ℃, then reacting for 10-15 min at 40-42 ℃, and then reacting for 10-20 min at 65-70 ℃;

(3) mixing the product obtained by synthesizing the first-strand cDNA with DNA polymerase, and synthesizing second-strand cDNA to obtain a cDNA/DNA compound;

the reaction conditions for the two-strand cDNA synthesis are as follows: firstly reacting for 20-40 min at 20-30 ℃, and then reacting for 10-15 min at 60-65 ℃;

(4) fragmenting the cDNA/DNA compound by using a fragmenting enzyme, and performing end repair, tailing reaction and joint connection, wherein the usage amount ratio of the fragmented DNA to the joint in a joint connection reaction system is (15-20) ng:1 muL;

after the joint connection is finished, PCR enrichment and purification are carried out to obtain a sequencing library constructed based on total nucleic acid, the concentration of the obtained sequencing library is more than or equal to 2.0 ng/mu L, and the length of the DNA fragment is 220 bp-400 bp.

10. A kit for constructing a sequencing library for a pathogenic microorganism using the method of any one of claims 1 to 9, the kit comprising:

DNA and RNA extraction fractions;

RNA enrichment components including Turbo DNase, Baseline DNase and RNA adsorption columns;

RNA reverse transcription component, including reverse transcriptase, reverse transcription reaction buffer solution and primer;

a fragmentation component comprising an RNA fragmentation enzyme and a DNA fragmentation enzyme;

the cDNA/DNA compound library building component comprises DNA fragmenting enzyme and corresponding premixed buffer solution, terminal repair-tailing enzyme A and corresponding premixed buffer solution, DNA fragmentation-tailing enzyme A enhancer, reinforced premixed buffer solution and a joint.