CN113980953A

CN113980953A - Method for efficiently and rapidly preparing double-stranded cDNA

Info

Publication number: CN113980953A
Application number: CN202111284967.3A
Authority: CN
Inventors: 陈凤; 舒小婷; 蔡昀; 魏少华
Original assignee: Zhongke Oumeng Weiyi Beijing Medical Technology Co ltd
Current assignee: Zhongke Oumeng Weiyi Beijing Medical Technology Co ltd
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2022-01-28

Abstract

The invention discloses a method for preparing double-stranded cDNA (complementary deoxyribonucleic acid) from RNA (ribonucleic acid) efficiently and quickly, which is used for macro-transcriptome analysis of clinical samples by a second-generation sequencing technology. The macro-transcriptome analysis is used for detecting pathogens on the RNA level, can detect RNA viruses and DNA pathogens with transcriptional activity in clinical samples, and is very important for detecting the pathogens in the clinical samples. The macro-transcriptome analysis scheme comprises the following parts: total RNA extraction, reverse transcription and double-strand cDNA synthesis, library preparation, library quantification and computer processing, data analysis and report interpretation. The invention optimizes the RNA reverse transcription and two-strand cDNA synthesis method of macro transcriptome analysis, transposase is adopted to construct a library, the construction of the RNA library is completed within 4 hours, the RNA reverse transcription and the two-strand cDNA synthesis are completed within 1 hour from the original 2-3 hours, the pathogen detection efficiency is improved, and the number of detected sequences of the pathogen is improved by 2-4 times.

Description

Method for efficiently and rapidly preparing double-stranded cDNA

Technical Field

The invention relates to a method for efficiently and quickly preparing double-stranded cDNA (complementary deoxyribonucleic acid), which can be used for macro-transcriptome analysis of clinical samples of a second-generation sequencing technology.

Background

Infectious diseases are key diseases threatening human health in today's world. The pathogeny presents the development trend of diversification and complication, various newly and recurrent infectious diseases, multiple infections which are not easy to discover and fever with unknown reasons all bring great threat to human health, so that higher requirements on the accuracy and timeliness of infectious disease detection are clinically provided.

There are two traditional methods of pathogen detection: one is a culture-based method, which is a gold standard for identifying pathogenic microorganisms, which relies on the sample and is very strict in experimental and technical conditions, limiting its wide application; the other method is a method based on specific primers, probes and antibodies, such as antigen-antibody reaction, PCR reaction and other specific rapid detection systems, and the method can only detect known pathogens and cannot deal with unknown and artificially modified pathogens.

The metagenome next generation sequencing (mNGS) technology directly detects pathogenic microorganism sequences aiming at nucleic acid in a specimen without bias. Different from the traditional detection, the metagenome detection has high sensitivity and high accuracy, and has unique value for etiological diagnosis of critical severe infection and new emergent infectious disease of unknown reasons.

Metagenomic sequencing is at the DNA level, while macrotranscriptome sequencing is at the RNA level to detect pathogens. Compared with the condition that the metagenome sequencing cannot distinguish whether the DNA is from an organism with life or without life, the macrotranscriptome sequencing can identify RNA viruses and microorganisms with transcriptional activity. Compared with the metagenome, the preparation of the macrotranscriptome sample is complex, reverse transcription and double-strand synthesis are required after RNA extraction, double-strand cDNA is converted and then library construction is carried out, the experiment cost is relatively high, and the experiment period is long.

The macro transcriptome process mainly comprises the following processes: preparing total RNA; reverse transcription and cDNA double-strand synthesis; constructing a library; high-throughput next-generation sequencing and bioinformatics analysis.

The macro transcriptome analysis has high requirement on the RNA purity of a sample, DNA pollution needs to be strictly removed after RNA extraction, and library construction can be carried out after the RNA is converted into a cDNA double chain through reverse transcription and double-chain synthesis. A commercial kit for RNA library construction is available from NEB corporation

Ultra II RNA Library Prep Kit, Nugen corporation Ovation RNAseq v2 Kit, Epicentre corporation Script Seq v2 RNA-Seq Kit etc. Of NEB Corp

The reaction time of reverse transcription and double-strand synthesis of the Ultra II RNA Library Prep Kit is long, and the reaction needs 2 to 3 hours to complete; after reverse transcription and two-strand synthesis, a conventional library construction method is adopted for library construction, and the RNA library construction experiment period is long. The subvertion RNAseq v2 kit reverse transcription and double-strand synthesis of Nugen company also need 2-3 hours to be finished, the Script Seq v2 RNA-Seq kit of Epicentre company and the reverse transcription and library construction are carried out simultaneously, the library construction time is short, but the RNA initial amount needs to be increased>0.5ng, not applicable to macrotranscriptional microsample.

Different from the conventional RNA library establishment, aiming at the macro-transcriptome analysis of pathogenic microorganisms, the concentration of RNA extracted from clinical samples such as cerebrospinal fluid, blood and the like is very low, and in addition, the experiment period required by the macro-transcriptome RNA library establishment is short. As mentioned above, similar products in the prior art have the defects of long cDNA synthesis time, long library construction time, difficulty in constructing a small reaction system, inapplicability to macrotranscriptome micro samples and the like. Therefore, there is a need for efficient and rapid reverse transcription and two-strand synthesis systems, as well as cDNA synthesis systems suitable for micro samples, which meet the requirement of micro RNA library construction for macro transcriptome.

Disclosure of Invention

Unless otherwise defined, all techniques and terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated, the techniques employed and covered herein are standard procedures well known to those skilled in the art to which the invention pertains. The materials, methods, and examples are illustrative only and are not intended to limit the scope of the present invention in any way.

In order to overcome the defects of the prior art, a reverse transcriptase reaction system with excellent performance on the market is screened for testing. According to the principle of double-strand synthesis, a double-strand synthesis system is optimized, including double-strand synthetase mix and reaction liquid of double-strand synthesis, and the reverse transcription and the double-strand synthesis time is shortened to be completed within 1 hour.

The invention provides a scheme for quickly establishing a micro-sample macro-transcriptome RNA, wherein a transposase is adopted for quickly establishing a library after RNA reverse transcription and two-strand synthesis. RNA reverse transcription and double-strand synthesis can be completed within 1 hour, RNA library construction can be completed within 4 hours, and the requirements of low initial quantity, high sensitivity and short experimental period of macrotranscription analysis of clinical samples are met.

In order to achieve the above objects, the present invention, in a first aspect, relates to a method for efficiently preparing double-stranded cDNA, the method comprising the steps of:

a) reverse transcription: carrying out reverse transcription by taking the prepared total RNA as a template to obtain a first chain of cDNA;

b) second strand cDNA Synthesis: synthesizing a second strand of the cDNA by using a double-strand synthesis reaction solution and a double-strand synthetase mix containing DNA polymerase and using a first strand of the cDNA synthesized by reverse transcription as a template;

c) double-stranded cDNA was obtained by purification.

In some embodiments, said step b) is performed at a suitable temperature, a second strand of cDNA can be synthesized in a short time, and said synthesizing step is adapted to a small volume reaction system; in some embodiments, the double-strand synthetase mix is a double-strand synthetase or a combination conventionally used in the art during cDNA synthesis; in some embodiments, the two-strand synthetase mix in step b) comprises one or more of the following: DNA polymerase I (Klenow large fragment), Sequenase V2.0DNA polymerase, Escherichia coli DNA polymerase I, RNaseH, T4 DNA ligase or Escherichia coli DNA ligase.

In other embodimentsIn step b), the reaction solution for the two-chain synthesis contains Mg²⁺、Mn²⁺、Ca²⁺One or more of; and one or more of Tris-HCl, 3(N morpholine) propanesulfonic acid and sodium citrate; DTT; KCl; dNTP, etc.

Preferably, the two-strand synthetase mix comprises Sequenase V2.0DNA polymerase and RNaseH; the reaction solution for the two-chain synthesis comprises Tris-HCl buffer solution and MgCl₂DTT, etc.

Further, the reagents of the reverse transcription step and the second strand synthesis step are respectively prepared into corresponding mixed solutions for each step.

In another aspect, the present invention also relates to a method for second-generation sequencing of a macro-transcriptome, comprising the steps of:

1) collecting a clinical patient sample;

2) total RNA extraction: extracting total DNA/RNA of the sample, and incubating with nuclease to remove DNA to obtain total RNA of the sample;

3) reverse transcription: preparing a reverse transcription reaction system which comprises a template RNA, a reverse transcription buffer, a random primer and a reverse transcriptase to obtain a cDNA first chain;

4) second strand cDNA Synthesis: synthesizing a second strand of cDNA by using a first strand of cDNA synthesized by reverse transcription as a template to obtain double-stranded cDNA, wherein the second strand of cDNA comprises a cDNA product synthesized by reverse transcription, a second strand reaction solution and a second strand synthetase mix;

5) purifying the double-stranded cDNA;

6) library construction: a transposase library building method is adopted, and the purified library is analyzed by a biological analyzer; (ii) a

7) Library quantification and Pooling;

8) high throughput sequencing and bioinformatic analysis.

As used herein, the term "sample" generally refers to a sample for sequencing or phasing in a biological fluid, cell, tissue, organ, or organism comprising a nucleotide or mixture comprising at least one nucleotide sequence, including, but not limited to sputum/oral fluid, amniotic fluid, blood, a portion of blood, a fine needle biopsy sample (e.g., surgical biopsy, fine needle biopsy, etc.), urine, peritoneal fluid, pleural fluid, tissue explant, organ or tissue culture, or cell preparation. The sample from an organism is typically taken from a human subject (e.g., a patient), or taken from any chromosome bearing organism, including but not limited to dogs, cats, horses, goats, sheep, cattle, pigs, and the like. Samples obtained from biological sources or from pre-treatment to modify their characteristics can also be used directly, such as for example the preparation of plasma from blood, the dilution of viscous liquids, etc.

In some embodiments, the clinical sample described herein is selected from one or more of: blood, lymph, cerebrospinal fluid, alveolar lavage, bronchial lavage, sputum, pleural effusion, urine, saliva, nasopharyngeal secretions or extracts or other tissue or body fluid samples.

In some embodiments, for trace clinical samples, the sample is stored in a virus collection tube, shaken, and then frozen at-80 ℃ or subjected to a second step of processing; the trace clinical sample such as a nasopharyngeal swab; preferably, the frozen stock is kept frozen at-80 ℃ for use within 1 hour with shaking for 30 s.

In other embodiments, e.g., nasopharyngeal secretion extracts, alveolar lavage fluid, sputum, etc., a liquefying agent is added first, and a second treatment or cryopreservation is performed; preferably, the frozen stock is stored at-80 ℃ within 1 hour.

In some embodiments, the total sample RNA obtained is 20-40. mu.L, preferably 20. mu.L.

In some embodiments, wherein the two-strand synthetase mix in step 4) comprises one or more of: DNA polymerase I (Klenow large fragment), Sequenase V2.0DNA polymerase, Escherichia coli DNA polymerase I, RNaseH, T4 DNA ligase or Escherichia coli DNA ligase.

In other embodiments, the reaction solution for the two-chain synthesis in step 4) contains Mg²⁺、Mn²⁺、Ca²⁺One or more of; and one or more of Tris-HCl, 3(N morpholine) propanesulfonic acid and sodium citrate; DTT; KCl; dNTP, etc.

In a preferred embodiment, the two-strand synthetase mix comprises Sequenase V2.0DNAPolymerase and RNaseH; the reaction solution for the two-chain synthesis comprises Tris-HCl buffer solution and MgCl₂DTT, etc.

In some embodiments, the reverse transcriptase is a reverse transcriptase system conventionally used in the art; the reverse transcriptase can be selected, for example, from SuperScirpt III reverse transcriptase (ThermoFisher), Takara reverse transcriptase or the NEB one-strand Synthesis Module (TM) ((TM))

Ultra^TMII RNA First Strand Synthesis Module E7771)。

In some embodiments, the reverse transcription reaction system further comprises a reverse transcription reaction solution (buffer), dntps, random primers and the like, wherein the reverse transcription reaction solution (buffer) is often a reagent for reverse transcriptase, for example, the reaction solution composition of the supersscirpt III reverse transcriptase comprises Tris-HCl, KCl, DTT, dntps and the like.

In another aspect, the present invention also relates to a kit for preparing double-stranded cDNA, comprising a reverse transcriptase, a double-stranded synthetase mix, a reverse transcription reaction solution, and a double-stranded synthesis reaction solution; wherein the two-strand synthetase mix comprises one or more of DNA polymerase I (Klenow large fragment), Sequenase V2.0DNA polymerase, E.coli DNA polymerase I, RNaseH, T4 DNA ligase, or E.coli DNA ligase.

In some embodiments, wherein said reaction solution for two-chain synthesis comprises Mg²⁺、Mn²⁺、Ca²⁺One or more of; and one or more of Tris-HCl, 3(N morpholine) propanesulfonic acid and sodium citrate, DTT, KCl, dNTP and the like.

In a preferred embodiment, in the kit, the two-strand synthetase mix comprises Sequenase V2.0DNA polymerase and RNaseH; the reaction solution for the two-chain synthesis comprises Tris-HCl buffer solution and MgCl₂DTT, etc.

The term "sequencing" refers to determining the identity of one or more nucleotides, i.e., whether a nucleotide is G, A, C, T or U.

As used herein, "second generation sequencing" includes sequencing by synthesis techniques (e.g., Illumina), pyrosequencing (454), Ion semiconductor technology (Ion Torrent sequencing), and the like.

In some embodiments, the sequencing is deep sequencing by a NextSeq CN500 sequencer; in a specific embodiment, the sequencing method is SE50, SE75, SE100, SE150, SE200, PE50, PE100, PE150 or PE200, preferably, SE 75.

In another aspect, the invention provides a complete macro-transcriptome next-generation sequencing method, which is implemented by the following technical scheme:

1) collecting clinical patient samples: fresh samples or frozen samples at-80 ℃. If the nasopharynx test sample is a trace amount, the nasopharynx test sample is firstly stored in a virus collecting tube, is shaken for 30s, and is subjected to the second step of treatment or is frozen and stored at minus 80 ℃ within 1 hour. Other nasopharyngeal secretion extracts, alveolar lavage fluid, sputum and the like need to be added with a liquefier for the second step of treatment or frozen storage at minus 80 ℃ within 1 hour.

2) Total RNA extraction: and extracting total DNA/RNA in the sample, and incubating with nuclease to remove DNA to obtain total RNA in the sample, wherein the RNA elution volume is 20 mu L.

3) Reverse transcription and second strand cDNA synthesis:

composition (I)	Volume (μ L)
		RNA template	5～10
Reverse transcription buffer (5X)	4
		Random Primers	5～10
Reverse transcriptase	1
		Ribozyme-free water	X
Total up to	20

Reverse transcription: and (2) preparing a reverse transcription reaction system as shown above, adding the template, the reverse transcription buffer and the random primer into a PCR tube, uniformly mixing, carrying out warm bath at 50-68 ℃ for 3-8 minutes, and then incubating on ice for 1-3 minutes. Adding reverse transcriptase by instantaneous centrifugation, uniformly mixing, incubating at 25-30 ℃ for 10 minutes, incubating at 40-50 ℃ for 15-30 minutes, incubating at 80 ℃ for 5 seconds-5 minutes to inactivate the reaction, and cooling at 4 ℃.

Synthesis of second Strand of cDNA: the cDNA product synthesized by reverse transcription is 10-50 mu L, and after the components shown in the specification are added on an ice bath, the cDNA product is incubated for 5-30 minutes at the temperature of 16-37 ℃, and then cooled at the temperature of 4-10 ℃.

Composition (I)	Volume (μ L)
		cDNA products	20
Double-chain reaction liquid	4～10
		Two-chain synthetase mix	1～4
Ribozyme-free water	Y
		Total up to	30～50

4) Double-stranded cDNA purification: the product was purified by magnetic bead method or column DNA purification kit, and eluted with 10. mu.L of nuclease-free water.

5) Library construction: using transposase library construction kit to construct the library, operating according to the instruction, and verifying the purified library by using a bioanalyzer, wherein the library is between 300 and 700 bp.

6) High-throughput sequencing and bioinformatic analysis: deep sequencing is carried out by a Berry Nextseq CN500 sequencer in a manner of SE75, SE150, PE150 and the like, and SE75 is preferred.

Compared with the conventional method, the method has the advantages that:

1) the method is suitable for, but not limited to, the detection of second-generation sequencing macrotranscriptome of samples such as cerebrospinal fluid, blood, alveolar lavage fluid, abscess tissue and the like of human and animals, can reduce the synthesis system of system cDNA to 30-50 mu L, and is more suitable for the reaction of a macrotranscriptome micro-system compared with the system of 80 mu L in the prior art;

2) reverse transcription and two-chain synthesis reagents are respectively prefabricated into mixed liquid, so that the steps of manual operation are reduced; the cost of the reagent is greatly reduced, the efficiency of the reverse transcription reaction and the two-chain synthesis reaction is improved, the reaction speed is accelerated, and the components in each mixed solution are not influenced by each other;

3) the time required by reverse transcription and two-chain synthesis is greatly reduced, the reaction can be completed within 1 hour, and compared with other products in the prior art, the reaction time is reduced by nearly 1 time; 4) the cDNA synthesis step of a micro system is also suitable for the subsequent micro RNA library building step, the reaction system is small and can be completed in a 200 mu L PCR reaction tube;

5) the invention improves the pathogen detection sensitivity, adopts improved reverse transcription and two-chain synthesis reagents, accelerates the cDNA step, and is matched with a rapid transposase library construction method, so that the positive pathogen Reads is improved by several times compared with the prior art method.

Drawings

FIG. 1: and (5) detecting a flow chart.

Detailed Description

The present invention will be described in further detail with reference to the following drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1: efficient reverse transcriptase system screening

The reverse transcription step compares several products: NEB one-chain Synthesis Module (

Ultra^TMII RNA First Strand Synthesis Module E7771), ThermoFisher corporation SSIII reverse transcriptase reaction system and Takara reverse transcriptase reaction system in three groups; the two-chain synthesis all uses NEB two-chain synthesis module (

Ultra^TMII RNA Second Strand Synthesis Module E6111) for double-stranded cDNA Synthesis. The reverse transcription reaction conditions are shown in Table 1.

1) The test is carried out by adopting 4 cases of simulated positive samples, and the respiratory syncytial virus A, the influenza A virus and the enterovirus are added into the 4 cases of negative samples when the simulated positive samples are prepared, and the concentration is 500-2000 copies/mL.

2) Each sample was tested for 3 reverse transcription conditions, so 4X 400. mu.L of each of the 4 samples were taken for RNA extraction.

3) Reverse transcription and second strand synthesis of cDNA: for a total of 4 samples, 3 reverse transcription comparisons were performed per sample for a total of 12 reactions.

Table 1: reverse transcription reaction System screening

4) Double-stranded cDNA purification: the product was purified using Zymo DNA Clean & concentrate Kits and eluted with 10. mu.L of nuclease-free water.

5) Library construction: and fragmenting the DNA fragment with a Novopoppy Kit DNA Library Prep Kit V2 for Illumina, and adding a specific linker at both ends of the fragmented DNA fragment.

6) And (3) quantifying and testing quality to obtain a DNA library: in the fragment selection, large fragments were removed by using 0.7-fold volume of magnetic beads of the purified product, and small fragments were removed by using 0.15-fold volume of magnetic beads of the purified product, thereby obtaining the optimal length of library fragments, and the average length of the library was about 325 bp.

7) And (3) performing on-machine sequencing, and analyzing off-machine data: after the data were de-humanized, low quality and low complexity sequences were removed, the sequence pathogens were annotated with Kraken rapid classification software and possible pathogenic pathogens were reported. The reverse transcriptase used in the invention has the best detection effect on pathogens with known low copy number, and the detection rate of the reaction on effective pathogens is 100%. The reverse transcriptase of Thermo company has high efficiency. The specific results are shown in Table 2. The mean of the numbers of detected pathogens in 4 parallel samples of each group was statistically analyzed and the results are shown in Table 2.

Table 2: detection of pathogen sequence number

Example 2: comparative testing of reverse transcription and two-chain Synthesis systems

RNA reverse transcription respectively adopts NEB single strand synthesis module (

Ultra^TMII RNA First Strand Synthesis Module E7771) and the SSIII reverse transcription system of ThermoFisher company, wherein the two-Strand Synthesis respectively adopts NEB two-Strand Synthesis Module(s) ((II)

Ultra^TMII RNA Second Strand Synthesis Module E6111) and Sequenase V2.0DNA polymerase reaction system, the reaction conditions are shown in Table 3.

1) The test was performed using 3 mock-positive samples, and the mock-positive sample preparation chamber added respiratory syncytial virus a, influenza a virus and enterovirus to the 3 negative samples at a concentration of 500-.

2) 4 conditions were tested per sample, so 4X 400. mu.L of each of 3 samples was taken for RNA extraction.

3) Reverse transcription and duplex synthesis: 3 samples, each of which was subjected to 4 reverse transcription/duplex synthesis reactions, and 12 reactions in total, the reaction conditions and reagents are shown in Table 3 below, and the duplex synthesis reaction solutions of test examples 1 and 2 included: Tris-HCl, MgCl₂KCl, DTT, dNTP, etc.; the reaction solution for two-chain synthesis of comparative example was the system used for NEB.

Table 3: reverse transcription and two-Strand cDNA Synthesis System test

4) The product was purified using a DNA purification kit from Zymo.

5) Library samples were prepared as in example 1, fragmented using the Novozam Kit DNA Library Prep Kit V2 for Illumina, and specific linkers were added to both ends of the fragmented DNA fragments.

7) And (3) performing machine sequencing, and analyzing the machine-unloading data: after the data is humanized, low-quality and low-complexity sequences are removed, the sequences are annotated and analyzed by Kraken rapid classification software to report possible pathogenic pathogens.

8) The results show that the effective pathogen reads number detected by the process of the invention can be higher than 4 times that of the table 4.

Table 4: pathogen detection Reads number

As can be seen from Table 3 of this example, whereas in the comparative example, the total time required was about 1 hour and 35 minutes, the two-strand synthesis reaction system was 80. mu.L, whereas in test examples 1 and 2, the total time required for completion of the reverse transcription step and the two-strand synthesis step was only about 50min, and the two-strand synthesis reaction system could be reduced to 30. mu.L; compared with the prior art (such as a comparative example), the method has the advantages that the reaction container does not need to be replaced, and the operation is more convenient and faster. Therefore, the method can finish the conversion of RNA into double-stranded cDNA in a micro reaction system, is convenient for subsequent purification and library construction of the double-stranded cDNA, and is more suitable for a micro sample macro transcriptome analysis process.

As can be seen from Table 4, the detection sensitivity of the test example 1 and 2 processes to the pathogen reads number is higher, and it can be seen that the process of the present invention has higher efficiency, higher speed and higher enzyme conversion efficiency.

Example 3: clinical sample full-flow detection

The NEB and the reverse transcription and two-strand cDNA synthesis embodiments of the present invention were compared by taking as an example the respiratory tract samples from patients with pulmonary disease, 9 samples diagnosed with human respiratory syncytial virus, rhinovirus and coronavirus collected from 10 months to 2021 months of 2020.

RNA reverse transcription and double-strand synthesis respectively adopt NEB module (

Ultra^TMII RNA First Strand Synthesis Module E7771 and

Ultra^TMII RNA Second Strand Synthesis Module E6111) and SSIII reverse transcription + Sequenase V2.0DNA polymerase reaction system, and the purified double-stranded cDNA is constructed by a transposase library construction kit.

1) Positive samples of 9 human respiratory syncytial viruses were used.

2) Reverse transcription and duplex synthesis: 9 samples were subjected to 2 reverse transcription/double-strand synthesis reactions each, and 18 reactions were carried out in total, and the reaction conditions and reagents were as in test example 1 and comparative example 1 of Table 3.

3) The product is purified using dsDNA purification beads or a kit.

4) Library samples were prepared as in example 1, fragmented using the Novozam Kit DNA Library Prep Kit V2 for Illumina, and specific linkers were added to both ends of the fragmented DNA fragments.

5) And (3) quantifying and testing quality to obtain a DNA library: in the fragment selection, large fragments were removed by using 0.7-fold volume of magnetic beads of the purified product, and small fragments were removed by using 0.15-fold volume of magnetic beads of the purified product, thereby obtaining the optimal length of library fragments, and the average length of the library was about 325 bp.

6) And (3) performing machine sequencing, and analyzing the machine-unloading data: after the data is humanized, low-quality and low-complexity sequences are removed, the sequences are annotated and analyzed by Kraken rapid classification software to report possible pathogenic pathogens.

7) The results show that the efficiency of converting RNA into double-stranded cDNA in the process of the invention is better than that of the control group, and the number of detected pathogen reads is higher than that of the control group, which is shown in Table 5.

Table 5: reads number for detection of positive pathogen

The above description is only an example of the present invention and is not intended to limit the present invention, and modifications and variations of the present invention may occur to those skilled in the art. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for efficiently preparing double-stranded cDNA, comprising the steps of:

b) second strand cDNA Synthesis: synthesizing a second strand of cDNA by using a double-strand synthesis reaction solution and a double-strand synthetase mix containing DNA polymerase and using a first strand of cDNA synthesized by reverse transcription as a template, wherein the synthesizing step is applicable to a small-volume reaction system;

c) purifying to obtain double-chain cDNA;

wherein the diastrase synthetase mix in step b) comprises one or more of the following: DNA polymerase I, Sequenase V2.0DNA polymerase, Escherichia coli DNA polymerase I, RNaseH, T4 DNA ligase, Escherichia coli DNA ligase; the DNA polymerase I is preferably Klenow large fragment DNA polymerase.

2. The method according to claim 1, wherein the reaction solution for the two-chain synthesis in step b) contains Mg²⁺、Mn²⁺、Ca²⁺One or more of; and one or more of Tris-HCl, 3(N morpholine) propanesulfonic acid and sodium citrate; DTT; KCl and dNTP.

3. A method for second-generation sequencing of a macrotranscriptome, comprising the steps of:

1) collecting a clinical patient sample;

4) second strand cDNA Synthesis: synthesizing a second strand of cDNA by using a first strand of cDNA synthesized by reverse transcription as a template to obtain double-stranded cDNA, wherein the second strand of cDNA comprises a cDNA product synthesized by reverse transcription, a double-strand reaction solution and a double-strand synthetase mix containing DNA polymerase;

5) purifying the double-stranded cDNA;

6) library construction: a transposase library building method is adopted, and the purified library is analyzed by a biological analyzer;

7) library quantification and Pooling;

8) high-throughput sequencing and bioinformatics analysis;

wherein the double-strand synthetase mix in the step 4) comprises one or more of the following: DNA polymerase I, Sequenase V2.0DNA polymerase, Escherichia coli DNA polymerase I, RNaseH, T4 DNA ligase, Escherichia coli DNA ligase; the DNA polymerase I is preferably Klenow large fragment DNA polymerase.

4. The method according to claim 3, wherein the reaction solution for the two-chain synthesis in the step 4) contains Mg²⁺、Mn²⁺、Ca²⁺One or more of; and one or more of Tris-HCl, 3(N morpholine) propanesulfonic acid and sodium citrate; DTT; KCl and dNTP.

5. The method of any one of claims 1-4, wherein the duplex synthetase mix comprises Sequenase V2.0DNA polymerase and RNaseH; the reaction solution for the two-chain synthesis comprises Tris-HCl buffer solution and MgCl₂、DTT。

6. The method of any one of claims 1 to 4, wherein the reagents of the reverse transcription step and the second strand cDNA synthesis step are each pre-prepared as a mixture for the respective synthesis step.

7. The method of any one of claims 3-6, wherein the clinical sample is from cerebrospinal fluid, blood, alveolar lavage, nasopharyngeal secretions/extracts, sputum, or abscess tissue of a human or animal.

8. The method according to any one of claims 3-6, wherein the sequencing step employs Nextseq CN500 sequencing in the manner of SE75, SE150 or PE150, preferably SE 75.

9. The method according to any one of claims 1 to 8, wherein in the reverse transcription step, the reverse transcriptase is selected from SuperScirpt III reverse transcriptase from ThermoFisher, Takara reverse transcriptase or

Ultra^TMII RNA First Strand Synthesis Module E7771; preferred SuperScirpt III reverse transcriptases and

Ultra^TMII RNA First Strand Synthesis Module E7771。

10. a kit for preparing double-stranded cDNA, characterized in that the kit comprises a reverse transcriptase, a double-stranded synthetase mix, a reverse transcription reaction solution and a double-stranded synthesis reaction solution; wherein the double-stranded synthetase mix comprises one or more of DNA polymerase I, Sequenase V2.0DNA polymerase, RNaseH, T4 DNA ligase or E.coli DNA ligase; wherein the DNA polymerase I is preferably Klenow large fragment DNA polymerase I or Escherichia coli DNA polymerase I.

11. The kit of claim 10, wherein the reaction solution for the two-chain synthesis comprises Mg²⁺、Mn²⁺、Ca²⁺One or more of; one or more of Tris-HCl, 3(N morpholine) propanesulfonic acid and sodium citrate; DTT; KCl and dNTP.

12. The kit according to claim 10 or 11, wherein the duplex synthetase mix comprises Sequenase V2.0DNA polymerase and RNaseH; the reaction solution for two-chain synthesisComprising Tris-HCl buffer, MgCl₂、DTT。