Method for detecting microbial genome RNA based on mNGS technology

Abstract

The invention discloses a method for detecting microbial genome RNA based on an mNGS technology. The invention provides a method for constructing an RNA sequencing library, which comprises the following steps: A1) fragmenting an RNA sample to obtain fragmented RNA; a2) carrying out reverse transcription and terminal repair reaction on the fragmented RNA in the following multi-enzyme reaction system to obtain double-chain cDNA (complementary deoxyribonucleic acid) with flat terminal and containing A basic group; and A3) sequentially carrying out joint connection and PCR amplification on the double-chain cDNA with the flat tail end and containing the A basic group to obtain the RNA sequencing library. According to the technical scheme, operation is easy, time consumption is short, reaction conditions and DNA library construction are the same, operation and reaction can be conducted at the same time, the initial amount of RNA is 100 pg, and daily requirements of microbiological detection are met.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for detecting microbial genome RNA based on mNGS technology. Background Art

[0002] The widespread application of high-throughput sequencing technology in the field of microbial detection has made microbial detection more and more convenient and faster. This technology can also accurately identify microorganisms (including newly discovered microbial species) that cannot be accurately identified by traditional detection methods. However, there are still some problems in the detection of microbial genomic RNA.

[0003] Most of the RNA library construction methods currently used in metagenomic next-generation sequencing (mNGS) are derived from the RNAseq method. The RNA obtained after extraction and purification needs to go through a reverse transcription step to obtain DNA. Reverse transcription includes two steps: synthesis of the first strand of cDNA and synthesis of the second strand of cDNA. Then the library is built in the same way as DNA. DNA library construction includes steps such as nucleic acid fragmentation, end repair, A addition, adapter connection, PCR amplification, etc. This method has the longest development time and the best library construction effect. However, the experimental cycle is long (possibly >6 hours). Because reverse transcription is required first, and then the library is built. The whole process is much more complicated than starting from DNA. If an experimenter has both DNA library construction needs and RNA library construction needs, since they cannot be carried out at the same time, the workload will be greatly increased.

[0004] Due to the natural instability of RNA, after the efforts of many practitioners, RNA fragmentation can be achieved by heating before reverse transcription, which greatly shortens the nucleic acid fragmentation step. At the same time, by mixing enzymes and optimizing the reaction system, some manufacturers have merged the second-strand synthesis of cDNA and the steps of end repair and A addition into one step, which also shortens the experimental cycle. This method is the method used by the most advertised "one-step" RNA library construction kit on the market, and is an evolution of the RNA library construction method used in the above-mentioned mNGS. However, its experimental cycle and operation process are still longer than the DNA process, and there are also situations where they cannot be operated together. Moreover, due to the integration of the second-strand synthesis and end repair steps, the reaction efficiency has decreased, and higher requirements are placed on the initial nucleic acid material (such as the total amount needs to be greater than 50ng, RIN>7, and the OD value is between 1.8 and 2.1).

[0005] Also for the purpose of shortening the experimental cycle, some researchers have introduced the following method: adding a binding site for a PCR primer to a section of the reverse transcription primer, and after the synthesis of one chain of reverse transcription is completed, PCR amplification is performed based on this site. This method is a relatively innovative method, which is far from the original method, so there is still the problem of not being able to operate simultaneously. Moreover, due to the longer primers for the synthesis of the first and second chains of cDNA and their low reaction efficiency, it requires a higher amount of starting RNA. It is more suitable for scientific research purposes. For clinical-level microbial testing, there may be insufficient nucleic acid. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems of multiple steps and long time for RNA library construction, RNA library cannot be operated together with DNA library, and the difficulty of library construction due to too low starting amount of clinical grade sample nucleic acid. The present invention provides a method for detecting microbial genomic RNA based on mNGS technology.

[0007] In a first aspect, the present invention provides a method for constructing an RNA sequencing library, comprising the following steps:

[0008] A1) fragmenting the RNA sample to obtain fragmented RNA;

[0009] A2) performing reverse transcription and end-repair reaction on the fragmented RNA in the following multi-enzyme reaction system to obtain a double-stranded cDNA with blunt ends and containing A bases;

[0010] The multi-enzyme reaction system comprises the following components: ribonuclease H, DNA polymerase I Klenow fragment, T4 DNA polymerase, polynucleotide kinase, thermostable DNA polymerase and reverse transcriptase;

[0011] A3) The double-stranded cDNA with blunt ends and containing A base is sequentially connected to an adapter and amplified by PCR to obtain an RNA sequencing library.

[0012] In the method described above, the activity unit ratio of the ribonuclease H, DNA polymerase I Klenow fragment, T4 DNA polymerase, polynucleotide kinase (T4 polynucleotide kinase in the embodiment of the present invention), thermostable DNA polymerase (rtaq polymerase in the embodiment of the present invention) and reverse transcriptase is 2:50:10:13:4:333.

[0013] In the method described above, the system further comprises the following components: Mg ions, Tris, dNTP, dATP and buffer.

[0014] The concentration of ribonuclease H in the system is 0.02U / μL

[0015] The concentration of the DNA polymerase I Klenow fragment in the system is 0.5 U / μL;

[0016] The concentration of the T4 DNA polymerase in the system is 0.1 U / μL;

[0017] The concentration of the T4 polynucleotide kinase in the system is 0.13 U / μL;

[0018] The concentration of the rtaq polymerase in the system is 0.04U / μL;

[0019] The concentration of the reverse transcriptase in the system is 3.3 U / μL;

[0020] The concentration of Mg ions in the system is >7.5mmol / L;

[0021] The concentration of Tris in the system is>20mmol / L;

[0022] The concentration of the dNTP in the system is 1.67 mmol / L each;

[0023] The concentration of dATP in the system is 3.33 mmol / L.

[0024] Invention content: Mg ion in the embodiment of the present invention is MgCl2.

[0025] In the method described above, the reaction conditions for reverse transcription and end repair are first incubating at 37° C. for 20 min-40 min, and then incubating at 65-72° C. for 10 min-20 min.

[0026] In the embodiments of the present invention, the temperature is 37° C. for 20 min and the temperature is 72° C. for 10 min.

[0027] In the method described above, the adapter is the adapter corresponding to the MGISEQ sequencing platform;

[0028] Furthermore, the sequencing platform is MGISEQ.

[0029] The RNA sample is derived from a microorganism;

[0030] Furthermore, the microorganism is a virus, a bacterium, a yeast, a mold or a combination thereof.

[0031] In the method described above, the RNA sample is prepared as follows: extracting nucleic acid from the sample to be tested, and then digesting DNA with the nucleic acid to obtain an RNA sample;

[0032] The initial amount of the RNA sample is 10pg-500ng.

[0033] In the examples of the present invention it is 10 pg-50 ng; for higher starting amounts, dilution is required.

[0034] In a second aspect, the present invention provides an RNA sequencing library prepared by the method described in the first aspect.

[0035] In a third aspect, the present invention provides a method for detecting whether a sample to be tested contains a microorganism or its genomic RNA, comprising the following steps:

[0036] B1) extracting nucleic acid from the sample to be tested, and then digesting DNA with the nucleic acid to obtain an RNA sample;

[0037] B2) treating the RNA sample according to step A1) of the method of the first aspect to obtain fragmented RNA;

[0038] B3) treating the fragmented RNA according to step A2) of the method described in the first aspect to obtain a double-stranded cDNA with blunt ends and containing base A;

[0039] B4) treating the double-stranded cDNA with blunt ends and containing base A according to step A3) of the method described in the first aspect to obtain an RNA sequencing library;

[0040] B5) sequencing the RNA sequencing library to detect whether the sample to be tested contains microorganisms or their genomic RNA.

[0041] In a fourth aspect, the present invention provides a kit for constructing the RNA sequencing library described in the second aspect, comprising the following components:

[0042] C1) the multi-enzyme reaction system of the first aspect;

[0043] C2) Reagents or instruments required for nucleic acid extraction;

[0044] C3) Reagents or instruments required for DNA digestion;

[0045] C4) Reagents or instruments required for RNA fragmentation;

[0046] C5) Reagents or instruments required for connecting the connector;

[0047] C6) Reagents or instruments required for PCR amplification.

[0048] In the above, the kit further comprises reagents for preparing a direction-specific RNA library.

[0049] In a fifth aspect, the present invention provides an application of the multi-enzyme reaction system described in the first aspect, the RNA sequencing library described in the second aspect, the kit described in the fourth aspect, or the components shown in C1-C6 in the fourth aspect in any of the following or in the preparation of a product having any of the following functions:

[0050] D1) RNA sequencing;

[0051] D2) Microbial genome RNA sequencing;

[0052] D3) Construction of RNA sequencing library

[0053] D4) Detecting whether the sample to be tested contains microorganisms;

[0054] D5) Detect whether the sample contains microbial genomic RNA.

[0055] The present invention experiments prove that the technical solution of the present invention brings the following beneficial effects:

[0056] 1. Simple operation steps and short time consumption. Reduce steps and reduce manual operations. The method of the present invention is more concise, and the three steps of the traditional method, namely, cDNA first-strand synthesis, cDNA second-strand synthesis, end repair and A addition, are combined into one step of reverse transcription and end repair.

[0057] 2. The reaction conditions are the same as those for DNA library construction (starting from reverse transcription and end repair, the reaction temperature and time are consistent with those of the DNA library, and can be operated on a PCR instrument), and the operation and reaction can be performed simultaneously. The present invention can react with the DNA library on the same PCR instrument under the same conditions. The difference between the two library constructions is reduced. This is more conducive to the arrangement of staff time and is conducive to saving equipment costs for small laboratories.

[0058] 3. While reducing the number of steps, the reaction efficiency is not reduced. The starting amount of 100pg is stable and is not inferior to common kits on the market. The starting amount of the present invention is 100pg RNA, which meets the daily needs of microbial detection; the experimental cycle is 3 hours, which meets the extreme time requirements of mNGS products under the current market model. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is a comparison chart of the microbial genome RNA detection process of the present invention and the traditional method.

[0060] Figure 2 It is the detection effect of the method of the present invention and the traditional detection method.

[0061] Figure 3 This is a screenshot of 2100 quality control. DETAILED DESCRIPTION

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0063] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0064] The following is a detailed description of the method steps, focusing on the reaction system composition of the improved steps. It should be understood that the embodiments are only exemplary and cannot be construed as limiting the scope of protection of the present invention.

[0065] Example 1. Microbial genome RNA detection method

[0066] Figure 1 The figure shows a comparison between the microbial genome RNA detection process in the embodiment of the present invention and the traditional method. The method of the present invention realizes the integration and simplification of steps by adjusting the reaction system such as enzymes and buffers. The method of the present invention includes: sample RNA extraction, DNA removal and RNA purification, fragmentation, reverse transcription and end repair, adapter connection and purification, PCR amplification and purification, and library construction and sequencing according to the standard process of the NGS sequencing platform, and then analyzing the microbial genome RNA.

[0067] In this embodiment, simulated samples were used to test the effect of the RNA library construction method of the present invention on the detection results. The simulated samples were prepared using Hela cells and contained RNA viruses such as Coxsackie virus and MS2 bacteriophage.

[0068] The simulated sample was prepared specifically according to the following method: the components listed in the first column of Table 1 were added to physiological saline, so that their respective final concentrations reached the concentrations indicated in the second column of Table 1.

[0069] The internal standard is an artificially synthesized RNA fragment, the universal internal standard of BGI Medicine's PMseq product, which is added to the simulated sample at a final concentration of 50pg / μL during detection.

[0070] Table 1 shows the contents required for the preparation of simulated samples.

[0071]

[0072]

[0073] The specific steps are as follows:

[0074] 1. Sample nucleic acid extraction

[0075] Use an RNA extraction kit using a column extraction method or a magnetic bead extraction method (the present invention uses QIAGEN's QIAamp Viral RNA Mini Kit, product catalog number Cat. No. 52906), extract nucleic acids from the simulated sample according to the instructions, and dissolve nucleic acids (RNA and DNA) back with nuclease-free water.

[0076] 2. DNA digestion and purification

[0077] The nucleic acid extracted in 1 above was digested with RNase free DNase I to remove DNA from the mixed nucleic acid.

[0078] The details are as follows: take 33 μL of the nucleic acid extracted in 1 above, add 4 μL buffer and 3 μL RNase free DNase I (Novozyme EN401), shake and mix well, incubate at 37°C for 10 minutes, and obtain the digested product.

[0079] After the reaction, Agencourt AMPure XP magnetic beads or any magnetic beads with the same function (in the present invention, Yisheng Bio, product catalog number 12601ES) are used for purification. The operation process is carried out according to the magnetic bead purification instructions, and the nucleic acid is re-dissolved with nuclease-free water to obtain purified RNA.

[0080] 3. RNA fragmentation

[0081] Take the purified RNA obtained in step 2 above and react with Mg-containing 2+ Ionic, homemade or commercial fragmentation buffer is heated for different time periods according to actual needs to achieve RNA fragmentation and obtain fragmented nucleic acids.

[0082] The specific reactions are as follows:

[0083] Take 10 μL of the purified RNA obtained in 2 above, add 12 μL of fragmentation buffer (Novozyme N402-01), shake and mix, incubate at 94°C for 3 min, and immediately cool on ice to obtain fragmented nucleic acid.

[0084] 4. Reverse transcription and end repair

[0085] Add 38 μL of the reaction system shown in Table 2 to the fragmented nucleic acid obtained in step 3 above, incubate at 37°C for 20 min and then at 72°C for 10 min to obtain a reaction product containing a double-stranded cDNA with blunt ends and A bases.

[0086] Table 2 shows the multi-enzyme reaction system for reverse transcription and end repair

[0087]

[0088]

[0089] 5. Adapter ligation and purification

[0090] To the 60ul reaction product obtained in step 4 (double-stranded cDNA with blunt ends and containing A base), add 38ul of the reaction system shown in Table 3 (Novel T4 DNA Ligase is yesen noval DNA ligase, Yisheng Bio 10298ES, 400U / μL), and then add 2μL of a label adapter with a concentration of 0.05μmol / L that matches the sequencing platform to make the final reaction concentration reach 1pmol / L for rapid DNA connection under the promotion of PEG.

[0091] The details are as follows Table 3:

[0092] Table 3 is the reaction system

[0093]

[0094] 23℃ for 15min

[0095] After the reaction, Agencourt AMPure XP magnetic beads or any magnetic beads with the same function (Yisheng Bio, product catalog number 12601ES) are used for purification, and the fragment selection is 200-500bp. The operation process is carried out according to the magnetic bead purification instructions, and the nucleic acid is re-dissolved with nuclease-free water to obtain the nucleic acid after adding the adapter.

[0096] 6. PCR amplification and purification

[0097] The nucleic acid obtained in the above step after adding the adapter was used as a template and amplified using high-fidelity PCR reagents (MGI platform PCR universal primers, reagents, MGI 1000027216).

[0098] After the reaction, use Agencourt AMPure XP magnetic beads or any magnetic beads with the same function for purification. At this point, the library construction is completed and the RNA sequencing library is obtained.

[0099] The RNA sequencing library constructed above was subjected to quality control by Qubit and Agilent 2100 detection.

[0100] Qualified libraries were subjected to subsequent sequencing tests.

[0101] 7. Sequencing

[0102] The RNA sequencing library constructed in 6 above was sequenced according to the sequencing instructions of the MGISEQ platform.

[0103] 8. Data Analysis

[0104] The offline data was analyzed using pathogenic microorganism analysis software to obtain the detection values ​​of the microorganisms added to the simulated samples. The analysis used the software "Pathogenic Microorganism Gene Detection Software" adapted for BGI's PMseq products to uniformly analyze the fq files obtained by sequencing. The above high-throughput sequencing offline data was compared with each viral genome, and the reads that could only be compared with the corresponding viral genome were counted. The results were plotted as shown in the figure. Figure 2 As shown (the vertical axis in the figure is the number of reads), it can be seen that the method of the present invention can obtain the genome sequence of the microorganism in the simulated sample and detect whether the sample to be tested contains microorganisms.

[0105] Comparative example: using traditional testing methods to test the above simulated samples

[0106] The simulated samples in Example 1 were detected using the BGI Z000100174PMseqTM RNA infection pathogen high-throughput detection kit. In order to fully reflect the parallel experiment and remote measurement, the analysis process was the same as the above method.

[0107] Example 1 The test results of the method of the present invention and the method of the comparative example are as follows Figure 2 It can be seen that the method of the present invention is not inferior to the traditional method in terms of detection effect.

[0108] The time of Example 1, the method of the present invention and the method of the comparative example are compared, as shown in Table 4 (timed by manual operation of 8 samples). It can be seen that the method of the present invention saves time cost.

[0109] Table 4 shows the time cost comparison of the two methods

[0110]

[0111] Example 2: Sample size exploration of the detection method of the present invention

[0112] Purified RNA was obtained from purchased Hela cells according to steps 1 and 2 in Example 1, and the purified RNA was diluted with nuclease-free water to obtain purified RNA with different concentration gradient dilutions shown in Table 5 as samples.

[0113] The purified RNA diluted in different concentration gradients was used to construct a library according to steps 3-6 of Example 1 to obtain an RNA sequencing library (the concentration of the library was detected by the Qubit method), and the quality of the library was detected by Agilent 2100.

[0114] 2100 Quality Control Test Results Figure 3As shown in A-3H, AH corresponds to 1-8 in Table 5. The results show that a library of qualified quality can be obtained with a starting amount of 100 pg for the next experimental detection.

[0115] Table 5 shows the starting nucleic acid amount and library concentration of purified RNA diluted at different concentration gradients

[0116] Serial number Starting nucleic acid amount Library concentration 2100 Quality Control Screenshot 1 10pg 2.43 ng / μL Figure 3 A 2 20pg 8.91 ng / μL Figure 3 B 3 50pg 11.5 ng / μL Figure 3 C 4 100pg 15.9 ng / μL Figure 3 D 5 1ng 28.7 ng / μL Figure 3 E 6 5ng 25.2 ng / μL Figure 3 F 7 10ng 29.5 ng / μL Figure 3 G 8 50ng 95ng / μL Figure 3 H

Claims

1. A method for constructing an RNA sequencing library, comprising the following steps: A1) fragmenting the RNA sample to obtain fragmented RNA; A2) performing reverse transcription and end-repair reaction on the fragmented RNA in the following multi-enzyme reaction system to obtain a double-stranded cDNA with blunt ends and containing A bases; The multi-enzyme reaction system comprises the following components: ribonuclease H, DNA polymerase I Klenow fragment, T4 DNA polymerase, polynucleotide kinase, thermostable DNA polymerase and reverse transcriptase; A3) The double-stranded cDNA with blunt ends and containing A base is sequentially connected to an adapter and amplified by PCR to obtain an RNA sequencing library.

2. The method according to claim 1, characterized in that: The activity unit ratio of the ribonuclease H, the DNA polymerase I Klenow fragment, the T4 DNA polymerase, the polynucleotide kinase, the thermostable DNA polymerase and the reverse transcriptase is 2:50:10:13:4:

333.

3. The method according to claim 2, characterized in that: The system further comprises the following components: Mg ions, Tris, dNTP, dATP and buffer.

4. The method according to any one of claims 1 to 3, characterized in that: The reaction conditions of the reverse transcription and end repair are: first, incubate at 37° C. for 20 min-40 min, and then incubate at 65-72° C. for 10 min-20 min.

5. The method according to any one of claims 1 to 4, characterized in that: The RNA sample is derived from a microorganism; Furthermore, the microorganism is a virus, a bacterium, a yeast, a mold or a combination thereof.

6. The method according to any one of claims 1 to 5, characterized in that: The initial amount of the RNA sample is 10pg-500ng.

7. An RNA sequencing library prepared by the method according to any one of claims 1 to 6.

8. A method for detecting whether a sample to be tested contains a microorganism or its genomic RNA, comprising the following steps: B1) extracting nucleic acid from the sample to be tested, and then digesting DNA with the nucleic acid to obtain an RNA sample; B2) treating the RNA sample according to step A1) of the method of any one of claims 1 to 6 to obtain fragmented RNA; B3) treating the fragmented RNA according to step A2) of the method of any one of claims 1 to 6 to obtain a double-stranded cDNA with blunt ends and containing A bases; B4) treating the double-stranded cDNA with blunt ends and containing A base according to step A3) of the method according to any one of claims 1 to 6 to obtain an RNA sequencing library; B5) sequencing the RNA sequencing library to detect whether the sample to be tested contains microorganisms or their genomic RNA.

9. A kit for constructing the RNA sequencing library of claim 7, comprising the following components: C1) the multi-enzyme reaction system according to any one of claims 1 to 6; C2) Reagents or instruments required for nucleic acid extraction; C3) Reagents or instruments required for DNA digestion; C4) Reagents or instruments required for RNA fragmentation; C5) Reagents or instruments required for connecting the connector; C6) Reagents or instruments required for PCR amplification.

10. The multi-enzyme reaction system according to any one of claims 1 to 6 or the RNA sequencing library according to claim 7 Or the use of the kit according to claim 9 or the components C1-C6 in claim 9 in any of the following or in the preparation of a product having any of the following functions: D1) RNA sequencing; D2) Microbial genome RNA sequencing; D3) Construction of RNA sequencing library D4) Detecting whether the sample to be tested contains microorganisms; D5) Detect whether the sample contains microbial genomic RNA.

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