Library building method applied to RNA virus detection in mNGS detection process

Abstract

The invention relates to a library building method applied to RNA virus detection in an mNGS detection process. Comprising the following steps: S1, preparing RNA nucleic acid to be detected; the method comprises the following steps: performing gDNA digestion reaction and digestion termination reaction on nucleic acid of a to-be-detected pathogen sample, and removing genome DNA to obtain mixed nucleic acid containing human RNA and virus RNA; s2, carrying out hybridization reaction on the human rRNA, and removing the human rRNA; s2, mixing a product obtained in the step S1 with a probe reagent without human rRNA, and performing hybridization reaction on a PCR instrument to complete combination of the probe reagent and the human rRNA; s3, performing reverse transcription and library building to synthesize cDNA (complementary deoxyribonucleic acid); s2, carrying out reverse transcription on a product obtained in the step S2 to finish reverse transcription of non-human rRNA, especially virus RNA, so as to generate cDNA (complementary deoxyribonucleic acid); step S4, library construction; and performing library construction on the cDNA generated by reverse transcription. According to the method, the human ribosome RNA can be removed in a targeted manner in a short time, so that the proportion of the human RNA is reduced, the proportion of the virus RNA is increased, the sequencing quality of the RNA virus is improved, the detection capability of the RNA virus is finally improved, and false negative results are reduced.

Description

Technical Field

[0001] The present invention belongs to the field of mNGS detection technology, and in particular relates to a library construction method applied to RNA virus detection in the mNGS detection process. Background Art

[0002] Metagenomic sequencing (mNGS) uses high-throughput sequencing technology to sequence all nucleic acids in a sample and, through bioinformatics analysis, to determine whether pathogens are present. Traditional clinical methods for detecting RNA viruses in pathogenic samples include reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative reverse transcription polymerase chain reaction (qRT-qPCR), in situ hybridization (ISH), enzyme-linked immunosorbent assay (ELISA), and nucleic acid test strips. These methods are often targeted for verification of highly suspected pathogens, and most require viral standards as internal standards. mNGS, however, can unbiasedly detect a wide range of microorganisms, including difficult-to-culture pathogens, pathogens whose growth is inhibited by antimicrobial drugs, and emerging pathogens such as the novel coronavirus. mNGS offers excellent application prospects, offering higher sensitivity and aligning with the concept of "precision diagnosis and treatment."

[0003] Clinical samples often contain both human cells and pathogens. Infectious samples (such as sputum, pus, and lung lavage) often contain a high proportion of human cells. When using mNGS for sequencing, human nucleic acid can account for over 99% of the total nucleic acid. This high level of human presence not only affects pathogen detection sensitivity and can lead to false-negative results, but also wastes sequencing resources (data volume). Therefore, minimizing human background interference while ensuring that pathogens are not affected is crucial. Currently, the primary approach to reducing the human contribution in RNA pathogen detection is rRNA removal. Traditional rRNA removal methods, such as chemical and physical methods, have numerous limitations, including complex procedures, time-consuming processes, and the potential loss of non-target RNA. Smith et al. proposed a specific antibody-based rRNA removal method. While this method offers high rRNA removal efficiency, the high cost of antibody preparation limits its application. Furthermore, enzymatic digestion methods, while simple to use, require stringent reaction conditions and can damage RNA samples.

[0004] The present invention aims to solve the problems existing in the prior art by highly specifically removing human ribosomal RNA (rRNA) during RNA library construction, thereby reducing the proportion of human RNA and enriching viral RNA, ultimately improving the detection performance of RNA viruses in the sample to be tested and increasing the utilization rate of sequencing data. Summary of the Invention

[0005] The purpose of the present invention is to provide a library construction method for RNA virus detection in the mNGS detection process, improve the detection performance of RNA viruses in the mNGS detection process, increase the utilization rate of sequencing data, greatly simplify the rRNA removal operation, improve the library construction efficiency, and reduce experimental costs.

[0006] The technical solution adopted by the present invention is: a library construction method for RNA virus detection in the mNGS detection process includes the following steps:

[0007] Step S1, preparing the RNA nucleic acid to be tested; performing a gDNA digestion reaction and a digestion termination reaction on the nucleic acid of the pathogen sample to be tested, removing the genomic DNA to obtain a mixed nucleic acid containing human RNA and viral RNA;

[0008] Step S2, human rRNA hybridization reaction to remove human rRNA; the product of step S1 is mixed with a probe reagent for removing human rRNA, and a hybridization reaction is performed on a PCR instrument to complete the binding of the probe reagent and the human rRNA, so that the human rRNA is blocked by the probe and does not participate in subsequent reverse transcription and RNA library construction;

[0009] Step S3, reverse transcription and library construction to synthesize cDNA; reverse transcription of the product of step S2 is performed to complete reverse transcription of non-human rRNA, especially viral RNA, to generate cDNA;

[0010] Step S4, library construction: the cDNA generated by reverse transcription in step S3 is used to construct a library.

[0011] Preferably, in step S1, gDNA digestion reaction is performed using gDNA enzyme. The digestion reaction conditions are as follows: 2 μL of gDNA enzyme and 1.6 μL of enzyme buffer are added to 12.4 μL of pathogen sample nucleic acid and mixed evenly for digestion reaction. The reaction conditions are as follows: the hot cover is closed, and the PCR instrument is reacted at 37°C for 15 minutes. After the program is completed and the temperature drops to 4°C, the processed product is taken out for use.

[0012] Preferably, in step S1, the gDNA digestion termination reaction is to add 2 μL of gDNA enzyme termination reaction buffer to the system after the gDNA digestion reaction and mix evenly to perform the digestion termination reaction. The reaction conditions are a hot cover at 105°C and a PCR instrument at 70°C for 10 minutes. After the program is completed and the temperature drops to 4°C, the treated product is taken out for use.

[0013] Preferably, the specific human rRNA probe reagent used in step S2 reacts with the product in step S1 to block the human rRNA in the system to form a human rRNA-probe double-stranded product, which does not participate in the subsequent RNA reverse transcription and library construction reaction, and ultimately achieves human rRNA removal and viral RNA enrichment. The probe reagent reaction conditions are 1 μL of probe reagent mixed with 17 μL of step S1 product, the reaction conditions are a hot cover at 105°C, and the PCR instrument reacts at 85°C for 5 minutes, at 72°C for 2 minutes, and at 60°C for 10 minutes. After the program is completed and the temperature drops to 4°C, the treated product is taken out for use.

[0014] Preferably, in step S2, the specific human rRNA probe reagent is a Golden Key brand RNA library enrichment reagent with a product number of 2072A.

[0015] Preferably, in step S3, the first step of cDNA synthesis is to reverse transcribe the product of step S2 to synthesize RNA-DNA double strands, that is, to complete the RNA single strand library construction, and the reaction conditions are:

[0016]

[0017] In step S3, the second step of cDNA synthesis is to synthesize cDNA from the first-strand library product to complete the RNA second-strand library construction. The reaction conditions are

[0018]

[0019]

[0020] After the second-strand library construction is completed, reverse transcription library construction is completed to synthesize cDNA.

[0021] Preferably, in step S4, cDNA library construction is completed using a commercial kit.

[0022] The advantages and positive effects of the present invention are:

[0023] The present invention provides a library construction method for RNA virus detection in the mNGS detection process. A human rRNA probe reagent is added to the RNA nucleic acid system to be tested. The optimal reaction conditions are set so that the probe reagent specifically binds to the human rRNA single strand to form a human rRNA-probe double-stranded product, while the RNA virus single strand in the system is not affected. The RNA library is constructed by reverse transcribing the single strand to form cDNA, and RNA virus detection is finally achieved. According to the library construction method proposed by the present invention, not only can the detection performance of RNA viruses in the mNGS detection process be improved and the utilization rate of sequencing data be increased, but also the rRNA removal operation can be greatly simplified, the library construction efficiency can be improved, and the experimental cost can be reduced.

[0024] (1) Efficient removal of human rRNA: The one-step method used in the present invention to rapidly remove human ribosomal RNA from the sample can efficiently remove human rRNA from the sample in a short period of time, thereby improving the accuracy and efficiency of subsequent library construction and sequencing.

[0025] (2) Simplified operation process: Compared with traditional rRNA removal methods, the present invention does not require additional purification steps, simplifies the operation process, and saves time and cost.

[0026] (3) Improving sequencing quality: By removing rRNA, the present invention can reduce human background interference in the sequencing process and improve the quality and reliability of sequencing data.

[0027] (4) Wide applicability: The method of the present invention is compatible with various library construction reagents on the market and is applicable to multiple fields such as industrial automated library construction and rapid disease diagnosis.

[0028] (5) Promote the development of pathogen detection technology: The application of this invention helps to improve the accuracy and efficiency of mNGS pathogen detection and provides new tools and means for the development of pathogen detection technology. DETAILED DESCRIPTION

[0029] To further understand the present invention, its features, and its effectiveness, the following examples are provided in detail. However, the present invention can be implemented in various forms and is not limited to the embodiments described herein. Rather, these examples are provided to provide a more thorough and comprehensive understanding of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0031] Unless otherwise specified, the reagents used in the following examples are all commercially available; the methods used in the following examples are all conventional methods unless otherwise specified.

[0032] In the embodiment, in the data analysis step, the read count of the RNA virus is normalized to 20M reads after the second-generation sequencing comparative analysis. The higher the read count after normalization of pathogen detection in the same sample, the higher the pathogen detection efficiency.

[0033] In one embodiment, the CT value of QPCR is introduced as an evaluation indicator. The lower the CT value, the smaller the number of cycles required to reach the threshold line after PCR amplification, which further indicates that the sample contains a higher concentration of the target nucleic acid.

[0034] Example 1

[0035] In this example, the human rRNA probe reagent was tested to perform a human rRNA removal reaction under different reaction conditions (such as temperature and time) to explore the optimal reaction conditions.

[0036] Step S1, preparing the RNA nucleic acid to be tested:

[0037] The nucleic acid extracted from the pathogen sample to be tested is subjected to gDNA digestion and digestion termination reaction to obtain a mixed nucleic acid containing human RNA and viral RNA, which serves as the starting material for subsequent experiments.

[0038] Step S2, human rRNA hybridization reaction, removal of human rRNA:

[0039] Take 17 μL of the above RNA nucleic acid and add 1 μL of the specific human rRNA probe reagent. Mix thoroughly with a vortex mixer and centrifuge briefly to ensure uniform mixing. Then, place the treated sample in a PCR machine and heat-treat according to the set program.

[0040] The specific human rRNA probe reagent is a Jinshi brand RNA library construction and enrichment reagent with the product number 2072A and a specification of 24 people / box. It is a commercially available product.

[0041] Test Group 1: Heat the lid to 105°C and run the PCR instrument at 85°C for 5 min, 72°C for 2 min, and 60°C for 10 min. After the program is completed and the temperature drops to 4°C, remove the processed product for use.

[0042] Test Group 2: Heat the lid to 105°C and run the PCR instrument at 75°C for 1 min and then at 50°C for 2 min. After the program is completed and the temperature drops to 4°C, remove the processed product and set aside.

[0043] Step S3, reverse transcription library synthesis of double-stranded cDNA:

[0044] Using the dehumanized rRNA product as a template, reverse transcription is performed using reverse transcriptase and enzyme buffer to synthesize a first-strand cDNA complementary to the RNA. Subsequently, using the first-strand cDNA as a template, a complementary second-strand cDNA is synthesized under the action of DNA polymerase to complete the cDNA library construction.

[0045] Specifically, the first step of cDNA synthesis is to reverse transcribe the product of step S2 to synthesize RNA-DNA double strands, that is, to complete the RNA single strand library construction. The reaction conditions are:

[0046]

[0047] In step S3, the second step of cDNA synthesis is to synthesize cDNA from the first-strand library product to complete the RNA second-strand library construction. The reaction conditions are:

[0048]

[0049] After the second-strand library construction is completed, reverse transcription library construction is completed to synthesize cDNA.

[0050] Step S4, library construction and sequencing:

[0051] The generated cDNA was used for subsequent library construction and sequencing analysis.

[0052] Step S5, data analysis:

[0053] Sequencing data were obtained, and pathogen gene sequences were compared. The number of pathogen reads in the control group and the number of pathogen reads in the test group without human rRNA were calculated and compared. The effective detection data volume was then evaluated.

[0054] Control group setup: The control group did not undergo human rRNA removal reaction, and the product of step S1 was directly used for the experiment of steps S3 to S5;

[0055] The control group had no technical repetitions, and the test group had two full-process technical repetitions. The results are shown in Tables 1 and 2.

[0056] Table 1 Differences in library concentration and sequencing QC under different reaction conditions

[0057]

[0058] Table 2 Differences in pathogen detection performance under different reaction conditions

[0059]

[0060] Table 1 shows that the key QC metric, Effective, for the same samples showed a significant improvement in Test Group 1, while the improvement was less pronounced in Test Group 2, indicating that the performance improvement in Test Group 2 was not significant in the sequencing data. Table 2 also shows that after normalization to 20M, the number of RNA virus detections in Test Group 1 increased by 98-95%, significantly improving detection efficiency. However, the increase in the number of detections in Test Group 2 after normalization ranged from 0.07 to 11.78%, a less significant improvement. Based on the above library construction data, it can be seen that the reaction conditions in Test Group 1 were more optimal.

[0061] Example 2 This example illustrates that the use of a human rRNA probe removal reagent to construct a library has an inhibitory effect on cDNA synthesis of human rRNA.

[0062] Step S1, prepare Homo sapiens 28S ribosomal RNA nucleic acid.

[0063] Step S2, human rRNA hybridization reaction, removal of human rRNA:

[0064] Take 17 μL of RNA from S1 and add 1 μL of the specific human rRNA probe reagent. Mix thoroughly with a vortex mixer and centrifuge briefly to ensure uniform mixing. Next, place the treated sample in a PCR instrument and heat-treat according to the programmed protocol: first, heat the lid to 105°C. The PCR instrument then incubates at 85°C for 5 minutes, 72°C for 2 minutes, and 60°C for 10 minutes. Once the protocol is complete and the temperature has dropped to 4°C, remove the treated sample and set aside.

[0065] Step S3, reverse transcription library synthesis of cDNA:

[0066] Using Homo sapiens 28S ribosomal RNA as a control group (i.e., the product of step S1), and 28S ribosomal RNA treated with a human rRNA probe reagent as a test group (i.e., the product of step S2), both nucleic acids were reverse transcribed using reverse transcriptase and enzyme buffer to synthesize a first-strand cDNA complementary to the RNA. Subsequently, using the first-strand cDNA as a template, a complementary second-strand cDNA was synthesized using DNA polymerase to complete the cDNA library construction.

[0067] Step S4, QPCR quantification:

[0068] Take 4 μL of cDNA obtained from the control group and test library, 2 μL of 28s primer qHM28S-FR, 4 μL of sterile distilled water, and 10 μL of VAHTS SYBR qPCR Master Mix (the commercial reagent brand is "vazyme" and the product number is "NQ101-NQ106"). After mixing, perform amplification quantitative reaction on the QPCR instrument. The program is set as Stage 1 pre-denaturation Reps: 1 Reps: 195℃ 5min; Stage 2 cycle reaction Reps: 35 Reps: 95℃ 30sec; 60℃ 45sec.

[0069] Step S5, CT value analysis of QPCR quantitative results:

[0070] Table 3 Effect of specific human rRNA probe reagent on CT value of cDNA product

[0071]

[0072] A higher CT value indicates a lower target concentration and a later peak in the amplification curve, indicating a lower 28S rRNA content in the sample. In the control group, all 28S rRNA was transcribed and synthesized into cDNA, resulting in a high rRNA concentration and a relatively low CT value. In the test group, the probe reagent specifically reacted with 28S rRNA, removing a significant amount of human rRNA, reducing rRNA content and leading to a higher CT value. This further demonstrates the effectiveness of the present method in removing human ribosomes.

[0073] Example 3

[0074] This example demonstrates that the use of a human rRNA-removed probe reagent method to build a library for testing real clinical samples can improve the performance of RNA virus detection results.

[0075] Step S1, prepare RNA nucleic acid sample:

[0076] The nucleic acid extracted from the pathogen sample to be tested needs to undergo gDNA digestion and digestion termination reaction to obtain a mixed nucleic acid containing human RNA and viral RNA as the starting material for subsequent experiments.

[0077] Step S2, human rRNA hybridization reaction, removal of human rRNA:

[0078] Take 17 μL of the above RNA nucleic acid, add 1 μL of specific human rRNA probe reagent, mix thoroughly with a vortex mixer, and centrifuge briefly to ensure uniform mixing. Subsequently, place the treated sample on a PCR instrument and perform heat treatment according to the set program. The reaction conditions are heated to 105°C, and the PCR instrument reacts at 85°C for 5 minutes, 72°C for 2 minutes, and 60°C for 10 minutes. After the program runs to the end and the temperature drops to 4°C, remove the treated product for use.

[0079] Step S3, reverse transcription library synthesis of double-stranded cDNA:

[0080] Using the dehumanized rRNA product as a template, reverse transcription is performed using reverse transcriptase and enzyme buffer to synthesize a first-strand cDNA complementary to the RNA. Subsequently, using the first-strand cDNA as a template, a complementary second-strand cDNA is synthesized under the action of DNA polymerase to complete the cDNA library construction.

[0081] Step S4, library construction and sequencing:

[0082] The generated cDNA was used for subsequent library construction and sequencing analysis.

[0083] Step S5, data analysis:

[0084] Sequencing data were obtained, and pathogen gene sequences were compared. The number of pathogen reads in the control group and the number of pathogen reads in the test group without human rRNA were calculated and compared. The effective detection data volume was then evaluated.

[0085] Control group setup: The control group did not undergo human rRNA removal reaction, and the product of step S1 was directly subjected to experiments S3 to S5; the results are shown in Tables 4 and 5.

[0086] Table 4 Differences in library output concentration and sequencing QC among different library construction methods

[0087]

[0088]

[0089] Table 5 Effects of different database construction methods on pathogen detection performance

[0090]

[0091] Table 4 shows that the sample concentration in the test group was significantly lower, indicating that human rRNA was not involved in library construction. The key QC indicator, Effectiveness, was significantly improved in the test group. Table 5 shows that after normalization to 20M, the number of RNA viruses detected in the test group increased by 104.97% to 2910.68%, significantly improving sample detection performance.

[0092] In summary, the three examples illustrate that using the human rNRA removal reagent proposed in the present invention to build a library can achieve a positive effect on RNA virus detection in the mNGS detection process.

Claims

1. A library construction method for RNA virus detection in the mNGS detection process, characterized by: The following steps are included: Step S1, preparing the RNA nucleic acid to be tested; performing a gDNA digestion reaction and a digestion termination reaction on the nucleic acid of the pathogen sample to be tested, removing the genomic DNA to obtain a mixed nucleic acid containing human RNA and viral RNA; Step S2, human rRNA hybridization reaction to remove human rRNA; the product of step S1 is mixed with a probe reagent for removing human rRNA, and a hybridization reaction is performed on a PCR instrument to complete the binding of the probe reagent and the human rRNA, so that the human rRNA is blocked by the probe and does not participate in subsequent reverse transcription and RNA library construction; Step S3, reverse transcription and library construction to synthesize cDNA; reverse transcription of the product of step S2 is performed to complete reverse transcription of non-human rRNA, especially viral RNA, to generate cDNA; Step S4, library construction: the cDNA generated by reverse transcription in step S3 is used to construct a library.

2. The library construction method for RNA virus detection in the mNGS detection process according to claim 1, characterized in that: In step S1, gDNA digestion reaction is performed using gDNA enzyme. The digestion reaction conditions are as follows: 2 μL of gDNA enzyme and 1.6 μL of enzyme buffer are added to 12.4 μL of pathogen sample nucleic acid and mixed evenly for digestion reaction. The reaction conditions are as follows: the hot cover is closed, and the PCR instrument is reacted at 37°C for 15 minutes. After the program is completed and the temperature drops to 4°C, the processed product is taken out for use.

3. The library construction method for RNA virus detection in the mNGS detection process according to claim 2, characterized in that: In step S1, the gDNA digestion termination reaction is to add 2 μL of gDNA enzyme termination reaction buffer to the system after the gDNA digestion reaction and mix it evenly to perform the digestion termination reaction. The reaction conditions are a hot cover at 105°C and a PCR instrument at 70°C for 10 minutes. After the program is completed and the temperature drops to 4°C, the processed product is taken out for use.

4. The library construction method for RNA virus detection in the mNGS detection process according to claim 1, characterized in that: The specific human rRNA probe reagent used in step S2 reacts with the product in step S1 to block the human rRNA in the system to form a human rRNA-probe double-stranded product. This product does not participate in the subsequent RNA reverse transcription and library construction reaction, and ultimately achieves human rRNA removal and viral RNA enrichment. The probe reagent reaction conditions are 1 μL probe reagent mixed with 17 μL of step S1 product, the reaction conditions are hot cover 105°C, the PCR instrument reacts at 85°C for 5 minutes, at 72°C for 2 minutes, and at 60°C for 10 minutes. After the program is completed and the temperature drops to 4°C, the processed product is taken out for use.

5. The library construction method for RNA virus detection in the mNGS detection process according to claim 4, characterized in that: In step S2, the specific human rRNA probe reagent is a Golden Key brand RNA library enrichment reagent with a product number of 2072A.

6. The library construction method for RNA virus detection in the mNGS detection process according to claim 1, characterized in that: In step S3, the first step of cDNA synthesis is to reverse transcribe the product of step S2 to synthesize RNA-DNA double strands, that is, to complete the RNA single strand library construction. The reaction conditions are In step S3, the second step of cDNA synthesis is to synthesize cDNA from the first-strand library product to complete the RNA second-strand library construction. The reaction conditions are After the second-strand library construction is completed, reverse transcription library construction and cDNA synthesis are completed.

7. The library construction method for RNA virus detection in the mNGS detection process according to claim 1, characterized in that: In step S4, cDNA library construction was completed using a commercial kit.

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