A kit for detecting 26 viruses and its usage method, along with targeted sequencing methods.

Abstract

This invention discloses a kit for detecting 26 viruses. The kit includes a mixture of probes capable of specifically binding to the nucleic acids of 26 target viruses, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.138. The 26 virus capture probes used in this invention comprehensively consider interference from human and non-target pathogens, exhibiting high sensitivity and high specificity.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically, it relates to a kit for detecting 26 types of viruses, its usage method, and a targeted sequencing method. Background Technology

[0002] Next-generation sequencing (NGS), also known as high-throughput sequencing, can sequence hundreds of thousands to millions of DNA fragments within a certain read length range in a single run. NGS technology allows for high-throughput sequencing of nucleic acids in clinical samples, followed by comparison with databases to enable gene mutation detection, tracing, detection, typing, and drug resistance assessment of infectious diseases. There are two main methods based on NGS technology: metagenomic NGS (mNGS) and targeted NGS (tNGS).

[0003] mNGS performs non-discriminatory, non-selective high-throughput sequencing of nucleic acids in clinical samples, providing a rapid, objective, and comprehensive reflection of the biological information of these samples. It is highly suitable for detecting novel or rare mutations and pathogens, playing an irreplaceable role in severe cases caused by rare or difficult-to-diagnose diseases. However, the sensitivity of mNGS results is significantly affected by non-target background nucleic acids, and the cost is relatively high. For example, in pathogen detection, tissue samples often contain over 99% human DNA; therefore, it is necessary to remove the human background from the sample to meet sequencing throughput requirements.

[0004] Compared to mNGS, tNGS detects target nucleic acid regions in samples through (hyper)multiplex PCR or hybridization capture, which can improve sensitivity and reduce sequencing costs. tNGS can specifically enrich the target genes being tested and is less affected by non-target background nucleic acids. Through the design of specific primers or probes, hundreds to thousands of target fragments can be enriched at once. This method is less expensive than mNGS sequencing and has higher sensitivity for detecting viruses and mutations with low abundance.

[0005] Conventional hybridization capture library construction methods are limited in their widespread application in pathogen detection due to their cumbersome procedures and the high cost of certain commercial reagents, such as Blocker. This invention utilizes single-primer amplification to convert a double-stranded library into a single-stranded DNA library, which then directly proceeds to the single-stranded library hybridization with the probe. This eliminates the need for Blocker and human cot I to block complementary regions of the library, significantly reducing experimental costs. Summary of the Invention

[0006] The purpose of this invention is to provide a kit and method of use for detecting 26 viruses, along with a targeted sequencing method. The 26 virus capture probes used in this invention comprehensively consider interference from human and non-target pathogens, exhibiting high sensitivity and high specificity.

[0007] The objective of this invention and the technical problem it solves are achieved by the following technical solutions.

[0008] One aspect of the present invention provides a kit for detecting 26 viruses, the kit comprising a mixture of probes capable of specifically binding to the nucleic acids of 26 target viruses, the nucleotide sequences of the probes being shown in SEQ ID NO.1 to SEQ ID NO.138.

[0009] Optionally, the 26 viruses include: Influenza A virus H1N1, Influenza A virus H1N1 (2009), Influenza B virus, Human parainfluenza virus type 1, Human parainfluenza virus type 2, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Herpes simplex virus type 1, Herpes simplex virus type 2, Human alpha herpesvirus type 3, Epstein-Barr virus, Human herpesvirus type 5, Human herpesvirus type 6A, Human herpesvirus type 6B, Human herpesvirus type 7, Human herpesvirus type 8, Rhinovirus type A, Rhinovirus type B, Rhinovirus type C, Respiratory syncytial virus, Human bocavirus type 1, Human bocavirus type 2, Human bocavirus type 3, Human bocavirus type 4, Human metapneumovirus, and Human adenovirus.

[0010] Optionally, the kit may also include TE buffer.

[0011] Optionally, the probe design principles are as follows: probe length 120 bp; GC content 30-70%; probes dispersedly distributed on the genome of the target pathogen; 15 human probes selected as internal controls.

[0012] Another aspect of the present invention provides a method for detecting 26 viruses, the method comprising the following steps:

[0013] S1: Viral nucleotide extraction: The VAMNE Magnetic Pathogen DNA / RNA Kit (RM601-C3) was used to extract viral DNA and RNA from the sample simultaneously. The required DNA + RNA concentration was not less than 5 ng / μL and the purity was qualified to meet the requirements for library construction.

[0014] S2: DNA / RNA co-construction library: rRNA was removed using a DNA / RNA co-construction library kit, followed by the synthesis of cDNA / double-stranded cDNA. After adapter ligation, the cDNA / double-stranded cDNA was purified and amplified by PCR. The amplified products were then purified to obtain the library.

[0015] S3: Probe hybridization capture;

[0016] S4: DNB is prepared and then placed on a sequencer.

[0017] Optionally, the samples may include plasma, bronchoalveolar lavage fluid, or cerebrospinal fluid.

[0018] Optionally, the DNA / RNA co-construction library includes the following steps:

[0019] 1) rRNA removal;

[0020] 2) cDNA synthesis;

[0021] 3) cDNA fragmentation, end repair, and dA tail addition;

[0022] 4) Connector connection;

[0023] 5) Purification of the ligation product;

[0024] 6) Library expansion;

[0025] 7) Purification of amplified products using magnetic beads.

[0026] Optionally, the probe hybridization capture includes the following steps:

[0027] 1) Metagenomic library mixing;

[0028] 2) Library amplification using single primers;

[0029] 3) Single-stranded library purification;

[0030] 4) Single-stranded library hybridization;

[0031] 5) Washing;

[0032] 6) Capture library PCR enrichment;

[0033] 7) Purification of amplification products.

[0034] By employing the above technical solution, the present invention has at least the following advantages:

[0035] The probe sequences of this invention. The 26 virus capture probes used in this invention comprehensively consider interference from human and non-target pathogens, exhibiting high sensitivity and high specificity.

[0036] This invention employs a probe hybridization capture method to obtain a hybridization capture library containing the target gene to be detected, and then amplifies and enriches the library. At the same time, it greatly reduces the influence of non-target background nucleic acids, resulting in a higher positive detection rate than traditional metagenomic libraries. It is particularly effective for low-load samples, thus compensating for the false negatives that easily occur in metagenomic libraries when the sample load is low.

[0037] For multiplex PCR library construction, the detection efficiency of different targets can be affected to varying degrees by primer amplification efficiency, inter-primer interference, or mutations in the primer-target binding region. This is especially true for high-pathogen loads where nucleic acids compete for reagents, affecting the detection of low-pathogen loads. Furthermore, PCR amplification of fixed regions can easily lead to aerosol contamination. Hybrid capture libraries avoid these problems associated with multiplex PCR libraries. There is no interference between probes, and the number of target pathogens can be directly added to the original protocol. Probes can tolerate mutations within 5% of the hybridization region, making them ideal for RNA virus detection. Moreover, through genomic analysis and probe design, the capture efficiency of each probe can be maintained at the same level, ensuring that high-pathogen loads do not affect the detection results of low-pathogen loads.

[0038] This invention transforms a double-stranded library into a single-stranded DNA library through single-primer amplification, and then directly proceeds to the hybridization step between the single-stranded library and the probe. This eliminates the need to use a blocker and human cot I to block the complementary regions of the library, greatly reducing experimental costs.

[0039] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Detailed Implementation

[0040] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0041] According to the present invention, a kit for detecting 26 viruses is provided, the kit comprising a mixture of probes capable of specifically binding to the nucleic acids of 26 target viruses, the nucleotide sequences of the probes being shown in SEQ ID NO.1 to SEQ ID NO.138.

[0042] The specific sequences of SEQ ID NO.1 to SEQ ID NO.138 are shown in Table 1 below:

[0043] Table 1. Probe nucleotide sequences corresponding to 26 viruses

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] The 26 viruses described in this invention include: Influenza A virus H1N1, Influenza A virus H1N1 (2009), Influenza B virus, Human parainfluenza virus type 1, Human parainfluenza virus type 2, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Herpes simplex virus type 1, Herpes simplex virus type 2, Human alpha herpesvirus type 3, Epstein-Barr virus, Human herpesvirus type 5, Human herpesvirus type 6A, Human herpesvirus type 6B, Human herpesvirus type 7, Human herpesvirus type 8, Rhinovirus type A, Rhinovirus type B, Rhinovirus type C, Respiratory syncytial virus, Human bocavirus type 1, Human bocavirus type 2, Human bocavirus type 3, Human bocavirus type 4, Human metapneumovirus, and Human adenovirus.

[0061] The kit according to the present invention also includes TE buffer.

[0062] The probe design principles described in this invention are as follows: probe length 120 bp; GC content 30-70%; probes dispersedly distributed on the genome of the target pathogen; 15 human probes selected as internal controls.

[0063] A method for detecting 26 viruses according to the present invention includes the following steps:

[0064] S1: Viral nucleotide extraction: The VAMNE Magnetic Pathogen DNA / RNA Kit (RM601-C3) was used to extract viral DNA and RNA from the sample simultaneously. The required DNA + RNA concentration was not less than 5 ng / μL and the purity was qualified to meet the requirements for library construction.

[0065] S2: DNA / RNA co-construction library: rRNA was removed using a DNA / RNA co-construction library kit, followed by the synthesis of cDNA / double-stranded cDNA. After adapter ligation, the cDNA / double-stranded cDNA was purified and amplified by PCR. The amplified products were then purified to obtain the library.

[0066] S3: Probe hybridization capture;

[0067] S4: DNB is prepared and then placed on a sequencer.

[0068] The samples described in this invention include plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid.

[0069] The DNA / RNA co-construction library according to the present invention includes the following steps:

[0070] 1) rRNA removal;

[0071] 2) cDNA synthesis;

[0072] 3) cDNA fragmentation, end repair, and dA tail addition;

[0073] 4) Connector connection;

[0074] 5) Purification of the ligation product;

[0075] 6) Library expansion;

[0076] 7) Purification of amplified products using magnetic beads.

[0077] The probe hybridization capture according to the present invention includes the following steps:

[0078] 1) Metagenomic library mixing;

[0079] 2) Library amplification using single primers;

[0080] 3) Single-stranded library purification;

[0081] 4) Single-stranded library hybridization;

[0082] 5) Washing;

[0083] 6) Capture library PCR enrichment;

[0084] 7) Purification of amplification products.

[0085] Example 1: Preparation of a Detection Kit for 26 Viruses

[0086] The kit consists of a probe combination working solution, the sequence of which is shown in SEQ ID NO.1 to SEQ ID NO.138, and a probe preservation solution TE buffer.

[0087] Example 2: Clinical detection and result analysis of the virus

[0088] Virus testing is performed according to the following steps:

[0089] 1. Extraction of viral nucleotides.

[0090] The VAMNE Magnetic Pathogen DNA / RNA Kit (RM601-C3) was used for simultaneous extraction of viral DNA and RNA. Sample types included plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid. The required DNA+RNA concentration for extraction was no less than 5 ng / μL, and the purity was sufficient for library construction.

[0091] 2. DNA / RNA co-construction library.

[0092] Using a DNA / RNA co-construction library kit, rRNA was first removed, followed by cDNA / double-stranded cDNA synthesis. After adapter ligation, purification and PCR amplification were performed. The amplification products were then purified to obtain the library. The specific steps are as follows:

[0093] 2.1 rRNA removal: Take 10-500 ng of total DNA + RNA into an enzyme-free PCR tube, add 2 μL of thawed and mixed rRNA Removal Buffer and 2 μL of rRNA Removal Probe Mix, and bring the total volume to 25 μL with enzyme-free water. Run the following program on the PCR instrument: 85℃ for 5 min, 72℃ for 2 min, 60℃ for 5 min. After the reaction is complete, immediately place the PCR tube on ice for 3 min.

[0094] 2.2 cDNA synthesis: Add 8 μL of cDNA Reaction Buffer, 2 μL of cDNAEnzyme Mix, and 10 μL of enzyme-free water to 25 μL of the product after removing rRNA. Run the following program on the PCR instrument: 25℃ for 5 min, 30℃ for 20 min, and 85℃ for 3 min. After the reaction is complete, place the PCR tube on ice.

[0095] 2.3 cDNA fragmentation, end repair and dA tail addition: Add 10 μL of Smearase Buffer and 5 μL of Smearase Enzyme Mix to 45 μL of cDNA synthesis product. Run the following program on the PCR instrument: 4℃ for 1 min, 30℃ for 15 min, 72℃ for 10 min. After the reaction, place the PCR tube on ice.

[0096] The segment durations for different types of samples can be found in Table 2 below.

[0097] Table 2. Fragmentation conditions for different nucleic acid types

[0098] Nucleic acid types Fragmentation temperature Fragmented time plasma 30℃ 5min Bronchoalveolar lavage fluid, cerebrospinal fluid, etc. 30℃ 15min

[0099] 2.4 Adapter ligation: Taking the ligation of a specific MGI adapter as an example, add 30 μL of Ligation Enhancer, 5 μL of T4 DNA Ligase, and 5 μL of 7.5 μM Adapter to 60 μL of the cDNA fragmentation, end repair, and dA tail addition product. Run the program on the PCR instrument at 20℃ for 15 min. Immediately after the reaction, place the PCR tube on ice for 3 min.

[0100] 2.5 Purification of ligation product: Add 60 μL of magnetic beads to 100 μL of PCR ligation product and mix well. Discard the supernatant, wash the magnetic beads twice with 80% ethanol, and wash with 22 μL of enzyme-free water. Carefully transfer 20 μL of supernatant to a new PCR tube.

[0101] 2.6 Library amplification: Add 25 μL of Super HF Mix and 5 μL of Primer Mix for MGI to 20 μL of purified ligation product. Run the following program on the PCR instrument: 98℃ for 1 min; 98℃ for 10 sec, 60℃ for 15 sec, 72℃ for 30 sec, 8–14 cycles; 72℃ for 1 min.

[0102] The number of library amplification step cycles can be found in Table 3 below.

[0103] Table 3 Number of Library Amplification Steps

[0104] Total RNA+DNA input Cycle number 20ng 12 50ng 10 100ng 8 Total dosage (21 μL, total amount ≤ 10 ng) 14

[0105] 2.7 Purification of Amplified Products with Magnetic Beads: Add 45 μL of magnetic beads to 50 μL of PCR amplified product and mix well. Discard the supernatant, wash the magnetic beads twice with 80% ethanol, and wash with 25 μL of enzyme-free water. Carefully transfer the supernatant to a new PCR tube for library concentration determination.

[0106] 3. Probe hybridization capture

[0107] 3.1 Metagenomic library mixing: Each hybridization reaction can perform operations on 1 to 8 metagenomic libraries, with an input of approximately 500 ng for each library and a total library volume of no more than 5 μg for each hybridization reaction.

[0108] 3.2 Library Single Primer Amplification: If the mixed library volume is ≤23μL, prepare the reaction solution directly. If the mixed library volume is >23μL, concentrate the mixed library to ≤23μL using a vacuum concentrator before preparing the reaction solution. Add 25μL of 2X PCR mix (containing Taq enzyme, dNTPs, and buffer) and 2μL of upstream adapter to the mixed library. Add enzyme-free water to bring the total volume to 50μL. Run the following program on the PCR instrument: 98℃ for 1 min; 98℃ for 10 sec, 60℃ for 25 sec, 72℃ for 30 sec, 25 cycles; 72℃ for 5 min.

[0109] 3.3 Single-stranded library purification: After the reaction, add 50 μL of PCR amplification product to 50 μL of magnetic beads and mix well. Discard the supernatant, wash the magnetic beads twice with 80% ethanol, wash with 20 μL of enzyme-free water, and carefully transfer 19 μL of supernatant to a new PCR tube.

[0110] 3.4 Single-stranded library hybridization: Add 50 μL of HYB-Buffer, 10 μL of Enhancer, and 4 μL of mixed probe to the purified single-stranded library. Run the following program on the PCR instrument: 98℃ for 1 min, 65℃ for 30 min. Then mix the library with streptavidin magnetic beads and run the PCR instrument at 65℃ for 30 min.

[0111] 3.5 Elution: First, the hybridization library was eluted with elution buffer at 65°C, followed by room temperature elution. 23 μL of magnetic bead suspension was obtained.

[0112] 3.6 Capture library PCR enrichment: Add 25 μL of 2X PCR mix and 2 μL of library adapter primer mix (5 mM each) to 23 μL of magnetic bead suspension. Run the following program on the PCR instrument: 95℃ for 1 min; 95℃ for 10 sec, 50℃ for 25 seconds, 72℃ for 25 seconds, 18 cycles; 72℃ for 2 min.

[0113] 3.7 Purification of Amplified Products: Add 50 μL of PCR amplified product to 50 μL of magnetic beads and mix well. Discard the supernatant, wash the magnetic beads twice with 80% ethanol, and then wash with 25 μL of enzyme-free water. Perform quality checks on the concentration and average length of the library.

[0114] 4. After DNB preparation, it is placed on a sequencer.

[0115] 5. Results and Analysis

[0116] The detection results of known positive bronchoalveolar lavage fluid, cerebrospinal fluid, and serum samples using this invention are shown in Table 4 below:

[0117] Table 4 Detection results for different samples

[0118] Sample number Sample type Q20 Q30 Known positive pathogens Pathogen detected Total reads A1 bronchoalveolar lavage fluid 97.60％ 94.05％ Influenza A virus Influenza A virus 1353 A2 Cerebrospinal fluid 97.67％ 94.31％ Human herpesvirus type 5 Human herpesvirus type 5 4765 A3 serum 97.40％ 93.58％ EB virus EB virus 2333

[0119] Note: 1) Q20: The proportion of bases with a mass value of 20 or higher;

[0120] 2) Q30: The proportion of bases with a mass value of 30 or higher;

[0121] 3) Total reads: The total number of pathogen sequences detected.

[0122] As can be seen from Table 4, the detection results for different types of samples are consistent with expectations, and the detection results are accurate.

[0123] Application Example 1

[0124] Based on the above-mentioned kit and detection method, we used positive control samples of three viruses—influenza A virus H1N1, respiratory syncytial virus, and enterovirus CA16—to verify the performance of this invention. We also performed targeted sequencing of the target regions of these microbial control samples using the MGISEQ-200 sequencer from BGI Genomics. Analysis showed that the probe designed in this invention can capture the target regions of the target microorganisms. The results are shown in Tables 5-7.

[0125] Table 5 Quality Control of Experimental Sequencing Data

[0126]

[0127] Note: 1) raw_reads: Number of raw sequences;

[0128] 2) raw_bases: The number of bases in the original sequence;

[0129] 3) clean_reads: Number of high-quality sequences;

[0130] 4) clean_bases: Number of high-quality sequence bases;

[0131] 5) Cleanrate filtering ratio: number of high-quality sequences / number of original sequences;

[0132] 6) Q20: The proportion of bases with a mass value of 20 or higher;

[0133] 7) Q30: The proportion of bases with a quality value of 30 or higher.

[0134] Table 6 Pathogen Detection Status

[0135]

[0136] Table 6 compares the detection results of three test quality control samples using tNGS and mNGS respectively. It can be seen that the sequence support number of tNGS is much higher than that of mNGS.

[0137] Table 7 Comparison of detection rates of samples with different pathogen concentrations using tNGS and mNGS.

[0138]

[0139] As can be seen from Table 7, the detection rate of tNGS is higher than that of mNGS.

[0140] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A kit for detecting 26 viruses, characterized in that, The kit includes a mixture of probes capable of specifically binding to the nucleic acids of 26 target viruses, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.

138. The 26 viruses include: Influenza A virus H1N1, Influenza A virus H1N1 (2009), Influenza B virus, Human parainfluenza virus type 1, Human parainfluenza virus type 2, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Herpes simplex virus type 1, Herpes simplex virus type 2, Human alpha herpesvirus type 3, Epstein-Barr virus, Human herpesvirus type 5, Human herpesvirus type 6A, Human herpesvirus type 6B, Human herpesvirus type 7, Human herpesvirus type 8, Rhinovirus type A, Rhinovirus type B, Rhinovirus type C, Respiratory syncytial virus, Human bocavirus type 1, Human bocavirus type 2, Human bocavirus type 3, Human bocavirus type 4, Human metapneumovirus, and Human adenovirus.

2. The kit for detecting 26 viruses according to claim 1, characterized in that, The kit also includes TE buffer.

3. The kit for detecting 26 viruses according to claim 1, characterized in that, The probe design principles are as follows: probe length 120bp; GC content 30-70%; probes dispersedly distributed on the genome of the target pathogen; 15 human probes selected as internal controls.

4. A method for detecting 26 viruses using the kit described in any one of claims 1-3, characterized in that, The method includes the following steps: S1: Viral nucleotide extraction: The VAMNE Magnetic Pathogen DNA / RNA Kit (RM601-C3) was used to extract viral DNA and RNA from the sample simultaneously. The required DNA + RNA concentration was not less than 5 ng / μL and the purity was qualified to meet the requirements for library construction. S2: DNA / RNA co-construction library: rRNA was removed using a DNA / RNA co-construction library kit, followed by the synthesis of cDNA / double-stranded cDNA. After adapter ligation, the cDNA / double-stranded cDNA was purified and amplified by PCR. The amplified products were then purified to obtain the library. S3: Probe hybridization capture; the probe hybridization capture includes the following steps: 1) Metagenomic library mixing; 2) Library amplification using single primers; 3) Single-stranded library purification; 4) Single-stranded library hybridization; 5) Washing; 6) Capture library PCR enrichment; 7) Purification of amplification products; S4: DNB is prepared and then placed on a sequencer; The method is used for non-diagnostic purposes.

5. The method for detecting 26 viruses using a reagent kit according to claim 4, characterized in that, The samples included plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid.

6. The method for detecting 26 viruses using a kit according to claim 4, characterized in that, The DNA / RNA co-construction library includes the following steps: 1) rRNA removal; 2) cDNA synthesis; 3) cDNA fragmentation, end repair, and dA tail addition; 4) Connector connection; 5) Purification of the ligation product; 6) Library expansion; 7) Purification of amplified products using magnetic beads.

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