CN111254190A - Nanopore third-generation sequencing detection method for plasma virology - Google Patents
Nanopore third-generation sequencing detection method for plasma virology Download PDFInfo
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Abstract
The invention provides a nanopore third-generation sequencing detection method of plasma virology. The nanopore three-generation sequencing detection and identification technology of the plasma virology comprises the following four parts: preparing blood sample virus nucleic acid, participating standard nucleic acid internal reference, constructing a nanopore sequencing library, sequencing a nanopore in real time, converting a real-time fastq file, analyzing bioinformatics and the like. The nanopore real-time detection technology can not only quickly detect the blood sample virus group, but also improve the coverage rate of the whole genome sequence of the virus, thereby achieving the purpose of quickly detecting the blood sample virus.
Description
The technical field is as follows:
the invention relates to the technical field of biology, and relates to a detection method of blood viruses.
Background art:
the blood biological safety is a great civil and public safety problem, and the supply safety guarantee of blood resources is the fundamental guarantee of the normal operation of the national medical health service system. The method is characterized in that the method comprises the steps of screening HBV, HCV, HIV and TP, screening HBV, HIV, screening HBV, screening HIV, screening TP, screening HBV, screening HIV, screening HBV, screening and screening.
The four items of routine blood pathogen screening HBV, HCV, HIV and TP are to confirm the type of blood virus infection by blood enzyme immunity method or PCR or RT-PCR quantitative or qualitative analysis of virus nucleic acid, but a sequence specific antibody or a specific primer is needed to detect the virus antigen or nucleic acid, virus leak detection is generated due to virus variation or low copy sample, and other possible pathogenic blood viruses are not in routine detection items, so blood virus metagenome detection is realized, and the extracted virus nucleic acid can detect not only known viruses but also unknown viruses through non-template dependent PCR amplification, thereby greatly improving detection sensitivity and specificity. The current Nanopore technology is mainly used for detecting cultured bacteria, viruses or biological genomes, and has the characteristics of single molecule sequencing, nucleic acid length of over 150kb, high sequencing speed, real-time sequencing data monitoring, convenience in carrying of a machine and the like. The invention applies the Nanopore third generation nano sequencing technology to the detection of blood virus spectra, is a brand new technical field, and has the core technical problems of the pollution of human host nucleic acid and large amount of target nucleic acid required by the detection. Therefore, the core problem to be solved by the invention is to enrich the blood sample virus and remove the host nucleic acid by ultracentrifugation and cocktail enzymolysis, then extract the virus nucleic acid, add the known nucleic acid internal reference and use the related reagent to construct the sequencing library, and simultaneously establish the localized virus database and the programmed bioinformatics analysis program to achieve real-time detection and analysis, and simultaneously, the standard nucleic acid internal reference is used as the quality control and sensitivity index of the whole experimental process, thus providing possibility for the confirmation of high-throughput blood virus detection.
Disclosure of Invention
The object of the present invention is to provide a method for the detection of blood viruses for non-diagnostic purposes.
The key problem to be solved by the invention is to enrich blood sample virus and remove host nucleic acid by using an ultracentrifugation and nucleic acid enzymolysis method, then extract virus nucleic acid and use related reagents to establish a sequencing library, simultaneously establish a standard nucleic acid internal reference as quality control and sensitivity indexes of an experiment and analysis whole process, and establish a localized virus database and a programmed bioinformatics analysis program, thereby achieving the purpose of detecting and analyzing blood virus in real time and providing possibility for the application of nanopore blood virus detection.
The detection method comprises the following steps: (1) the method comprises the following four steps of (1) blood sample virus enrichment and library nucleic acid preparation and standard nucleic acid internal reference addition, (2) nanopore sequencing library construction and verification, (3) library on-machine and nanopore real-time sequencing, and (4) bioinformatics analysis.
Wherein, (1) blood sample virus enrichment and nucleic acid preparation of library and standard nucleic acid internal reference are added: enriching viruses and nucleic acid of a sample to be detected based on blood, extracting viral nucleic acid, preparing library-establishing nucleic acid and adding standard nucleic acid;
wherein, (2) nanopore sequencing library construction and verification: constructing a nucleic acid library for nanopore sequencing by using a PCR (polymerase chain reaction) barcode Kit (PCR Barcoding Kit), and verifying;
wherein, (3) library computer and nanopore real-time sequencing: adding Nanopore sequencing kit (such as EXP-FLP002) into mixed sample library containing mixed internal reference, and performing real-time detection on the sample library;
wherein, (4) bioinformatics analysis: firstly, a local database containing 300 Reseq standard viruses is established, a Guppy software is used for removing barcode groups from nanopore sequencing data, BlastN (parameters are e-5 to e-10) is compared with the local database, the result is introduced into MEGAN software, the virus composition in a blood sample is accurately analyzed, and a standard nucleic acid internal reference is used as a quality control and sensitivity index of an evaluation result.
Wherein, (1) blood sample virus enrichment and nucleic acid preparation of library and standard nucleic acid internal reference are added: aiming at the characteristics of low virus content and high blood viscosity of a sample to be detected in a blood sample, the invention adopts a set of blood sample virus enrichment method, and the method comprises the steps of firstly enriching the sample virus by using an ultracentrifugation method, then removing host free nucleic acid by using a composite nuclease digestion method, then extracting virus nucleic acid, then synthesizing virus nucleic acid dsDNA, and finally adding standard nucleic acid internal reference.
Specifically, the blood sample virus enrichment and library nucleic acid preparation in the step (1) comprises the following three steps: 1) viral enrichment and host free nucleic acid removal of plasma samples, 2) extracting viral nucleic acid DNA or RNA and synthesizing double-stranded DNA (dsDNA), 3), viral nucleic acid purification and standard internal reference nucleic acid.
1) Plasma sample virus enrichment and host free nucleic acid removal: taking 6-8ml of healthy adult blood containing EDTA anticoagulant without clinical main symptoms, centrifuging 3000g for 15min, taking supernatant as plasma, centrifuging 10000g of plasma for 10min, removing cell debris, taking 0.5-6ml of plasma sample, adding Hank's solution into 12ml of plasma sample, carefully and uniformly mixing, rotating 28800g of blood sample by SW41, centrifuging at 4 ℃ for 3h, sucking and removing supernatant, adding Hank's solution with certain volume, transferring the mixture into a 1.5ml centrifuge tube, and digesting by nuclease to remove host cell nucleic acid.
The representatives are mixed with different samples or can be single samples, if each sample is 0.5-6ml, 1-12 samples can be mixed, and the like, because the abundance of the blood virus is low, the cost can be saved by mixing the samples, and the abundance of the virus can be improved.
2) Extracting DNA or RNA from virus nucleic acid and synthesizing double-stranded DNA (dsDNA), wherein the nucleic acid of the DNA or RNA virus mainly comprises DNA or RNA, converting virus RNA into cDNA which is more stable and easier to store, and further synthesizing double-stranded DNA (dsDNA).
3) Viral nucleic acid purification and standard reference nucleic acids: the viral nucleic acid obtained in the second step is purified, and 100 copy standard internal reference nucleic acids (prepared in laboratory, with fragment size of 3560, containing 495bp insert, and capable of PCR confirmation and quantification) are added to each nucleic acid sample as quality control and sensitivity indexes for whole detection and biological information analysis. Nucleic acid samples (S-dsDNA) containing standard internal controls were prepared for subsequent pooling and sequencing and bioinformatic analysis.
(2) Nanopore sequencing library construction and validation
After the quality of the S-dsDNA prepared above was quantified, a nucleic acid library for nanopore sequencing was constructed with a PCR Barcoding Kit (PCR Barcoding Kit) and verified by electrophoresis with Angilent2100 nucleic acid.
Specifically, the method comprises the following steps: 1) accurately quantifying and diluting the virus nucleic acid purified in the step (1), 2) repairing DNA and preparing ends, 3) adding a bar code, amplifying and verifying, and 4) mixing a sample and a DNA library.
1) Accurate quantification and dilution of purified viral nucleic acid: detecting the virus nucleic acid purified in the step (1) by using Nanodrop, determining that the virus nucleic acid meets the requirements that OD260/280 is 1.8-1.9 and OD260/230 is 2.2-2.5, quantifying the virus nucleic acid purified in the step (1) by using a Qubit method, and diluting to the nucleic acid concentration of 100ng/50 ul.
2) DNA repair and end preparation: repair and dA-tail addition were performed by NEBNext end repair/dA-tail addition module (NEBNext EndRepiair/dA-tail module) and S-dsDNA-A was purified with magnetic beads (Agencour AMPure XPbeads, Beckman).
3) Adding a bar code, amplifying and verifying: amplifying each sample by using 12 pairs of primers of the PCR bar code kit, adding a barcode joint into nucleic acid of each sample to obtain S-dsDNA-A-BC after bar code, purifying by using magnetic beads, carrying out PCR amplification on the S-dsDNA-A-BC after bar code, selecting and purifying S-dsDNA-A-BC in the third step by using magnetic beads (Agencour AMPure XP beads, Beckman), quantifying by using qubit, running glue by using Agilent 2100, diluting the library between 1k and 10k into 200pg/ul purified samples before computer sequencing.
4) Mixed sample and DNA library: and calculating the amount of the sample required for mixing, ensuring that the number of moles of the mixed sample is equal and the total amount is between 10 and 50fmol, mixing the sample into a new 1.5ml centrifuge tube according to the calculation result of the sample, and if the volume of the mixed sample is more than 11 mu l, purifying and concentrating by using 2X magnetic beads (Agencour AMPure XP beads, Beckman) and eluting to 11 mu l. Taking 1 mul for quantification, taking 10 mul of DNA sample in the mixing pool, adding 1 mul of RAP, mixing uniformly at room temperature for 5min, and placing on ice for S-dsDNA-A-BC-R-Bank for computer sequencing.
(3) Library processing and nanopore real-time sequencing
Adding the mixed sample library of step 2 connected with a sequencing knot and a mixed internal reference into a Nanopore sequencing kit (such as EXP-FLP002) for on-machine real-time detection;
specifically, the nucleic acid mixed library S-dsDNA-A-BC-R-Bank connected with a sequencing knot and an internal reference by the step (2) is about 30-50fmol, and the on-machine sample is further perfected. The method comprises the following steps: preparation of the on-machine library, preparation of the Nanopore chip, on-machine and real-time sequencing.
1) Preparing a computer library: and (3) using a sequencing reagent kit, adding sample Beads and a nucleic acid mixed library with a sequencing knot in a sequencing buffer, and completing all work of the computer library in one step in one tube.
2) Preparation of the Nanopore chip: firstly connecting a sequencer and a computer, starting sequencing software, judging whether a sequencing chip is qualified, and if so, preparing a cleaning reagent of the chip and cleaning the chip for next machine use.
3) Computer and real-time sequencing: the method mainly comprises the steps of completely adding 75 mu l of an upper computer library in the first step into a sample hole of a chip prepared in the second step, connecting the chip to a sequencer, setting a file name and a sequencing program, selecting and converting an electric sequencing signal fast5 file into a nucleic acid sequence information fast q file in real time, realizing real-time sequencing, and observing sample sequencing information and the using condition of the sequencing hole at any time.
(4) Bioinformatics analysis
Firstly, a local database containing 300 Reseq standard viruses is established, a Guppy software is used for removing barcode groups from nanopore sequencing data, BlastN (parameters are e-5 to e-10) is used for comparing with the local database, the result of the comparison 5 is introduced into MEGAN software, the virus composition in a blood sample is accurately analyzed, and the internal reference of standard nucleic acid is used as the sensitivity index of an evaluation result.
Preferably, the detection method of the present invention comprises the following steps:
(1) blood sample virus enrichment, nucleic acid extraction and library nucleic acid preparation
1) And (3) enriching the plasma sample virus: centrifuging 6-8ml of healthy adult blood containing EDTA anticoagulant for 15min at 3000g, taking supernatant as plasma, centrifuging 10000g of the plasma for 10min, removing cell debris, adding 0.5-6ml of the supernatant into a super-centrifuge tube, adding Hank's solution into the super-centrifuge tube with the volume of 12ml, carefully mixing the plasma and the plasma, balancing the plasma by a balance, selecting W41 to turn the head, centrifuging the plasma at 28800g at 4 ℃ for 2h, carefully removing the supernatant, adding the Hank's solution to the bottom of the tube, transferring the tube to a 1.5ml centrifuge tube, and removing host cell nucleic acid by a nuclease digestion method. Wherein the nucleases include Turbo Dnase deoxyribonuclease, RNase one is ribonuclease, and Nuclease, digested at 37 ℃ for 30 min.
2) Extraction of viral nucleic acid DNA or RNA viral nucleic acid was extracted using the QIamp viral RNA mini Kit from Qiagen, 30. mu.l of AVE was eluted and the procedure was as described. Using superscriptTMIV First-Strandsynthesis system and Klenow used to complete the second strand of cDNA synthesis of dsDNA, according to the instructions.
3) Viral nucleic acid purification and standard internal reference nucleic acids: purifying the virus nucleic acid obtained in the step 2) by using magnetic beads (Agencour AMPure XP beads, Beckman), recovering by using 25 mu l of nuclease-free water, adding 100 copies of standard internal reference nucleic acid (prepared in a laboratory, the fragment size is 3560, the fragment contains 495bp of insertion fragments, and can be used for PCR confirmation and quantification) into each nucleic acid sample, and using the nucleic acid sample as the quality control and sensitivity index of the whole detection and biological information analysis. A nucleic acid sample (S-dsDNA) containing a nucleic acid standard internal reference was prepared for subsequent pooling and sequencing and bioinformatic analysis.
(2) Nanopore sequencing library construction and validation, the approximate process is as follows:
1) accurate quantification and dilution of purified viral nucleic acid: detecting the virus nucleic acid purified in the step (1) by using Nanodrop, determining the nucleic acid which meets the requirements that OD260/280 is 1.8-1.9 and OD260/230 is the attached requirement, quantifying the virus nucleic acid purified in the step (1) by using a Qubit method, and diluting to the nucleic acid concentration of 100ng/50 ul.
2) DNA repair and end preparation: repair and dA tail addition were performed by NEBNext end repair/dA tail addition module (NEBNext EndRepiair/dA-tailing module), the procedure was as described, followed by purification with 1-magnetic beads (Agencour AMPure XP beads, Beckman), the procedure was as described, and S-dsDNA-A was obtained with 16. mu.l RNase-free water recovery.
3) Add barcode (Adaper) and zoom in: taking PCR Barcoding kit (SQK-PBK004) as an example, amplifying each sample of the purified S-dsDNA-A by using a 12x primer pair of a PCR barcode kit, adding a barcode linker into nucleic acid of each sample to obtain the S-dsDNA-A-BC after barcode, purifying by using magnetic beads, carrying out PCR amplification on the S-dsDNA-A-BC after barcode, and carrying out PCR reaction after adding adaptors as follows:
extension time is determined by the size of the fragment length of the nucleic acid, typically 50sec/kb of enzymatic activity.
The PCR product was finally sorted and purified by using 0.6 ratio magnetic beads, and after PCR, the S-dsDNA-A-BC with Barcode was eluted with 10. mu.l of 50mM NaCl +10mM Tris.HCl pH8.0, and 1. mu.l was quantified with qubit for the following mixed sample calculation.
4) Pool mixing and DNA library: and calculating the amount of the sample required for mixing, ensuring that the number of moles of the mixed sample is equal and the total amount is between 10 and 50fmol, mixing the sample into a new 1.5ml centrifuge tube according to the calculation result of the sample, and if the volume of the mixed sample is more than 11 mu l, purifying and concentrating by using 2X magnetic beads (Agencour AMPure XP beads, Beckman) and eluting to 11 mu l. Taking 1 mul for quantification, taking 10 mul of DNA sample in the mixing pool, adding 1 mul of RAP, mixing uniformly at room temperature for 5min, and placing on ice for S-dsDNA-A-BC-R-Bank for computer sequencing.
(3) Library on-machine and nanopore real-time sequencing
1) Preparing a machine library:
preparing before loading, and mixing to ensure no bubble.
2) Preparation of Nanopore chips: firstly connecting a sequencer and a computer, starting sequencing software, judging whether a sequencing chip is qualified, and if so, preparing a chip cleaning reagent and cleaning the chip for next machine use.
3) And (3) computer loading and real-time sequencing: the method mainly comprises the steps of completely adding 75 mu l of an upper computer library in the first step into a prepared chip sample hole (SpotON sample port) in the second step, connecting the chip to a sequencer, setting a file name and a sequencing program, and selecting a file which converts an electric sequencing signal fast5 file into a nucleic acid sequence information fast q file in real time, so that real-time sequencing is realized, and the use conditions of the sample sequencing information and the sequencing hole are observed at any time.
(4) Bioinformatics analysis
Firstly, a local database containing 300 Reseq standard viruses is established, a Guppy software is used for removing barcode groups from nanopore sequencing data, BlastN (parameters are e-5 to e-10) is used for comparing with the local database, the result of the comparison 5 is introduced into MEGAN software, the virus composition in a blood sample is accurately analyzed, and the internal reference of standard nucleic acid is used as the sensitivity and quality control index of an evaluation result.
Compared with the existing detection method, the invention has the advantages that:
1) it is suitable for, but not limited to, the detection and identification of human and animal blood viruses;
2) the method has the advantages that real-time detection and real-time analysis can be realized, the purpose of rapid detection is achieved, the composition of a blood virus spectrum can be found through data analysis within 1 minute, and the distribution of the whole genome of most viruses reaches more than 90% within 30 minutes;
3) the enrichment of blood virus provides possibility for the sensitivity of blood virus detection, and the reference of nucleic acid standard can be used as the sample detection sensitivity and quality control index;
4) the establishment of a local database and the establishment of a biological information method provide software support for rapid detection; in view of these advantages of the present invention, it can be used for accurate medical analysis of, but not limited to, blood donated people and clinical blood samples, and is of great significance for the rapid identification of the blood etiology of newly developed emergent infections.
According to the results of example 1, fig. 1 detects the virus spectrum of blood, which includes reference sequence, hepatitis b virus and various parvoviruses, the reference can be used as detection sensitivity and quality control index, fig. 2 represents the detected sequence number of Alpha parvoviruses and viruses found by hepatitis b virus over time in different time, the sequence length is between 1k and 3k, taking hepatitis b virus as example, the result shows that 20 HBV nucleic acid sequences of blood hepatitis b virus can be detected in 1 minute, thus proving that HBV infection of blood can be detected, 172 nucleic acid sequences can be detected in 10 minutes, and HBV full nucleic acid sequence can be spliced. Compared with the second-generation sequencing time which is at least 20 hours, the analysis can be carried out only after the shutdown, and the time is at least 24 hours, so the research shows that the distribution of the whole genome of most viruses in 30 minutes reaches more than 90 percent; the analysis can be detected in real time.
Drawings
FIG. 1 is a classification chart and a reference sequence chart of an example of a blood virus spectrum (after removing a human genome)
FIG. 2 is a graph showing the number of nucleic acid sequences of hepatitis B virus and parvovirus in blood detected over time.
The specific implementation mode is as follows:
the following describes embodiments of the present invention by way of specific examples. The selection and adjustment of the analysis paths and the adjustment of specific parameters according to different specific examples without departing from the spirit of the present invention, which are disclosed by the present specification, belong to the protection scope of the present invention.
Example 1 detection method
We used an example of 24 cases of asymptomatic conventional four-item test negative adult blood collected at the red cross blood center of Beijing city at 4 months 2015 to sequentially illustrate the specific embodiment of the present invention.
Blood sample virus enrichment, nucleic acid extraction and library nucleic acid preparation
1) And (3) enriching the plasma sample virus: taking 6-8ml of healthy adult blood containing EDTA anticoagulant without clinical main symptoms, centrifuging 3000g for 15min, taking supernatant as plasma, centrifuging 10000g of plasma for 10min, removing cell fragments, adding the supernatant which is 0.5-6ml of plasma sample into a super-separation tube, adding Hank's solution into a volume of 12ml, carefully mixing uniformly, balancing by a balance, selecting W41 to turn a head, centrifuging at a speed of 28800g, centrifuging at 4 ℃ for 2h, carefully removing the supernatant, adding Hank's solution into the bottom of the tube to dissolve, transferring into a 1.5ml centrifuge tube, simultaneously adding Turbo Dnase deoxyribonuclease, Nuclease Nuclease and RNaseone ribonuclease, and then digesting at 37 ℃ for 30min to remove host cell nucleic acid.
2) Extracting DNA or RNA included in nucleic acid of DNA or RNA virus with QIampviral RNA miniKit from Qiagen, eluting viral RNA in a sample with 30. mu.l of AVE, performing the following procedures, and subjecting viral RNA to superscriptptTMIV First-strand synthesis system, using Klenow synthesis to synthesize double-stranded DNA, i.e., dsDNA, according to the instructions.
3) Purifying the virus dsDNA obtained in the step 2) by using magnetic beads (Agencour AMPure XP beads, Beckman), recovering by using 25 mu l of nuclease-free water, adding 100 copy of standard internal reference nucleic acid (prepared in a laboratory, the fragment size is 3560, the fragment contains 495bp of insertion fragment, and can be used for PCR confirmation and quantification) into each nucleic acid sample, and using the purified nucleic acid as a quality control and sensitivity index of the whole detection and biological information analysis. A nucleic acid sample (S-dsDNA) containing a nucleic acid standard internal reference was prepared for subsequent pooling and sequencing and bioinformatic analysis.
Secondly, constructing and verifying a nanopore sequencing library: a nucleic acid sample (S-dsDNA) containing a nucleic acid standard internal reference is subjected to library construction by using a Nanopore library construction kit, which comprises the following specific steps:
1) accurate quantification and dilution of purified viral nucleic acid: detecting the virus nucleic acid purified in the step (1) by using Nanodrop, determining nucleic acid which meets the requirements that OD260/280 is 1.8-1.9 and OD260/230 is compatible, quantifying the S-dsDNA of the virus nucleic acid purified in the step one by using a Qubit method, and diluting to the nucleic acid concentration of 100ng/50 ul.
2) DNA repair and end preparation: repair and dA tail addition are performed by NEBNext end repair/dA tail addition module (NEBNext EndRepiair/dA-tailing module).
The following procedure was performed: 5min at 20 ℃; 65 ℃ for 5 min.
3) And (3) purification: and (3) purifying the DNA in the previous step by using magnetic beads (Agencour AMPure XP beads, Beckman), purifying the virus nucleic acid by using the method in the step 6 in the step (1), and finally, recovering 16 mu l of RNase-free water to obtain S-dsDNA-A.
4) Add barcode (Adaper) and zoom in: PCR Barcoding kit (SQK-PBK004) is carried out according to the operation instruction to obtain Barcode DNA, magnetic beads with the proportion of 0.6 are used for sorting, finally 10 mu l of 50mM NaCl +10mM Tris. HCl PH8.0 is used for elution, 1 mu l of the Barcoding DNA is quantified by qubit, Agilent 2100 is used for running gel, the size of a target fragment is preliminarily determined, the method is mainly used for calculating the concentration after mixing samples, and the sample is diluted into 200pg/ul purified sample S-dsDNA-A-BC.
5) Pool mixing and DNA library: and calculating the amount of the sample required for mixing, ensuring that the number of moles of the mixed sample is equal and the total amount is between 10 and 50fmol, mixing the sample into a new 1.5ml centrifuge tube according to the calculation result of the sample, and if the volume of the mixed sample is more than 11 mu l, performing purification concentration by using 2X purified magnetic beads (Agencour AMPure XP beads, Beckman) and eluting to 11 mu l. Taking 1 ul of the quantification, uniformly mixing 10 ul l S-dsDNA-A-BC and 1 ul RAP, performing rapid sequencing knot (adapter) connection at room temperature for 5min, placing on ice for machine sequencing, and finally completing the DNA library construction to 11 mu l S-dsDNA-A-BC-R.
Thirdly, library processing and nanopore real-time sequencing
1) Preparing a machine library:
and preparing before loading, and uniformly mixing to ensure no bubbles.
2) Preparation of Nanopore chips: firstly connecting a sequencer and a computer, starting sequencing software, judging whether a sequencing chip is qualified, and if so, preparing a chip cleaning reagent and cleaning the chip for next machine use. The cleaning of the chip is carried out according to the specification, and the whole operation avoids introducing air bubbles into the chip
3) Loading and real-time sequencing: the method mainly comprises the steps of completely adding 75 mu l of an upper computer library in the first step into a prepared chip sample hole (SpotON sample port) in the second step, connecting the chip to a sequencer, setting a file name and a sequencing program, and selecting a file which converts an electric sequencing signal fast5 file into a nucleic acid sequence information fast q file in real time, so that real-time sequencing is realized, and the use conditions of the sample sequencing information and the sequencing hole are observed at any time.
Fourth, bioinformatics analysis
Establishing a local database containing 300 Reseq standard viruses, using Guppy software to remove barcode grouping from nanopore sequencing data, using BlastN (parameters are e-5 to e-10) to compare with the local database, taking the result of 5 before comparison, introducing the result into MEGAN software, accurately analyzing the virus composition in a blood sample, and using internal reference of standard nucleic acid as the sensitivity and quality control index of an evaluation result. The results are shown in FIG. 1, the viral profile at different time points in blood, in which HBV, hepatitis B virus, was found.
Claims (10)
1. A method for detecting a blood virus, the method being for non-diagnostic purposes, the method comprising the steps of: (1) the method comprises the steps of (1) blood sample virus enrichment and library nucleic acid preparation and standard nucleic acid internal reference addition, (2) nanopore sequencing library construction and verification, (3) library on-machine and nanopore real-time sequencing, and (4) bioinformatics analysis.
2. The detection method according to claim 1,
wherein, (1) blood sample virus enrichment and nucleic acid preparation of library and standard nucleic acid internal reference are added: enriching viruses and nucleic acid of a sample to be detected based on blood, extracting viral nucleic acid, preparing library-establishing nucleic acid and adding standard nucleic acid;
wherein, (2) nanopore sequencing library construction and verification: constructing a nucleic acid library for nanopore sequencing by using a PCR (polymerase chain reaction) bar code kit, and verifying;
wherein, (3) library computer and nanopore real-time sequencing: adding a Nanopore sequencing kit into a mixed sample library containing a mixed internal reference, and performing real-time detection on the mixed sample library;
wherein, (4) bioinformatics analysis: firstly, a local database containing 300 Reseq standard viruses is established, a Guppy software is used for removing barcode groups from nanopore sequencing data, a BlastN is compared with the local database, the result is led into MEGAN software, the virus composition in a blood sample is accurately analyzed, and the internal reference of the standard nucleic acid is used as the quality control and sensitivity index of an evaluation result.
3. The method for detecting virus of claim 1, wherein the virus enrichment and nucleic acid library preparation of the blood sample in step (1) comprises the following three steps: 1) viral enrichment and host free nucleic acid removal of plasma samples, 2) extracting viral nucleic acid DNA or RNA and synthesizing double-stranded DNA (dsDNA), 3), viral nucleic acid purification and standard internal reference nucleic acid.
4. The method for detecting virus according to claim 3, wherein the virus enrichment and nucleic acid library preparation of the blood sample in step (1) comprises the following three steps: 1) plasma sample virus enrichment and host free nucleic acid removal: centrifuging 6-8ml of whole blood containing EDTA anticoagulant for 15min at 3000g for healthy adults without clinical main symptoms, taking supernatant as plasma, centrifuging 10000g of plasma for 10min, removing cell debris, taking 0.5-6ml of plasma sample, adding Hank's solution into a volume of 12ml, carefully mixing the plasma, rotating 28800g of the sample by SW41, centrifuging at 4 ℃ for 3h, sucking and removing supernatant, adding Hank's solution with a certain volume, transferring the mixture into a 1.5ml centrifuge tube, digesting and removing host cell nucleic acid by nuclease,
2) extracting virus nucleic acid DNA or RNA and synthesizing double-strand DNA: mainly extracting DNA or RNA virus nucleic acid including DNA or RNA, converting virus RNA into more stable and storable cDNA, further synthesizing double-stranded DNA,
3) viral nucleic acid purification and standard reference nucleic acids: and (3) purifying the virus nucleic acid obtained in the second step, adding 100 copy standard internal reference nucleic acids into each nucleic acid sample, using the nucleic acid samples as quality control and sensitivity indexes of the whole detection and biological information analysis, and preparing the nucleic acid samples containing the standard internal reference for subsequent library construction, sequencing and biological information analysis.
5. The detection method according to claim 1, wherein (2) nanopore sequencing library construction and validation comprises the following steps: 1) accurately quantifying and diluting the virus nucleic acid purified in the step (1), 2) repairing DNA and preparing ends, 3) adding a bar code, amplifying and verifying, and 4) mixing a sample and a DNA library.
6. The detection method according to claim 5, wherein (2) nanopore sequencing library construction and validation comprises the following steps:
1) accurate quantification and dilution of purified viral nucleic acid: detecting the virus nucleic acid purified in the step (1) by using Nanodrop, determining that the virus nucleic acid meets the requirements that OD260/280 is 1.8-1.9 and OD260/230 is 2.2-2.5, quantifying the virus nucleic acid purified in the step (1) by using a Qubit method, and diluting to the nucleic acid concentration of 100ng/50ul,
2) DNA repair and end preparation: repair and dA tail addition were performed by NEBNext end repair/dA tail addition module and S-dsDNA-A was purified with magnetic beads.
3) Adding a bar code, amplifying and verifying: amplifying each sample by using 12 pairs of primers of a PCR bar code kit, adding a barcode joint into nucleic acid of each sample to obtain S-dsDNA-A-BC after bar code, purifying by using magnetic beads, performing PCR amplification on the S-dsDNA-A-BC after bar code, performing sorting and purification by using magnetic beads to obtain S-dsDNA-A-BC in the third step, quantifying by using qubit, running gel by using Agilent 2100, diluting the library between 1k and 10k to 200pg/ul purified sample before computer sequencing,
4) mixed sample and DNA library: calculating the amount of the sample required for mixing, ensuring that the number of moles of the mixed sample is equal and the total amount is between 10 and 50fmol, mixing the sample into a new 1.5ml centrifuge tube according to the calculation result of the sample, if the volume of the mixed sample is more than 11 mu l, performing purification and concentration by using 2X magnetic beads, eluting to 11 mu l, taking 1 mu l for quantification, taking 10 mu l of the DNA sample in the mixed pool, adding 1 mu l of RAP, mixing uniformly for 5min at room temperature, and placing on ice for preparing S-dsDNA-A-BC-R-Bank for computer sequencing.
7. The detection method according to claim 1, wherein (3) the library is subjected to in-machine and nanopore real-time sequencing, and the method comprises the following steps:
1) preparing a computer library: using a sequencing reagent kit, adding sample Beads and a nucleic acid mixed library with a sequencing knot in a sequencing buffer to complete all work of the computer library in one step in one tube,
2) preparation of the Nanopore chip: firstly connecting a sequencer and a computer, starting sequencing software, determining whether a sequencing chip is qualified, and if the sequencing chip is qualified, preparing a cleaning reagent of the chip and cleaning the chip for next machine loading,
3) computer and real-time sequencing: the method mainly comprises the steps of completely adding 75 mu l of an upper computer library in the first step into a sample hole of a chip prepared in the second step, connecting the chip to a sequencer, setting a file name and a sequencing program, selecting and converting an electric sequencing signal fast5 file into a nucleic acid sequence information fast q file in real time, realizing real-time sequencing, and observing sample sequencing information and the using condition of the sequencing hole at any time.
8. The detection method according to claim 1, wherein (4) bioinformatics analysis includes the steps of: firstly, a local database containing 300 Reseq standard viruses is established, a Guppy software is used for removing barcode groups from nanopore sequencing data, BlastN is used for comparing with the local database, the result of comparison 5 is introduced into MEGAN software, the virus composition in a blood sample is accurately analyzed, and a standard nucleic acid internal reference is used as a sensitivity index for evaluating the result.
9. The detection method according to claim 1, characterized by comprising the steps of:
(1) blood sample virus enrichment, nucleic acid extraction and library nucleic acid preparation
1) And (3) enriching the plasma sample virus: centrifuging 6-8ml of whole blood containing EDTA anticoagulant for 15min at 3000g for 15min from healthy adult without clinical main symptoms, taking supernatant as plasma, centrifuging 10000g of plasma for 10min, removing cell debris, adding 0.5-6ml of supernatant into a super-centrifuge tube, adding Hank's solution into a volume of 12ml, carefully mixing, balancing by a balance, selecting W41 to turn the head, centrifuging at 28800g at 4 ℃ for 2h, carefully removing supernatant, adding Hank's solution to the bottom of the tube, transferring into a 1.5ml centrifuge tube, removing host cell nucleic acid by Nuclease digestion, wherein Turbo Dnase deoxyribonuclease, RNase one is ribonuclease and Nuclease are digested at 37 ℃ for 30min,
2) extracting viral nucleic acid DNA or RNA: DNA or RNA viral nucleic acid was extracted using the QIamp viral RNA mini Kit from Qiagen, 30. mu.l AVE was eluted, and the protocol was followed using superscriptTMIV First-Strandsynthesis system and Klenow to complete the second strand of cDNA synthesis of dsDNA, according to the instructions,
3) viral nucleic acid purification and standard internal reference nucleic acids: purifying the virus nucleic acid obtained in the step 2) by using magnetic beads, recovering by using 25 mu l of nuclease-free water, adding 100 copy standard internal reference nucleic acids into each nucleic acid sample as the quality control and sensitivity indexes of the whole detection and biological information analysis, preparing the nucleic acid sample containing the nucleic acid standard internal reference for subsequent library construction, sequencing and biological information analysis,
(2) constructing and verifying a nanopore sequencing library, and comprising the following steps of:
1) accurate quantification and dilution of purified viral nucleic acid: detecting the virus nucleic acid purified in the step (1) by using Nanodrop, determining the nucleic acid which meets the requirements that OD260/280 is 1.8-1.9 and OD260/230 is compatible, quantifying the virus nucleic acid purified in the step (1) by using a Qubit method, diluting to the nucleic acid concentration of 100ng/50ul,
2) DNA repair and end preparation: repair and dA tail addition were performed by NEBNext end repair/dA tail addition module, as per instructions, followed by 1 magnetic bead purification, methods referenced to the instructions, and recovery with 16. mu.l RNase-free water to obtain S-dsDNA-A,
3) adding a bar code and amplifying: taking a PCR Barcoding kit as an example, amplifying each sample of the purified S-dsDNA-A by using a 12x primer pair of a PCR barcode kit, adding a barcode linker into nucleic acid of each sample to obtain the barcoded S-dsDNA-A-BC, purifying by using magnetic beads, carrying out PCR amplification on the barcoded S-dsDNA-A-BC, and carrying out PCR reaction after adding adaptors as follows:
4) pool mixing and DNA library: calculating the amount of the sample required for mixing, ensuring that the number of moles of the mixed sample is equal and the total amount is between 10 and 50fmol, mixing the sample into a new 1.5ml centrifuge tube according to the calculation result of the sample, if the volume of the mixed sample is more than 11 mu l, purifying and concentrating by using 2X magnetic beads, eluting to 11 mu l, taking 1 mu l for quantification, taking 10 mu l of the DNA sample in the mixed pool, adding 1 mu l of RAP, mixing uniformly for 5min at room temperature, placing on ice for preparing S-dsDNA-A-BC-R-Bank for computer sequencing,
(3) library on-machine and nanopore real-time sequencing
1) Preparing a machine library:
preparing before loading, mixing uniformly to ensure no bubble,
2) preparation of Nanopore chips: firstly connecting a sequencer and a computer, starting sequencing software, determining whether a sequencing chip is qualified, and if the sequencing chip is qualified, preparing a chip cleaning reagent and cleaning the chip for next machine loading,
3) and (3) computer loading and real-time sequencing: mainly comprises the steps of completely adding 75 mu l of the on-board library in the first step into a sample hole of the chip prepared in the second step, connecting the chip to a sequencer, setting a file name and a sequencing program, selecting a file which converts an electric sequencing signal fast5 file into a nucleic acid sequence information fast q file in real time, realizing real-time sequencing, and observing the sample sequencing information and the using condition of the sequencing hole at any time,
(4) bioinformatics analysis
Firstly, a local database containing 300 Reseq standard viruses is established, a Guppy software is used for removing barcode groups from nanopore sequencing data, BlastN is used for comparing with the local database, the result of comparison 5 is introduced into MEGAN software, the virus composition in a blood sample is accurately analyzed, and a standard nucleic acid internal reference is used as the sensitivity and quality control index of an evaluation result.
10. The detection method according to claim 1, characterized by comprising the steps of:
(1) enriching blood sample virus, extracting nucleic acid and preparing nucleic acid for establishing library
1) And (3) enriching the plasma sample virus: centrifuging 6-8ml of whole blood containing EDTA anticoagulant for 15min at 3000g for a healthy adult without clinical main symptoms, taking supernatant as plasma, centrifuging 10000g of the plasma for 10min, removing cell debris, adding 0.5-6ml of the supernatant as a plasma sample into a super-separation tube, adding Hank's solution into the super-separation tube with the volume of 12ml, carefully and uniformly mixing, balancing by a balance, selecting W41 to turn the head, centrifuging at the speed of 28800g at 4 ℃ for 2h, carefully removing the supernatant, adding Hank's solution into the bottom of the tube to dissolve, transferring into a 1.5ml centrifuge tube, simultaneously adding Turbo DNase deoxyribonuclease, Nuclease Nuclease and RNase one ribonuclease, and then digesting for 30min at 37 ℃ to remove host cell nucleic acid,
2) extracting DNA or RNA included in nucleic acid of DNA or RNA virus with QIamp viral RNA miniKit from Qiagen, eluting viral RNA in sample with 30. mu.l AVE, performing the procedures as described in the specification, and subjecting viral RNA to superscriptptTMIV First-strand synthesis system, synthesizing cDNA into double-stranded DNA, i.e. dsDNA, by Klenow synthesis, the operation is performed according to the instruction,
3) purifying the virus dsDNA obtained in the step 2) by using magnetic beads, recovering by using 25 mu l of nuclease-free water, adding 100 copies of standard internal reference nucleic acid into each nucleic acid sample, using the nucleic acid samples as quality control and sensitivity indexes of whole detection and biological information analysis, preparing nucleic acid samples containing the nucleic acid standard internal reference for subsequent library construction, sequencing and biological information analysis,
(2) nanopore sequencing library construction and validation: the nucleic acid sample containing the nucleic acid standard internal reference is subjected to library construction by using a Nanopore library construction kit, which comprises the following specific steps:
1) accurate quantification and dilution of purified viral nucleic acid: detecting the virus nucleic acid purified in the step (1) by using Nanodrop, determining the nucleic acid which meets the requirements that OD260/280 is 1.8-1.9 and OD260/230 is compatible, quantifying the S-dsDNA of the virus nucleic acid purified in the step one by using a Qubit method, and diluting to the nucleic acid concentration of 100ng/50ul,
2) DNA repair and end preparation: repair and dA tail addition are performed by a NEBNext end repair/dA tail addition module,
the following procedure was performed: 5min at 20 ℃; the temperature is 65 ℃, the time is 5min,
3) and (3) purification: purifying the DNA in the previous step by using magnetic beads, purifying the virus nucleic acid in the same way as in the step 6) in the step (1), and finally recovering 16 mu l of RNase-free water to obtain S-dsDNA-A,
4) adding a bar code and amplifying: obtaining DNA with Barcode by PCR Barcoding kit according to the operation instruction, sorting by using magnetic beads with 0.6 proportion, finally eluting by using 10 mul of 50mM NaCl +10mM Tris.HCl PH8.0, quantifying 1 mul by using qubit, running gel by using Agilent 2100, preliminarily judging the size of a target fragment, mainly used for calculating the concentration after mixing samples, diluting into 200pg/ul purified sample S-dsDNA-A-BC,
5) pool mixing and DNA library: calculating the amount of the sample required by mixing, ensuring that the number of moles of the mixed sample is equal and the total amount is between 10 and 50fmol, mixing the sample into a new 1.5ml centrifuge tube according to the calculation result of the sample, if the volume after mixing is more than 11 mu l, purifying and concentrating by using 2X purified magnetic beads, eluting to 11 mu l, taking 1 mu l for quantification, uniformly mixing 10 mu l S-dsDNA-A-BC and 1 mu l RAP for quick sequencing knot connection at room temperature for 5min, placing on ice for on-machine sequencing, and finally completing the construction of a DNA library to 11 mu l S-dsDNA-A-BC-R,
(3) library on-machine and nanopore real-time sequencing
1) Preparing a machine library:
preparing before loading, mixing uniformly to ensure no bubble,
2) preparation of Nanopore chips: firstly connecting a sequencer and a computer, starting sequencing software, determining whether a sequencing chip is qualified, and if the sequencing chip is qualified, preparing a chip cleaning reagent and cleaning the chip for later use, wherein the chip is cleaned according to a specification, and the whole operation avoids introducing bubbles into the chip
3) Loading and real-time sequencing: mainly comprises the steps of completely adding 75 mu l of the on-board library in the first step into a sample hole of the chip prepared in the second step, connecting the chip to a sequencer, setting a file name and a sequencing program, selecting a file which converts an electric sequencing signal fast5 file into a nucleic acid sequence information fast q file in real time, realizing real-time sequencing, and observing the sample sequencing information and the using condition of the sequencing hole at any time,
(4) bioinformatics analysis
Establishing a local database containing 300 Reseq standard viruses, using Guppy software to remove barcode grouping from nanopore sequencing data, using BlastN to compare with the local database, introducing the result of comparison 5 into MEGAN software, accurately analyzing the virus composition in a blood sample, and using a standard nucleic acid internal reference as the sensitivity and quality control index of an evaluation result.
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