CN111662959A

CN111662959A - High-throughput rapid detection method for fungi

Info

Publication number: CN111662959A
Application number: CN202010708234.7A
Authority: CN
Inventors: 刘涛; 徐星晔; 金奇
Original assignee: Institute of Pathogen Biology of CAMS
Current assignee: Institute of Pathogen Biology of CAMS
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2020-09-15

Abstract

The invention relates to a fungus high-flux rapid detection method, which comprises the following steps: step 1, obtaining and processing a sample; step 2, extracting fungal nucleic acid; step 3, constructing and verifying a high-throughput sequencing library; 4, high-throughput sequencing; step 5, bioinformatics analysis; by using the method of the invention, more than 95% of host nucleic acid can be removed, and the interference of the host nucleic acid is effectively eliminated; the fungus reads obtained in the detection is increased from about 0.1 percent to 1 to 3 percent and is increased by more than 10 times compared with the fungus reads obtained by directly extracting nucleic acid by the conventional method. The optimization of the database building method and the localization of the fungus detection database are combined, so that the fungus in the clinical sample can be detected without isolated culture.

Description

High-throughput rapid detection method for fungi

The technical field is as follows:

the invention relates to the technical field of biology, and relates to a high-throughput rapid detection method for trace fungi in a sample.

Background art:

fungi are various in variety, and widely exist in the environment, and are a type of microorganism which is closely related to human beings. Fungi are also important infectious disease pathogens of human beings, and with the aging population, the large application of broad-spectrum antibiotics, antitumor drugs and immunosuppressive agents, the wide implementation of radiotherapy and organ transplantation, the general development of catheters and cannulas and the rapid increase of immunodeficiency patients, particularly AIDS patients, fungal infection, particularly deep fungal infection, is greatly increased. Deep fungal infection is now one of the leading causes of death in major diseases such as AIDS and tumors. In addition, the emergence of some highly pathogenic fungi and multi-drug resistant mutant fungal strains in recent years has also raised social and public concerns about the potential for fungi to initiate public health emergencies.

The current main technology for clinical fungal infection is still very weak, and the traditional microscopic examination and isolated culture are still the gold standards for fungal detection and fungal infection diagnosis. These techniques have the defects of low positive rate and long time consumption. In recent years, various PCR or RT-PCR quantitative or qualitative analysis techniques for common fungal infections have been established and applied in clinical detection and diagnosis, but these techniques require sequence-specific primers for detection of fungal nucleic acids. Thus, the current fungal detection techniques are far from meeting the need for early detection, characterization of variation and determination of transmission pathways of new or prevalent infectious fungal infections.

With the rapid development of Next Generation Sequencing (NGS), the sequencing cost is also rapidly reduced, and the sequencing quality and depth are greatly improved. NGS has been applied to the detection of bacteria and viruses, and has found a number of applications in human microbiology research. NGS can detect all viruses and bacteria present in a sample with high efficiency. This method is particularly important for the detection and analysis of some unknown pathogens. But the use of NGS for the detection of fungal infections in clinical specimens and the variety of fungi in clinical specimens remains difficult. The invention mainly aims to establish a rapid and effective fungus detection method by utilizing NGS in trace samples including clinical samples and the like without separation culture.

The inventor knows that the application of the NGS in the clinical sample is difficult, and the reason is mainly in several aspects, firstly, the content of the fungus in the clinical sample is low even if the sample is infected by the fungus, and the nucleic acid which is enough for detection is difficult to obtain by using the conventional extraction method under the limit of the total amount of the clinical sample due to the existence of the cell wall of the fungus. Moreover, because the total amount of some clinical samples is small, sufficient nucleic acid for effectively detecting and analyzing the existence amount and the types of fungi in the clinical samples cannot be obtained. Single cell sequencing is one way to solve the problem of fungus detection in micro sample. Single cell sequencing requires that the cells of the fungus to be detected are first isolated and therefore detection of a particular fungus in a sample is feasible. However, in clinical samples, it is difficult to separate various fungal cells, even all different fungal cells, and then perform single cell sequencing detection, and the requirement of high-throughput rapid detection cannot be met.

On the other hand, both humans and fungi are eukaryotes, and the relatively large number of human cells and tissues in clinical samples interferes with subsequent high throughput methods of specific methods, such as 18S and ITS sequencing, as well as data analysis. In addition, since the host cell tissue is much higher than that of the fungal cells in clinical samples, after nucleic acid extraction, more than 99% of the data (reads) are often found to be human in data analysis through subsequent high-throughput sequencing. Due to the low abundance of fungal nucleic acid, in order to increase the detection sensitivity, the sequencing depth can only be increased, but the overall data volume is greatly increased, so that the subsequent bioinformatics analysis is long in time consumption, occupies a large amount of resources, and has a limited effect on improving the detection sensitivity.

Therefore, the invention establishes a set of technical scheme by researching the problems, and comprises the steps of filtering and removing large tissue fragments by using a large-aperture filter membrane in the initial treatment stage of a sample; and (3) repeatedly freezing and thawing the small tissue fragments and the cells by liquid nitrogen to fully break the host cells, effectively release the host nucleic acid and change the host nucleic acid into free nucleic acid. Then digested with DNA/RNA enzymes. Through the sample processing stage, more than 95% of human nucleic acid can be removed, the proportion of host data in high-throughput sequencing data is effectively reduced, and the problem of interference of the host nucleic acid on fungus detection in clinical samples is solved. In the nucleic acid extraction stage, cell walls of fungi are removed by a combined enzymolysis method, and then nucleic acid extraction is carried out, so that the fungal nucleic acid can be released and extracted; clinical samples were used for NGS detection of fungi without isolation and culture. Meanwhile, the public database is localized, redundancy and errors are removed, a special fungus detection database is constructed, and the time and efficiency of detection and bioinformatics analysis are improved.

In clinical sample fungus detection by using the NGS technology, a picogram (pg) grade nucleic acid sample can meet the requirements of library construction and subsequent sequencing according to the Nextera technology of Illumina. The above invention can obtain nucleic acid in high flux for fungus detection. Under the condition of extremely low sample volume, random primers and an equal proportion amplification technology can be utilized to carry out independent sequence amplification on a detection sample and then establish a library for detection. In this case, if the RNA of the fungus is detected in the sample, it can be amplified by PCR by reverse transcription into cDNA using SMART-Seq v4Ultra Low Input RNA Kit for Sequencing Kit from Takara/Clontech, or using MALBACPlatinum Single Cell RNA Amplification Kit from Yikang Gene. When the extracted DNA is detected and the amount of the extracted nucleic acid can not meet the library building requirement, a Kit REPLI-g Single Cell WGA Kit of Qiagen is used. Amplification is performed, but the amplification time is optimized to be more suitable for detection of clinical fungal samples.

Disclosure of Invention

The invention aims to provide a method for directly detecting fungi in a clinical sample by using a high-throughput sequencing technology without isolated culture, and the method can be used for clinical diagnosis and non-diagnostic purposes.

The invention relates to a fungus high-flux rapid detection method, which comprises the following steps:

step 1, obtaining and processing a sample;

step 2, extracting fungal nucleic acid;

step 3, constructing and verifying a high-throughput sequencing library;

4, high-throughput sequencing;

and 5, bioinformatics analysis.

Step 1, obtaining and processing a sample, wherein the method comprises the following steps: first obtaining a sample comprising nasopharyngeal secretions, alveolar lavage, sputum, pus, blood, urine, feces, and body fluids or tissues of a person that may be infected with a fungus; secondly, processing the sample, including storing, protecting, culturing and washing the sample by any method suitable for preserving the sample, such as culture solution, water, physiological saline, buffer solution, diluent, marking solution, isotonic solution and fixing solution; separating the sample, repeatedly freezing and thawing the separated sample, and violently shaking to break host cells and tissues in the sample and release host nucleic acid; filtering to remove host tissue residue to obtain filtrate containing fungus and part of host free nucleic acid; the free host nucleic acid filtrate is adsorbed by DNA and RNA magnetic beads to remove host free nucleic acid, and then the nuclease digestion method is used for further removing host cell nucleic acid.

Wherein, the fungus nucleic acid (DNA/RNA) extraction in the step 2 comprises the following steps of removing sample filtrate obtained in the step 1 after host nucleic acid enzymolysis, and digesting fungus cell walls by using mixed enzyme to obtain a sample containing the fungus nucleic acid; then, extracting the fungal nucleic acid by any (DNA/RNA) extraction method for fungal high-throughput sequencing detection;

step 3, constructing and verifying a high-throughput sequencing library, wherein the method comprises the following steps: constructing a high-throughput sequencing library by using an enzyme digestion method;

wherein, step 4, the high-throughput sequencing method comprises the following steps: high throughput sequencing with any one of the sequencing platforms, preferably the Miseq, Hiseq platform;

wherein, the bioinformatics analysis in the step 5 comprises the following steps: information about the fungi in the sample was obtained from high throughput sequencing data.

Preferably, the method of the invention comprises the following steps:

step 1, obtaining and processing a sample; the method comprises the following steps:

first, sampling and storing of samples: including human or animal samples, fresh samples or samples frozen at-80 ℃, for trace samples such as nasopharyngeal test specimens, firstly storing in a collecting tube, shaking for 30s, and carrying out the second step of treatment or freezing at-80 ℃ within 1 hour; various body fluids can be directly frozen at-80 ℃; for other nasopharyngeal secretion extracts, alveolar lavage fluid, sputum and pus, adding a liquefying agent, and performing second-step treatment or freezing and storing at-80 ℃ within 1 hour;

secondly, primary treatment of the sample: filtering with 12-20 μm filter membrane to remove residual human tissue;

thirdly, taking 50-500ul of the virus collection tube sample, adding pure water without RNase, repeatedly freezing and thawing twice at minus 80 ℃ or by liquid nitrogen, and repeatedly and violently shaking and cracking tissues and cells during the period;

and fourthly, removing host and environmental free nucleic acid in the sample: adsorbing the filtered sample by using DNA and RNA magnetic beads to remove host free nucleic acid;

fifthly, further removing the host cell nucleic acid of the sample: removing host cell nucleic acid by enzymatic digestion; wherein the enzyme comprises a deoxyribonuclease; a nuclease; ribonucleases; deoxyribonuclease;

step 2, extracting fungal nucleic acid; the method comprises the following steps:

a first step of removing the cell walls of the fungus in the sample by a combined enzyme treatment, including, but not limited to, a combination of helicase (Snailase), Chitinase (Chitinase), Cellulase (celluloase), crashease (Driselase), beta-Glucosidase (beta-D-glucopyranosase), and resolvase (macrozyme), enabling the subsequent extraction of fungal nucleic acids by conventional methods; in the range of 1% -10% of the final concentration, these enzymes can be combined to carry out enzymolysis on cell walls of various fungi, including yeast-like and filamentous fungi;

step two, extracting fungal nucleic acid in the sample: the extraction process comprises DNA and RNA extraction; the sample after the first step of treatment can be directly extracted by a conventional method to obtain DNA or RNA; if RNA is extracted, reverse transcription is needed to synthesize cDNA; if the amount of DNA is insufficient, amplification is required: amplifying cDNA by using a non-sequence-dependent primer based on Polymerase Chain Reaction (PCR) until the initial amount of 100-200pg required by next high-throughput sequencing sample library construction is met; the DNA can also obtain the initial quantity meeting the high-throughput sequencing library building by using an equal-proportion amplification method; for ITS sequencing, amplification can be performed with ITS-specific primers;

purification of the amplified nucleic acid: if the sample is processed through the PCR amplification in the third step, the obtained amplification product needs to be purified for library construction and verification preparation before subsequent sequencing;

step 3, constructing and verifying a high-throughput sequencing library; comprises the following steps

Wherein, the construction and verification of the high-throughput sequencing library refer to the accurate quantification of the nucleic acid of the purified clinical sample obtained by the steps and the dilution according to the library construction requirement; constructing a sequencing library of 300-700bp by a Nextera enzymolysis fragmentation and labeling step; the quality of the library was verified by electrophoresis on angioent 2100 nucleic acids; the method specifically comprises the following steps:

in the first step, the purified nucleic acid is accurately quantified: quantifying the purified nucleic acid in the step (2) by using a Qubit method, and diluting to the nucleic acid concentration of 100-200 pg/ul;

second, Nextera enzymatic hydrolysis fragments and splices the nucleic acids: taking 1ul of the nucleic acid diluted to 100-200pg/ul in the first step, adding Nextera enzyme to fragment the nucleic acid and adding a joint

Third, purification of the tagged fragmented nucleic acids: purifying and constructing a library by the labeled fragmented nucleic acid;

fourthly, verifying the constructed library: the purified library is verified by Angilent2100 electrophoresis, so that the library is between 300 and 700 bp;

4, high-throughput sequencing; the method comprises the following steps:

the high-throughput sequencing can select second-generation or third-generation sequencing according to the length of detection required; high throughput sequencing comprises two steps: high-throughput sequencing, converting the image sequencing signal into nucleic acid sequence information;

step 5, bioinformatics analysis; the method comprises the following steps:

before analyzing high-throughput sequencing data of fungus detection in a clinical sample, firstly establishing a special database of a fungus detection database; then obtaining, comparing and analyzing fungus-related reads;

the first step is as follows: establishing a fungus detection database: downloading and arranging the information containing all fungal nucleic acids, protein sequence information, structure information and evolution information from a public biological information database; the data is subjected to redundancy removal and error checking; the establishment of the fungus detection database reduces the capacity of the database on the basis of not reducing information, reduces the analysis time from the original 120 hours to within 20 hours, and can quickly finish obtaining a high-throughput analysis result;

the second step is that: and obtaining, comparing and analyzing the relevant reads of the fungi to obtain the information related to the detection of the fungal species.

Most preferably, the detection method of the present invention comprises the following steps:

step 1, obtaining and processing a sample:

1) the samples comprise various clinical samples such as respiratory tract secretion, digestive tract secretion, blood, urine, excrement and the like.

2) Primary treatment of a sample: for a sufficient amount of liquid samples, directly centrifuging at 15000rpm and 4 ℃ for 10min, and discarding the supernatant to obtain samples; for insufficient liquid samples, 500ul of pure water without RNase can be added firstly, and the mixture is shaken and mixed evenly; centrifuging at 15000rpm and 4 deg.C for 10min, and removing supernatant to obtain sample; adding 500ul of pure water without RNA enzyme into solid samples such as feces, and mixing uniformly by shaking; centrifuging at 15000rpm and 4 deg.C for 10min, and removing supernatant to obtain sample;

3) adding 500ul of pure water without RNase into the precipitate, shaking, mixing, filtering with 12-20 μm filter membrane, and removing residual human tissue.

4) Lysis of residual cells and debris in the sample: adding a proper amount of pure water without RNase into the sample, freezing and thawing the sample twice at the temperature of liquid nitrogen or minus 80 ℃, repeatedly shaking the sample during the freezing and thawing, and breaking cells and fragments remained in the filtrate to release human nucleic acid in the filtrate; centrifuging at 15000rpm and 4 deg.C for 10min, and removing supernatant. Removal of host-free nucleic acids in the sample: adding 50ul of pure water without RNA enzyme into the precipitate, shaking and mixing uniformly, adding 100ul of each of DNA clean XP and RNA clean XP magnetic beads, freezing for 5min, and removing the magnetic beads by using a magnetic frame. Then, the mixture was centrifuged at 15000rpm at 4 ℃ for 5min, the supernatant was discarded, and 40. mu.l of RNase-free purified water was added to the precipitate to mix well.

5) The sample host nucleic acids were further removed by enzymatic digestion:

then digesting for 30min at 37 ℃, wherein the Turbo Dnase is deoxyribonuclease named Turbo; nucleic is a Nuclease; rnase a is ribonuclease a; the Turbo DNase Buffer is a Buffer solution of Turbo DNase;

step 2, extracting fungal nucleic acid:

1) enzymatic removal of fungal cell walls in samples: adding 50 μ l mixed enzyme (containing 1-5% chitinase, 1-5% helicase and 1-5% lywallzyme) into the digested sample to 100 μ l, and continuously digesting at 37 deg.C for 30 min;

2) extracting sample nucleic acid DNA or RNA: RNA or DNA was extracted using the RNeasy Plant Mini Kit or DNeasy Plant Mini Kit from QIAGEN according to the instructions. If RNA is detected, further reverse transcription into cDNA is required; if the quantity of the nucleic acid can not meet the sequencing requirement, the nucleic acid can be amplified in equal proportion, purified and then subjected to library construction for sequencing. Purification was carried out using QIAGEN-PCR recovery kit, as described.

Step 3, constructing and verifying high-throughput sequencing library

The extracted DNA is quantified by using the Qubit fluorescence, diluted into 100-one 200pg/ul purified sample before machine sequencing, and subjected to Agilent2100 gel running to preliminarily determine the concentration and quality of a target fragment.

Such as: taking 1-3ul of about 200pg of the extracted DNA sample, performing nucleic acid fragmentation and joint building according to the instruction in accordance with a kit of Illumina company, Nextera XTDNA sample kit and Nextera XT index kit, and screening a 300-700bp fragment and purifying by using AMPure XP magnetic beads.

The purified library was verified by angioent 2100 electrophoresis. The qualified library fragment sizes should be centered between 300-700 bp.

Step 4, high-throughput sequencing:

deep sequencing is carried out by a MiniSeq or Hiseq sequencer of Illumina, and the sequencing length can be selected from reading length of 150bp to 300 bp.

And 5, biological information analysis:

after the obtained sequencing reads are subjected to quality control and host sequence removal, the obtained sequencing reads are compared with a constructed special fungal detection database; the BLAST results were converted to species composition information using MEGAN for further analysis.

Wherein, the RNA is extracted and detected in the step 2. Fungal RNA needs to be reverse transcribed into cDNA and PCR amplification is required if the amount of nucleic acid is not sufficient for subsequent high throughput sequencing and pooling. Reverse transcription and PCR Amplification of cDNA can be performed using SMART-Seq v4Ultra Low Input RNA Kit for Sequencing Kit from Takara/Clontech, or using MALBAC platform Single Cell RNA Amplification Kit from Yikang Gene.

The method comprises the following steps:

firstly, synthesizing a chain:

total RNA 5. mu.L

RT Buffer 13.3μL

Total volume 18.3. mu.L

Mixing and centrifuging the mixture for a short time,

(ii) the RNA and RT Buffer mixture was incubated at 72 ℃ for 5 minutes, then on ice for at least 1 minute,

adding 1.7 mu L of RT Enzyme Mix into the mixture, and gently mixing the mixture evenly;

and thirdly, adding 30 mu L of PCR Mix into the reverse transcription cDNA to carry out PCR amplification. The initial quantity requirement of 100-200pg for subsequent library construction can be met by 3-8 cycles of PCR amplification according to the initial quantity. The following are the conditions for PCR amplification.

Aiming at the characteristics of small clinical sample amount and low fungal content in clinical samples, the invention utilizes a physical method to firstly treat the samples, then removes host free nucleic acid by an enzyme digestion method, then removes fungal cell walls by combined enzyme treatment, and finally extracts the fungal nucleic acid according to a conventional method.

The key problem to be solved by the invention is to solve the problem that the clinical sample is interfered by host nucleic acid in the detection due to low content of fungus; and the fungus is difficult to break effectively due to the presence of the cell wall, and the conventional method cannot obtain enough fungus nucleic acid for high-throughput detection. The invention integrates physical and biological enzymolysis methods to remove host nucleic acid and free nucleic acid; the method is used for high-throughput rapid detection of fungi in clinical samples by digesting and cracking the cell walls of the fungi by using combined enzyme and further extracting nucleic acid in the samples. By using the method, the number of the fungus reads obtained in the detection can be increased from less than 0.1% to 1% -5% compared with the fungus reads obtained by directly extracting nucleic acid by the conventional method. The optimization of the database building method and the localization of the fungus detection database are combined, so that the fungus in the clinical sample can be detected.

Compared with the existing detection method, the invention has the advantages that:

1) it is suitable for, but not limited to, detection and identification of human and animal respiratory fungi; 2) through the filtration of a large-aperture filter membrane, repeated freeze thawing and magnetic bead adsorption, after host nucleic acid is removed, the percentage of reads of the host in a detection sample is greatly reduced, the complexity of subsequent detection data analysis is reduced, and the analysis time is shortened; 3) the problems of difficult fungal cell wall breakage, low extraction rate and direct detection in clinical samples, particularly trace samples are solved; 4) the proportion of the fungus reads in the whole sequencing reads is improved; 5) the requirement of the whole detection on the initial sample amount is very low, the sample does not need to be cultured, and the detection time is greatly shortened. In view of these advantages of the present invention, it can be used for, but not limited to, fungal infection analysis of clinical specimens, rapid identification of the etiology of newly emergent infectious diseases.

Drawings

FIG. 1 shows two methods for detecting RNA in a sample. Plots of the number of reads for the fungi from Spa1, Spa2 as a percentage of the total number of reads (after removal of the human genome) were used to detect sample RNA using the present invention. The proportion of fungal sequencing reads to the total reads is improved by more than 10 times.

FIG. 2 two methods detect sample DNA. The species of fungi were detected in the samples from Spb1, Spb 2. The fungus species and reads detected by the method of the invention are obviously improved compared with the prior method. Spb1 can detect 9 fungi of 5 families by sequencing; a total of 19 fungi of the 7 genera can be detected by the method Spb2 of the present invention.

Detailed Description

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.

Examples 1,

We illustrate embodiments of the present invention in order by taking the virus detection of 4 respiratory tract samples of human lung disease taken in 6 months of 2018 as an example.

Step 1, obtaining and processing a sample:

1) first, pharyngeal test-tubes were rapidly stored in virus collection tubes (about 3ml) from inpatients diagnosed as lung cancer patients (Spa) and pulmonary embolism patients (Spb), respectively, and shaken for 30 s.

1ml of each of the two Sp1 and Sp2 samples was aspirated. Spa and Spb were each split into two 500ul aliquots, labeled Spa1, Spa2, and Spb1 and Spb2, respectively. Spa1 and Spa2 extract RNA for detection. Spb1, Spb2 detect DNA.

For ease of comparison, two samples were treated in two ways. Wherein, the samples Spa1 and Spb1 are processed according to the conventional method, and the nucleic acid is directly extracted without the wall breaking process and the host nucleic acid removing process of the method. For detecting; samples Spa2 and Spb2 were processed at various steps in the method of the invention.

2) Spa1, Spa1, Spa2 and Spa2, four samples 15000rpm, at 4 ℃, centrifuged for 10min, and the supernatant discarded. Spa1, Spb1 were transferred directly to 2) in step 2 for nucleic acid extraction according to the conventional method. Spa2 and Spa2 then proceed with the following processing in accordance with the method of the present invention.

3) Adding 500ul of RNase-free pure water into the precipitates of the samples Spa2 and Spb2, centrifuging at the temperature of 4 ℃ at the rpm of 15000rpm for 10min, shaking and mixing uniformly, and filtering by a 12-20um filter membrane to remove human tissue fragments in the samples.

4) Freezing and thawing the filtrate at least twice in liquid nitrogen or-80 deg.C, shaking repeatedly, and breaking the residual cells and debris to release human nucleic acid. Centrifuging at 15000rpm and 4 deg.C for 10min, and removing supernatant.

5) Adding 50ul of pure water without RNA enzyme into the precipitate, shaking and mixing uniformly, adding 100ul of each of DNA clean XP and RNA clean XP magnetic beads, freezing for 5min, and removing the magnetic beads by using a magnetic frame. Then, the mixture was centrifuged at 15000rpm at 4 ℃ for 5min, the supernatant was discarded, and 40. mu.l of RNase-free purified water was added to the precipitate to mix well.

6) Further removal of host nucleic acids in the sample:

then digesting for 30min at 37 ℃, wherein the Turbo Dnase is deoxyribonuclease named Turbo; nucleic is a Nuclease; RNase A is ribonuclease A; turbo DNase Buffer is Buffer solution of Turbo DNase,

step 2, extracting fungal nucleic acid:

1) enzymatic removal of fungal cell walls:

mu.l of enzyme mixture (containing 1.5% chitinase, 1.5% helicase and 1.5% lywallzyme) was added to the sample in a total of 100. mu.l, and digestion was continued at 37 ℃ for 30 min.

2) Extracting nucleic acid DNA or RNA:

RNA (Spa1, Spa2) or DNA (Spa1, Spa2) was extracted with the RNeasy Plant Mini Kit or DNeasy Plant Mini Kit from QIAGEN according to the instructions, respectively, and the samples were stored at-80 ℃.

3) Reverse transcription and PCR amplification of cDNA were performed using SMART-Seq v4Ultra Low Input RNA Kit for sequencing Kit from Takara/Clontech or using MALBAC Platinum Single Cell RNAAmplification Kit from Yikang Gene.

The method comprises the following steps:

firstly, synthesizing a chain:

total RNA 5. mu.L

RT Buffer 13.3μL

Total volume 18.3. mu.L

Mixing and centrifuging the mixture for a short time,

and thirdly, adding 30 mu L of PCR Mix into the reverse transcription cDNA to carry out PCR amplification. The initial quantity requirement of 100-200pg for subsequent library construction can be met by 3-8 cycles of PCR amplification according to the initial quantity.

The following are the conditions for PCR amplification.

4) Sample nucleic acid purification: nucleic acids were purified as described for subsequent preparation for library construction and validation prior to sequencing using the QIAGEN-PCR recovery kit.

Step 3, constructing and verifying a high-throughput sequencing library

The RT-PCR products (Spa1, Spa2) and the extracted sample DNA (Spb1, Spb2) were quantified by Qubit fluorescence, run using Agilent2100, preliminarily determined the concentration and amount of the nucleic acid fragments, and diluted to 100-200pg/ul for library construction.

1-3ul of about 100-200pg of the above samples, Illumina kit, NexteraXT DNA sample kit, and NexteraXT index kit were taken to fragment and ligate nucleic acids: the operation is as described in the specification.

The purified library was verified by Angilent2100 electrophoresis to yield a library of between 300 and 700 bp.

Step 4, high throughput sequencing

The library was deep sequenced using a MiSeq sequencer from Illumina. High-throughput sequencing, converting image sequencing signals into nucleic acid sequence information.

Step 5 bioinformatics analysis

And after the obtained sequencing reads are subjected to quality control and host sequence removal, the obtained sequencing reads are compared with the obtained host sequence to construct a special fungal detection database. The BLAST results were converted to species composition information using MEGAN for further analysis.

Claims

1. A fungus high-throughput rapid detection method is characterized by comprising the following steps:

step 1, obtaining and processing a sample;

step 2, extracting fungal nucleic acid;

step 3, constructing and verifying a high-throughput sequencing library;

4, high-throughput sequencing;

and step 5, the analysis of bioinformatics,

step 1, obtaining and processing a sample, wherein the method comprises the following steps: first obtaining a sample comprising nasopharyngeal secretions, alveolar lavage, sputum, pus, blood, urine, feces, and body fluids or tissues of a person that may be infected with a fungus; secondly, processing the sample, including storing, protecting, culturing and washing the sample by any method suitable for preserving the sample, such as culture solution, water, physiological saline, buffer solution, diluent, marking solution, isotonic solution and fixing solution; separating the sample, repeatedly freezing and thawing the separated sample, and violently shaking to break host cells and tissues in the sample and release host nucleic acid; filtering to remove host tissue residue to obtain filtrate containing fungus and part of host free nucleic acid; adsorbing the free host nucleic acid filtrate with magnetic DNA and RNA beads to remove host free nucleic acid, further removing host cell nucleic acid by nuclease digestion,

2. The method of claim 1, wherein,

secondly, primary treatment of the sample: large-aperture screen filtration of the sample: filtering with 12-20 μm filter membrane to remove residual human tissue;

step two, extracting fungal nucleic acid in the sample: the extraction process comprises DNA and RNA extraction; the sample after the first step of treatment can be directly extracted by a conventional method to obtain DNA or RNA; if RNA is extracted, reverse transcription is needed to synthesize cDNA; if the amount of DNA is insufficient, amplification is required: amplifying the cDNA by using a non-sequence-dependent primer based on Polymerase Chain Reaction (PCR) until the sample loading amount required by next high-throughput sequencing sample library construction is 1 pg; the DNA can also obtain the sample loading quantity meeting the requirement of high-throughput sequencing library building by using an equal-proportion amplification method; for ITS sequencing, amplification can be performed with ITS-specific primers;

in the first step, the purified nucleic acid is accurately quantified: quantifying the purified nucleic acid in the step (2) by using a Qubit method, and diluting to a nucleic acid concentration of 200 pg/ul;

second, Nextera enzymatic hydrolysis fragments and splices the nucleic acids: taking 1ul of the nucleic acid diluted to 200pg/ul in the first step, adding Nextera enzyme to fragment the nucleic acid and adding a connector

4, high-throughput sequencing; the method comprises the following steps:

step 5, bioinformatics analysis; the method comprises the following steps:

the first step is as follows: establishing a fungus detection database: downloading and arranging the information containing all fungal nucleic acids, protein sequence information, structure information and evolution information from a public biological information database; the data is subjected to redundancy removal and error checking; the establishment of the fungus detection database reduces the capacity of the database on the basis of not reducing information, reduces the analysis time from the original 72 hours to 10 hours, and can quickly finish obtaining a high-throughput analysis result;

3. The method of claim 2, wherein,

step 1, obtaining and processing a sample:

1) the samples comprise various clinical samples such as respiratory tract secretion, digestive tract secretion, blood, urine, excrement and the like,

3) adding 500ul of pure water without RNase into the precipitate, shaking, mixing, filtering with 12-20 μm filter membrane, removing residual human tissue,

4) lysis of residual cells and debris in the sample: adding a proper amount of pure water without RNase into the sample, freezing and thawing the sample twice at the temperature of liquid nitrogen or minus 80 ℃, repeatedly shaking the sample during the freezing and thawing, and breaking cells and fragments remained in the filtrate to release human nucleic acid in the filtrate; 15000rpm, 4 ℃, centrifugation for 10min, supernatant removal, removal of host free nucleic acids in the sample: adding 100ul of each of DNA clean XP and RNAclean XP magnetic beads, ice-cooling for 5min, removing the magnetic beads by using a magnetic rack, then centrifuging at 15000rpm and 4 ℃ for 5min, removing supernatant, adding 40ul of RNase-free pure water into the precipitate, mixing uniformly,

5) the sample host nucleic acids were further removed by enzymatic digestion:

then digesting for 30min at 37 ℃, wherein the Turbo Dnase is deoxyribonuclease named Turbo; nucleic is a Nuclease; rnase a is ribonuclease a; turbo DNase Buffer is Turbo DNase Buffer.

4. The method of claim 2, wherein,

step 2, extracting fungal nucleic acid:

2) extracting sample nucleic acid DNA or RNA: respectively extracting RNA or DNA by using an RNeasy Plant Mini Kit or DNeasy Plant Mini Kit of QIAGEN company according to the instruction, and if detecting RNA, further performing reverse transcription to obtain cDNA; if the amount of nucleic acid can not satisfy the sequencing, the nucleic acid can be amplified in equal proportion, purified and then subjected to library construction sequencing, and the purification is carried out by using a QIAGEN-PCR recovery kit according to the instructions.

5. The method of claim 4, wherein in step 2, the fungal nucleic acid is extracted, the RNA of the fungus is reverse-transcribed into cDNA, if the amount of nucleic acid is not enough for the subsequent high-throughput Sequencing library construction, PCR Amplification is performed, the reverse transcription and PCR Amplification of cDNA can be performed by using SMART-Seq v4Ultra Low Input RNA Kit for Sequencing Kit from Takara/Clontech, or by using MALBAC Platinum Single Cell RNA Amplification Kit from Yikang Gene,

the method comprises the following steps:

firstly, synthesizing a chain:

total RNA 5. mu.L

RT Buffer 13.3μL

Total volume 18.3. mu.L

Mixing and centrifuging the mixture for a short time,

thirdly, adding 30 mu L of PCR Mix into the reverse transcription cDNA for PCR amplification, and satisfying the subsequent initial quantity requirement of 100-200pg for constructing the library according to 3-8 cycles of initial quantity PCR amplification, as follows is the condition of PCR amplification,

6. the method of claim 2, wherein,

step 3, constructing and verifying high-throughput sequencing library

Quantifying the extracted DNA by using the Qubit fluorescence, diluting the DNA into 100-one 200pg/ul purified samples before machine sequencing, and preliminarily judging the concentration and quality of a target fragment by using Agilent2100 gel running;

such as: taking 1-3ul about 200pg of the extracted DNA sample, performing nucleic acid fragmentation and joint building according to the instruction in accordance with the kit of Illumina company, Nextera XT DNA sample kit and Nextera XT index kit, screening a 300-700bp fragment by using AMPure XP magnetic beads and purifying,

the purified library was verified by Angilent2100 electrophoresis and the qualified library fragment sizes should be centered between 300 and 700 bp.

7. The method of claim 2, wherein,

step 4, high-throughput sequencing:

8. The method of claim 2, wherein,

and 5, biological information analysis: