WO2020248110A1 - Method and kit for detecting echinococcus multilocularis infection - Google Patents

Abstract

Disclosed is the detection of Echinococcus multilocularis infection. Particularly, the detection comprises detecting free DNA from Echinococcus multilocularis in the blood of a subject to determine the infection status thereof. Also disclosed are a kit and a system for the detection method.

Description

Method and kit for detecting alveolar hydatid infection Technical field

The present invention relates to the detection of alveolar hydatid infections, in particular to determining the infection status of the subject by detecting free DNA from alveolar hydatid in blood. The present invention also relates to a kit used in this detection method and its use for detecting alveolar echinococcosis.

Background technique

Echinococcosis (echinococcosis), also known as echinococcosis, is caused by the infection of intermediate hosts (cattle, sheep, pigs, humans, etc.) with the larvae of Echinococcus parasitic in the small intestine of the final host (dog, wolf, fox, etc.) Of zoonotic diseases. Hydatid disease has a wide distribution worldwide, seriously threatening public health safety and animal husbandry production, and it has been listed as one of the major infectious diseases for free treatment by China. Hydatid disease in China is mainly distributed in western pastoral and semi-agricultural pastoral areas such as Qinghai, Tibet and Xinjiang. There are 4 species of Echinococcus that can infect humans. In China, Echinococcus granulosus is mainly caused by Cystic Echinococcosis (CE) and Echinococcosis multilocularis, which cause Alveolar echinococcosis. Echinococcosis, AE). China is dominated by cystic echinococcosis (CE), but its fatality rate is not high, ranging from 2% to 4%. The deaths are mainly caused by complications; while cystic echinococcosis (AE) is similar to malignant tumors. Invasive growth and distant metastasis, so it is also called "carcinoma", a highly lethal disease. Without treatment, the 10-year mortality rate can reach 90%.

The onset of alveolar echinococcosis is insidious, and the incubation period is asymptomatic. It usually lasts for 5-10 years. Once the patient is found, it is mostly at an advanced stage when re-seeing. Almost all of the pathogenic lesions of alveolar hydatid disease are located in the liver, and may spread locally and transfer to the brain and other important organs through the blood. Liver infections are usually treated by surgical resection, but many patients often have their lesions invaded important blood vessels or other organs when they are discovered, making the operation difficult and missing the best opportunity for surgery. The diagnosis of alveolar hydatid disease currently mainly relies on imaging examinations supplemented by serological tests. However, some patients lack typical imaging features and it is difficult to make a clear diagnosis before surgery. Serological tests have poor specificity for the diagnosis of the disease. At present, there is no mature technology to detect early alveolar echinococcosis.

In 2018, French scholar Alice Baraquin et al. found for the first time that AE free nucleic acid (AE-cfDNA) can be detected in circulating free DNA (cfDNA) in the peripheral blood of patients with alveolar hydatid disease through PCR and digital PCR technology, confirming the existence of AE and Like solid tumors, AE-cfDNA will be produced (see Baraquin et al., Circulating cell-free DNA in patients with alveolar echinococcosis, Mol Biochem Parasitol. 2018 Jun; 222:14-20). However, the conclusion of the study found that the positive rate of AE-cfDNA in AE patients was too low and the amount of detection had little correlation with clinical symptoms. It was considered that it could not be used as a biomarker for AE detection and diagnosis. We speculate that this may be related to the limitations of the technology itself. Since PCR or digital PCR is a targeted detection of a specific gene fragment, and the release of broken gene fragments from dead hydatid worms to the circulatory system is random, it may appear that "peripheral blood does contain free nucleic acid of hydatid. , But it does not contain the nucleic acid targeted by PCR". The limitations of this technology have caused the detection of free nucleic acid in peripheral blood of AE patients to be too low.

In recent years, with the emergence and wide application of the second-generation sequencing technology of metagenomics, the detection of very low levels of AE-cfDNA in peripheral blood has become possible. This technology can perform unbiased sequencing of all nucleic acids, but in practical applications there are also the problems of "extremely high detection sensitivity" and "excellent detection specificity".

Therefore, there is an urgent need in the art for a method based on metagenomics second-generation sequencing technology to detect alveolar hydatid free nucleic acid in peripheral blood of patients with suspected AE.

Summary of the invention

The present invention provides a method for specifically detecting free DNA derived from alveolar hydatid in the blood of a host, thereby determining whether the host has alveolar hydatid infection. Even before the clinical symptoms are found, the free DNA derived from the alveolar hydatid can be detected in the blood of the host infected with alveolar hydatid by the method of the present invention. Therefore, vesicular echinococcosis can be detected early in the infection, and therapeutic intervention can be implemented for vesicular echinococcosis, and the effect of the treatment of vesicular echinococcosis can be monitored.

Specifically, the present invention provides methods for enrichment and extraction, library construction, high-throughput sequencing and bioinformatics analysis of free nucleic acids in the peripheral blood of subjects, and realizes the detection and detection of AE-cfDNA in peripheral blood of patients. Determination of abundance. AE-cfDNA can be used as a molecular marker for clinical AE diagnosis. The method of the invention has high detection sensitivity, good specificity, accurate and reliable detection results, and can correlate the abundance of AE-cfDNA in the patient's peripheral blood with the patient's condition.

In the first aspect, the present invention provides a method for detecting AE-cfDNA in a sample of a subject, which comprises:

a) Extract and enrich free DNA in the subject's sample;

b) Library construction and sequencing of the free DNA obtained in step a); and

c) Perform bioinformatics analysis on the sequencing data of step b) to obtain AE-cfDNA abundance.

In a specific embodiment, the bioinformatics analysis in step c) includes:

1) Remove the linker sequence, repetitive sequence, and sequence less than 60bp, use the remaining sequence as data for AE sequence analysis, and record the number as a;

2) Align the sequence obtained in step 1) with the host reference genome sequence to remove the sequence matching the host genome;

3) Compare the sequence obtained in step 2) with the AE reference genome to obtain the AE sequence;

4) Align the AE sequence obtained in step 3) with the sequence in the NCBI nucleic acid database again, remove the sequence that can match with other species other than AE, and obtain the AE-specific sequence, the number of which is recorded as b;

5) Calculate the AE-cfDNA abundance according to the following formula, which means the content of AE-cfDNA in every tens of millions of free nucleic acid sequences in peripheral blood;

Abundance _(AE-cfDNA) = b/a×10 ⁸

6) Judging the test results through the results obtained in step 5):

i) Abundance _(AE-cfDNA) = 0, indicating that the sample of the subject does not contain AE-cfDNA, which is negative for AE-cfDNA;

ii) 0 <Abundance _(AE-cfDNA) ≤ 5, indicating that the subject sample may contain AE-cfDNA, which is weakly positive for AE-cfDNA;

iii) Abundance _(AE-cfDNA) >5, indicating that the subject sample contains AE-cfDNA and is AE-cfDNA positive.

In a second aspect, the present invention provides a method for diagnosing whether a subject is suffering from alveolar hydatid infection, which includes:

a) Provide blood samples from subjects;

b) Use the method described in the first aspect of the present invention to detect the presence of AE-cfDNA in the sample. If AE-cfDNA is present, it indicates that the subject is suffering from alveolar hydatid infection; and

c) If the subject is suffering from alveolar hydatid infection, treatment for alveolar hydatid infection is performed on the subject.

In a third aspect, the present invention provides a kit for diagnosing whether a subject is suffering from alveolar hydatid infection, which contains a kit for detecting alveolar hydatid free nucleic acid (AE-cfDNA ) Reagents and at least one reagent selected from the following: reagents for extracting and enriching free DNA in subject samples, reagents for DNA library construction, reagents for high-throughput sequencing, and instructions for use .

In the fourth aspect, the present invention provides a reagent for detecting AE-cfDNA in a sample of a subject in the preparation of a kit for diagnosing whether the subject is suffering from echinococcosis infection. Use, wherein the diagnosis includes the following steps:

a) Extract and enrich free DNA in the subject's sample;

b) Library construction and sequencing of the free DNA obtained in step a); and

c) Perform bioinformatics analysis on the sequencing data of step b) through the following steps:

1) Remove the linker sequence, repetitive sequence, and sequence smaller than 60bp, use the remaining sequence as the data for AE sequence analysis, and record the number as a;

2) Align the sequence obtained in step 1) with the host reference genome sequence to remove the sequence matching the host genome;

3) Compare the sequence obtained in step 2) with the AE reference genome to obtain the AE sequence;

4) Align the AE sequence obtained in step 3) with the sequence in the NCBI nucleic acid database again, remove the sequence that can match with other species other than AE, and obtain the AE-specific sequence, the number of which is recorded as b;

5) Calculate the AE-cfDNA abundance according to the following formula, which means the content of AE-cfDNA in every tens of millions of free nucleic acid sequences in peripheral blood;

Abundance _(AE-cfDNA) = b/a×10 ⁸ ,

6) Judging the test results through the results obtained in step 5):

i) Abundance _(AE-cfDNA) = 0, indicating that the sample of the subject does not contain AE-cfDNA, which is negative for AE-cfDNA;

ii) 0 <Abundance _(AE-cfDNA) ≤ 5, indicating that the subject sample may contain AE-cfDNA, which is weakly positive for AE-cfDNA;

iii) Abundance _(AE-cfDNA) >5, indicating that the subject sample contains AE-cfDNA and is AE-cfDNA positive.

In a fifth aspect, the present invention provides a system for determining whether a subject has a vesicular hydatid infection, which includes:

A) A peripheral blood sample obtained from the subject;

B) Tools for extracting and enriching free DNA in subject samples;

C) Tools for library construction and sequencing of free DNA; and

D) A computer-readable medium with multiple instructions for performing the following steps:

1) Remove the linker sequence, repetitive sequence, and sequence less than 60bp, use the remaining sequence as data for AE sequence analysis, and record the number as a;

2) Align the sequence obtained in step 1) with the host reference genome sequence to remove the sequence matching the host genome;

3) Compare the sequence obtained in step 2) with the AE reference genome to obtain the AE sequence;

4) Align the AE sequence obtained in step 3) with the sequence in the NCBI nucleic acid database again, remove the sequence that can match with other species other than AE, and obtain the AE-specific sequence, the number of which is recorded as b;

5) Calculate the AE-cfDNA abundance according to the following formula, which means the content of AE-cfDNA in every tens of millions of free nucleic acid sequences in peripheral blood;

Abundance _(AE-cfDNA) = b/a×10 ⁸

6) Judging the test results through the results obtained in step 5):

i) Abundance _(AE-cfDNA) = 0, indicating that the sample of the subject does not contain AE-cfDNA, which is negative for AE-cfDNA;

ii) 0 <Abundance _(AE-cfDNA) ≤ 5, indicating that the subject sample may contain AE-cfDNA, which is weakly positive for AE-cfDNA;

iii) Abundance _(AE-cfDNA) >5, indicating that the subject sample contains AE-cfDNA and is AE-cfDNA positive.

The detection object of the method of the present invention is cfDNA. After the alveolar hydatid infects the human host, its own degraded DNA fragments will enter the human peripheral blood. Therefore, the deep sequencing of the peripheral blood cfDNA is performed and combined with efficient and sensitive biological Informatics analysis methods can achieve the purpose of pathogen detection. Compared with the known method for detecting alveolar echinococcosis in the prior art, the technical solution of the present invention does not need to extract DNA from alveolar echinoid bodies, and only needs to extract free DNA in the blood sample of the subject to detect A trace amount of free DNA derived from alveolar hydatid simplifies the work of collecting samples during detection to only a small amount of peripheral blood samples. In addition, the method of the present invention has high detection sensitivity, good specificity, accurate and reliable detection results, realizes the detection of AE-cfDNA in peripheral blood of patients and the determination of its abundance, and can determine the abundance of AE-cfDNA in peripheral blood of patients. Related to the patient's condition.

Detailed ways

Unless otherwise specified, the terms used herein have the usual meanings understood by those skilled in the art. The explanations of some terms used in this article are listed below, unless otherwise specified, the explanations of these terms are subject to the definitions in this article.

The term "free DNA", also known as Circulating Cell-free DNA (cfDNA) is a kind of cell-free extracellular DNA, which is widely present in human body fluids and is usually fragmented from its natural state. DNA. In a preferred embodiment of the invention, circulating cell-free DNA from a plasma sample of a subject suspected of being infected with Echinococcus is used. The term AE-cfDNA refers to circulating cell-free DNA from Echinococcus multilocularis (which causes Alveolar Echinococcosis (AE)) isolated from a plasma sample of a subject.

cfDNA can be isolated and extracted from body fluids using a variety of techniques known in the art. In some cases, commercially available kits such as the Qiagen Circulating Nucleic Acid kit can be used to isolate, extract, and prepare cell-free nucleic acids. In other examples, the Qiagen Qubit ^™ dsDNA HS assay kit protocol, Agilent ^™ DNA 1000 kit, or TruSeq ^™ Sequencing Library Preparation; Low-Throughput (LT) protocol can be used to quantify nucleic acids. In a specific embodiment of the present invention, the QIAamp ccfDNA extraction kit is used to enrich and extract free DNA.

In one embodiment, the sample is a blood sample, in particular a plasma sample. The sample may be from a host infected with Echinococcus, such as cattle, sheep, pigs, humans, etc., preferably humans.

In the method of the present invention, after the free DNA in the sample from the subject is extracted and enriched, a sequencing library is constructed and sequenced. The library construction is performed by methods known in the art, and the process includes: repairing the ends of the extracted free DNA, adding linkers, and performing PCR library enrichment to obtain a nucleic acid library. In addition, the quality and concentration of the library can be detected and identified by Qubit and Agilent2100.

According to some embodiments of the present invention, sequencing is performed using high-throughput sequencing technology. Those skilled in the art can understand that sequencing can be performed by any high-throughput sequencing technology known in the art. According to specific examples of the present invention, sequencing platforms such as the Iontorrent sequencing platform and the Illumina sequencing platform are preferably used for sequencing.

Using the method for bioinformatics analysis of sequencing data according to the present invention can determine whether the sample contains AE-cfDNA and determine its abundance, and can correlate the abundance of AE-cfDNA in the patient's peripheral blood with the patient's condition.

Specifically, the method of the present invention performs bioinformatics analysis on the obtained sequencing data including the following steps.

1) Remove low-quality sequences

"Low-quality sequences" mainly include linker sequences, repetitive sequences (including continuous single base repeats), and reads with a quality value lower than Q20, and reads with a length of less than 60 bp. After removing these data, valid data for AE sequence analysis can be obtained, and the number of these valid data is recorded as a.

2) Remove host sequence

In this step, the valid data is compared with the host genome data, the reads on the comparison are removed, and the non-host data is retained. In a specific embodiment, the valid data is aligned with the human reference genome sequence (hg19) through the software Bowtie2 or bwa to remove the sequence matching the human genome.

3) Obtain AE sequence

In this step, compare the valid data with the host sequence removed and the reference genome sequence of Echinococcus multilocularis (for example, through NCBI's Blastn) to obtain a sequence that matches the reference genome of Echinococcus multilocularis, referred to as AE sequence;

4) Obtain AE specific sequence

Due to the extremely low content of AE sequences in cfDNA, it is necessary to increase sensitivity to detect them. However, as the sensitivity increases, the specificity becomes worse. Some samples contain low AE-cfDNA content or do not contain AE-cfDNA specific sequences. If the secondary comparison is not performed, it may be misjudged as positive, leading to false positives. In order to solve this problem, the inventors re-aligned the AE sequence obtained in step 3) with the sequence in the NCBI nt database, and removed the sequence matching other species other than AE. The remaining sequence is the AE-specific sequence. Count the number and mark it as b.

5) Calculate the abundance of AE-cfDNA

The abundance of AE-cfDNA is calculated according to the following formula, which means the content of AE-cfDNA in free nucleic acid per tens of millions of peripheral blood.

Abundance _(AE-cfDNA) = b/a×10 ⁸

(6) Obtain the result through step (5), and judge the detection result:

i) Abundance _(AE-cfDNA) = 0, indicating that the sample of the subject does not contain AE-cfDNA, which is negative for AE-cfDNA;

ii) 0 <Abundance _(AE-cfDNA) ≤ 5, indicating that the subject sample may contain AE-cfDNA, which is weakly positive for AE-cfDNA;

iii) Abundance _(AE-cfDNA) >5, indicating that the subject sample contains AE-cfDNA and is AE-cfDNA positive.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples usually follow the conventional conditions or the conditions recommended by the manufacturer.

Example 1. Detection of free nucleic acid of alveolar hydatid in peripheral blood based on the Ion Torrent platform

1. Enrichment and extraction of free nucleic acid

The kit used is QIAamp MinElute ccfDNA Mini Kit, and the process is as follows.

(1) Take 1-4mL plasma sample, add the corresponding amount of magnetic beads, Proteinase K and binding buffer, and mix well

(2) Place it on the sample mixer, mix for 10 minutes, and centrifuge immediately;

(3) Place the tube on a magnetic stand and let it stand for 2 minutes until the solution becomes clear, then remove the supernatant;

(4) Add 200 μL of washing buffer to the tube, vortex to mix, transfer the mixture to a new 1.5 mL centrifuge tube, and incubate at room temperature for 5 minutes;

(5) Place the tube on the magnetic stand and let it stand for 2 minutes until the solution becomes clear. Transfer the supernatant to a new 1.5 mL centrifuge tube;

(6) Add 300 μL of buffer ACB to the above supernatant, and mix;

(7) Transfer the above mixed solution to the MinElute adsorption column, centrifuge at 6000g for 1 min, and transfer the adsorption column to a new collection tube;

(8) Add 500μL ACW2 to the adsorption column, centrifuge at 6000g for 1min, and transfer the adsorption column to a new collection tube

(9) Centrifuge at 14000g for 3min, transfer the adsorption column to a new 1.5mL elution tube, open the lid, and incubate at 56°C for 5min;

(10) Add 60 μL of nuclease-free water to the adsorption column, incubate at room temperature for 1 min, centrifuge at 14000 g for 1 min, discard the adsorption column, and recover the solution to enrich and extract free nucleic acid.

2. Library construction

The kit used is Ion Xpress Fragment Library Kit. The library construction process includes three steps: end repair, adaptor addition and library amplification.

Step 1: End repair

(1) Take 20-39.5 μL of the extracted free DNA into a new 200 μL PCR tube, add 10 μL end repair buffer and 0.5 μL end repair enzyme, and make up to 50 μL with nuclease-free water;

(2) Shake and mix the prepared reaction solution for 15 seconds, centrifuge briefly, and react at room temperature for 20 minutes;

(3) After the reaction is over, add 90 μL (1.8×) AMPure XP magnetic beads, vortex to mix, and place at room temperature for 5 minutes;

(4) Place the reaction tube on a magnetic stand and let it stand for 3 minutes until the solution is clear, then remove the supernatant;

(5) Add 500μL of freshly prepared 70% ethanol to the tube, incubate at room temperature for 30s, and remove the supernatant;

(6) Repeat step 5 once;

(7) Dry at room temperature for 3-5 minutes, and remove the tube punch from the magnetic stand;

(8) Add 14 μL of nuclease-free water to the tube, resuspend the magnetic beads, and place them at room temperature for 2 minutes;

(9) Place the tube on the magnetic stand for 2 minutes, until the solution becomes clear, transfer 12.5 μL of supernatant containing nucleic acid to a new 200 μL PCR tube for use.

Step 2: Connect the connectors

(1) Add 24.5μL of nuclease-free water to the 12.5μL of nucleic acid solution obtained in the previous step;

(2) Continue to add 5μL 10× ligation buffer, 1μL dNTP, 1μL P1 adapter, 1μL Ion Xpress Barcode X adapter (X is the adapter number), 1μL DNA ligase and 4μL shell repair polymerase in order to the tube;

(3) Mix the above system and centrifuge briefly

(4) Put the PCR tube containing the above system in the PCR machine, and run the following program:

25℃, 15min, 1 cycle; 72℃, 5min, 1 cycle; 4℃, hold, 1 cycle;

(5) After the reaction is over, add 75μL (1.5×) AMPure XP magnetic beads, vortex to mix, and place at room temperature for 5 minutes;

(6) Place the reaction tube on a magnetic stand and let it stand for 3 minutes until the solution is clear, then remove the supernatant;

(7) Add 500μL of freshly prepared 70% ethanol to the tube, incubate for 30s at room temperature, and remove the supernatant;

(8) Repeat step 5 once;

(9) Dry at room temperature for 3-5 minutes, and remove the tube punch from the magnetic stand;

(10) Add 14 μL of nuclease-free water to the tube, resuspend the magnetic beads, and place them at room temperature for 2 minutes;

(11) Place the tube on the magnetic stand for 2 minutes, until the solution becomes clear, transfer 12.5 μL of supernatant containing nucleic acid to a new 200 μL PCR tube for use.

Step 3: Library amplification

(1) Add 50 μL of PCR amplification solution and 2.5 μL of PCR primer mixture to the PCR tube containing 12.5 μL of nucleic acid obtained in the second step;

(2) Vortex and shake to mix, and centrifuge immediately;

(3) Put the PCR tube containing the above system in the PCR machine, and run the following program:

(4) 95℃, 5min, 1 cycle; 95℃, 15s→58℃, 15s→70℃, 1min, 5 cycles; 4℃, hold, 1 cycle;

(5) After the reaction is over, add 97.5μL (1.5×) AMPure XP magnetic beads, vortex to mix, and place at room temperature for 5 minutes;

(6) Place the reaction tube on a magnetic stand and let it stand for 3 minutes until the solution is clear, then remove the supernatant;

(7) Add 500μL of freshly prepared 70% ethanol to the tube, incubate for 30s at room temperature, and remove the supernatant;

(8) Repeat step 5 once;

(9) Dry at room temperature for 3-5 minutes, and remove the tube punch from the magnetic stand;

(10) Add 20 μL of nuclease-free water to the tube, resuspend the magnetic beads, and place at room temperature for 2 minutes;

(11) Place the tube on the magnetic stand for 2 minutes, until the solution becomes clear, transfer 18 μL of supernatant containing nucleic acid to a new 200 μL PCR tube, which is the constructed library;

(12) Use Qubit to quantify the constructed library, record the library concentration, use Agilent 2100 to identify the library, and determine the fragment size

(13) Calculate the amount and concentration of library substances based on the results of quantification and fragment size, and mix the same amount of substances to obtain a mixed library with a final concentration of 100 pM for use.

3. Sequencing template preparation

The kit used is Ion PI ^TM Hi-Q ^TM OT2 200 Kit, and the instrument used is One Touch2. The template preparation process includes two steps: emulsion PCR and template enrichment.

Step 1: Emulsion PCR

(1) Turn on One Touch2, and clean according to the instrument cleaning requirements;

(2) After washing, unload the old amplification plate, install the new amplification plate, collection tube and collection bridge, add 150μL of Emulsion II to each collection tube, combine OT2 oil and Emulsion I The solution in the bottle is inverted 3 times and mixed evenly, and the position of the emulsion I to 1/4 is added.

(3) Thaw the emulsion PCR buffer at room temperature, vortex and shake for 1 min, then centrifuge briefly

(4) Add 170 μL of nuclease-free water, 120 μL of emulsion PCR enzyme mixture, and 100 μL of bead solution to the reaction tube.

(5) Take a new reaction filter and place it on the shelf with the sample hole facing up. Vortex the amplification reaction solution prepared in step (4) for 5 seconds, centrifuge it to the bottom of the tube, and immediately use an 800μL pipette to pipette and mix 3 times, and slowly move the amplification reaction solution from the sample hole to vertical Add to the filter. Use a 1000μL pipette to draw 200μL of isolation oil, slowly and vertically add it into the filter from the sample hole.

(6) Remove the cleaning connector on One Touch2, slowly turn over the reaction filter in the previous step until the sample hole is facing down, and install it on One Touch2.

(7) Click "Run" on the One Touch2 screen, select the corresponding program: IonPI ^TM Hi-QOT2200Kit, select "Assisted" to confirm each step again, click "Next" to run the program, the program running time is about 5 hours.

(8) At the end of the program, click "Final Spin" on the screen and centrifuge the collection tube for 10 minutes. After centrifugation, the following steps should be performed immediately.

(9) After the centrifugation of One Touch2 is finished, click "open lid" on the screen, and the lid will open. Remove the collection bridge on the rotor, take out the two collection tubes, use a 1000 μL pipette, with the tip of the pipette facing the non-precipitating area, and carefully aspirate the supernatant against the liquid surface, and keep about 150 μL of solution in each tube.

(10) Use a pipette to repeatedly blow the remaining solution in the pipette, mix well, and transfer all the liquid in the two collection tubes to a new 1.5 mL low-adsorption centrifuge tube.

(11) Add 200 μL of nuclease-free water to each collection tube and mix by pipetting, and transfer all the nuclease-free water to the 1.5 mL low-adsorption centrifuge tube in the previous step.

(12) Repeat (11) once.

(13) Vortex the solution obtained from (10) to (12) for 30 seconds, and centrifuge at 15500 g for 8 minutes. Point the pipette tip to the non-precipitating area, carefully aspirate the supernatant, and keep about 100μL of solution in the tube.

(14) Add 900 μL of nuclease-free water to the 1.5 mL low-adsorption centrifuge tube in the previous step to reach a total volume of 1000 μL.

(15) Vortex the sample bead solution in the previous step for 30 seconds, and centrifuge at 15500g for 8 minutes. Point the pipette tip to the non-precipitating area, carefully aspirate the supernatant, and keep about 20μL of solution in the tube.

(16) Add 80μL of template resuspension solution to the sample bead solution from the previous step to make the total volume in the tube reach 100μL, vortex and shake for 30 seconds, and then centrifuge to the bottom of the tube.

(17) Clean the One Touch2 instrument and turn off the power if it is not used for a long time.

Step 2: Template enrichment

(1) Equilibrate the streptavidin C1 magnetic beads at room temperature for 30 minutes.

(2) Prepare the eluent: add 280 μL of Tween solution and 40 μL of 1M NaOH solution in a 1.5 mL centrifuge tube, vortex and shake for 10 seconds, and centrifuge briefly.

(3) The streptavidin C1 magnetic beads are vortexed for 2 minutes and mixed thoroughly. Take 100μL and add it to a new 1.5mL low-adsorption centrifuge tube; put the low-adsorption centrifuge tube on the magnetic stand, let it stand for 2 minutes, and carefully aspirate the supernatant.

(4) Add 1000μL C1 magnetic bead cleaning solution to the low-adsorption centrifuge tube, vortex for 30 seconds, and centrifuge immediately; place the low-adsorption centrifuge tube on the magnetic rack, stand for 2 minutes, and carefully aspirate the supernatant.

(5) Add 130μL of C1 magnetic bead capture solution to the low-adsorption centrifuge tube, vortex and shake for 30 seconds, and centrifuge briefly. Follow the steps below to add streptavidin C1 magnetic beads to the second well of the 8-well reaction strip

(6) Take an 8-well reaction strip and place it on the experimental table in the direction of the left and right circles, and add the reaction solution in the following order.

Well 1: 100μL sample bead solution + 130μL streptavidin C1 magnetic beads (obtained in step 5 above)

Hole 2: empty

Well 3: 300μL purified cleaning solution

Well 4: 300μL purified cleaning solution

Well 5: 300μL purified cleaning solution

Hole 6: empty

Well 7: 300μL of eluent (obtained in step 3 above)

Hole 8: empty

(7) Place the reaction strip in the ES card slot, install the ES tip on the robotic arm, place a new 200μL PCR tube in the collection hole, press the "Start/Stop" button, and run the machine. The machine runs about 40 minute.

(8) After the ES run is over, check the liquid volume in the PCR tube. If the liquid is greater than 250μL or obviously less than 200μL, you must perform template amplification again; if the liquid volume is not less than 200μL, continue to the next step.

(9) Remove the 200μL PCR tube containing the sample bead solution from the ES, mark the sample information on the tube cap, centrifuge at 15500g for 5 minutes, point the pipette to the non-precipitating area, carefully aspirate the supernatant, and keep 10μL in the tube Solution.

(10) Add 200μL of nuclease-free water, vortex for 60 seconds, centrifuge at 15500g for 5 minutes, and check if there are brown residual magnetic beads at the bottom of the 200μL PCR tube.

a) If there are no brown residual magnetic beads at the bottom of the 200μL PCR tube: proceed to the following step 11).

b) If there are brown residual magnetic beads at the bottom of the 200μL PCR tube: After aspirating 20μL of supernatant, pipette 10 times to mix the sample bead solution, put the PCR tube on the magnetic stand and let it stand for 4 minutes. Transfer everything to a new 200μL PCR tube. Add 20μL of nuclease-free water to the original PCR tube and mix by pipetting 10 times. After the beads are clarified, transfer all the supernatant to the new 200μL PCR tube and centrifuge at 15500g for 5 minutes.

(11) Point the pipette tip to the non-precipitating area, carefully aspirate the supernatant, keep 10 μL of liquid in the tube, add 90 μL of nuclease-free water, vortex for 60 seconds, centrifuge briefly, and set aside.

4. On-machine sequencing

The kit used is the Ion PI Hi-Q Sequencing 200 Kit and PI chip, the sequencer used is the Ion Proton sequencer, and the operation process is as follows;

(1) Instrument cleaning: Follow the instrument cleaning instructions to clean the sequencer

(2) Instrument initialization: initialize the sequencer according to the operating requirements of the kit manual;

(3) Set Plan: Set the corresponding Plan according to requirements;

(4) Prepare the computer library:

1) Take out the quality control microbead solution and shake for 30s, 15500g, and centrifuge for 5min; carefully aspirate the supernatant and keep 10μL of the solution in the tube;

2) Add 15μL of annealing buffer to the above sample bead solution;

3) Add 20μL of sequencing primers to the above solution, vortex and shake for 60s, and centrifuge briefly

4) Put the above reaction tube on the PCR machine, and run the program for annealing: 95°C, 2min, 1 cycle; 37°C 2min, 1 cycle, 20°C, hold;

5) After annealing, add 10μL of loading buffer to the above reaction tube. I choose to shake for 10s and centrifuge immediately.

(5) Chip loading and sequencing

1) Place the chip in the hanging basket, draw 55 μL of the library solution prepared in step (4), and inject it into the chip injection tank;

2) Place the chip on a centrifuge and centrifuge for 10 minutes;

3) Prepare 50% annealing buffer: take a 1.5mL centrifuge tube, add 0.5mL annealing buffer and 0.5mL nuclease-free water, and mix;

4) Prepare 50% rinsing solution: take a 1.5mL centrifuge tube, add 0.5mL 100% isopropanol solution and 0.5mL annealing buffer, and mix;

5) Prepare enzyme reaction solution: Take a 1.5mL centrifuge tube, add 60μL of 50% annealing buffer and 6μL of sequencing polymerase, and mix;

6) Preparation of foaming agent: Take a 1.5mL centrifuge tube, add 49μL of 50% annealing buffer and 1μL of foaming solution, mix well, use a 100μL pipette to pour 100μL of air into the foaming agent, quickly and repeatedly pipette the liquid 5 Seconds, make big bubbles turn into small bubbles, use a 100μL pipette again to inject 100μL of air into the foaming agent, quickly and repeatedly blow the liquid for 5 seconds to make the foaming agent become dense small bubbles;

7) After the centrifugation is completed, take out the chip, inject 100μL of small bubbles into the sample hole at a constant speed, and aspirate the overflow liquid from the sample outlet; add 55μL of 50% annealing buffer to the sample slot, and put the chip back into the centrifuge Machine, centrifuge for 30s;

8) Repeat step 7) once

9) Slowly add 100μL rinse solution to the injection hole of the chip twice. Each time the solution is added to the chip, the drained liquid is sucked in another hole.

10) Vertically slowly add 100 μL of 50% annealing buffer to the sample hole of the chip three times to avoid bubbles. Each time the solution is added to the chip, the drained liquid is sucked in another hole.

11) Slowly add 65μL of enzyme reaction solution vertically to the injection hole of the chip to avoid bubbles. In the other hole, suck up the discharged liquid.

12) After 5 minutes of enzymatic incubation at room temperature, install the chip on the chip slot of the sequencer, run the program, select the Plan set in advance, and after the check is correct, start sequencing, the running time is about 2.5 hours.

13) Within 72 hours after the program runs, return to the main menu for washing. If it exceeds 72 hours, perform a chlorine wash and then a water wash on the instrument. After washing, turn off the instrument on the main menu, and put tubes on the 4 dNTP holes to prevent dust. If not used for a long time, close the nitrogen cylinder valve

5. Bioinformatics analysis

Analyze the measured data according to the following procedure to obtain the abundance of AE-cfDNA in the sample and perform interpretation of the result.

(1) Remove low-quality sequences: Calculate the quality of sequencing data through Trimmomatic, remove low-quality sequences, linker sequences, repeated sequences, and sequences less than 60bp, and count the remaining sequences as data for AE sequence analysis. And count its number, denoted as a;

(2) Removal of host sequence: The remaining sequence will be analyzed through step 1), and aligned with the human reference genome sequence (hg19) through Bowtie2 to remove sequences that can match the human genome;

(3) Obtain AE sequence: The remaining sequence will be analyzed through step 2), and it will be compared with the AE reference genome through Blastn of NCBI to obtain a sequence that can be matched with the AE reference genome, which is the AE sequence;

(4) Obtain AE specific sequence: align the AE sequence obtained in step 3) again with the sequence in the NCBI nt library, remove the sequence that can match with other species other than AE, and the remaining sequence is AE specific Sequence, and count its number as b.

(5) Calculate the abundance of AE-cfDNA: According to the number of AE-specific sequences obtained in 4) and the number of measured sequences, calculate the abundance of AE-cfDNA according to the following formula, which means every tens of millions of peripherals The amount of AE-cfDNA in blood free nucleic acid.

Abundance _(AE-cfDNA) = b/a×10 ⁸

(6) Obtain the result through step (5), and judge the detection result:

1) Abundance _(AE-cfDNA) = 0, indicating that the peripheral blood sample tested does not contain AE-cfDNA and is AE-cfDNA negative;

2) 0 <abundance _(AE-cfDNA) ≤ 5, indicating that the peripheral blood sample tested may contain AE-cfDNA, which is the technical gray area of the test and is weakly positive for AE-cfDNA.

3) Abundance _(AE-cfDNA) > 5, indicating that the peripheral blood sample tested clearly contains AE-cfDNA and is positive for AE-cfDNA.

Example 2. Detecting free nucleic acid of alveolar hydatid in peripheral blood based on Illumina platform

1. Enrichment and extraction of free nucleic acid

The method is the same as the enrichment and extraction method of free nucleic acid in Example 1.

2. Library construction

The kits used are KAPA Library Preparation Kits. The library construction process includes four steps: end repair, end adding A, adding adaptor, and library amplification.

Step 1: End repair

(1) Take 50μL of free nucleic acid DNA extracted from the enrichment, add 8μL nuclease-free water, 7μL end repair buffer, 5μL end repair enzyme, and mix well;

(2) Put the reaction tube in the PCR machine and perform the reaction according to the following procedure: 20°C, 30min;

(3) Add 120 μL (1.7×) KAPA purified magnetic beads to the reaction tube at the end of the reaction, vortex to mix, and place at room temperature for 5 minutes;

(4) Place the reaction tube on the magnetic plate and let it stand for 3 minutes until the solution is clear, then remove the supernatant;

(5) Add 200μL of freshly prepared 80% ethanol to the tube, incubate at room temperature for 30s, and remove the supernatant;

(6) Repeat step 5 once;

(7) Dry at room temperature for 3-5 minutes, and remove the tube from the magnetic plate;

Step 2: Add A at the end

(1) Prepare the end plus A mixture: take a 200 μL PCR tube, add 42 μL nuclease-free water, 5 μL end plus A buffer, 3 μL end plus A enzyme, mix well, and centrifuge immediately

(2) Add the prepared reaction solution to the first step, dry the tube containing magnetic beads at room temperature, and vortex to mix;

(3) Place the reaction tube in the PCR machine and run the following program: 30°C, 30min;

(4) After the reaction is over, add 90μL PEG/NaCl solution to the reaction solution, vortex to mix, and incubate at room temperature for 10-15 minutes;

(5) Place the reaction tube on the magnetic plate and let it stand for 3 minutes until the solution is clear, then remove the supernatant;

(6) Add 200μL of freshly prepared 80% ethanol to the tube, incubate at room temperature for 30s, and remove the supernatant;

(7) Repeat step 6 once;

(8) Dry at room temperature for 3-5 minutes, and remove the tube from the magnetic plate;

Step 3: Add connectors

(1) Prepare the joint connection mixture: Take a 200μL PCR tube, add 30μL nuclease-free water, 10μL ligation buffer, 5μL DNA ligase in sequence, and vortex to mix well.

(2) Add the joint connection mixture prepared above to the dried magnetic beads in the second step, and add 5 μL of the corresponding joint storage solution;

(3) Put the reaction tube in the PCR machine for collection, and carry out the reaction according to the following procedure, 20℃, 15min;

(4) After the reaction is over, add 50μL of PEG/NaCl solution to the reaction solution, vortex to mix, and incubate at room temperature for 10-15 minutes;

(5) Place the reaction tube on the magnetic plate and let it stand for 3 minutes until the solution is clear, then remove the supernatant;

(6) Add 200μL of freshly prepared 80% ethanol to the tube, incubate at room temperature for 30s, and remove the supernatant;

(7) Repeat step 6 once;

(8) Dry at room temperature for 3-5 minutes, and remove the tube from the magnetic plate;

(9) Resuspend the magnetic beads with 50μL elution buffer, and incubate for 2min at room temperature

(10) Add 50μL of PEG/NaCl solution to the above magnetic bead solution pool

(11) Vortex to mix, incubate at room temperature for 5-15min,

(12) Centrifuge instantaneously, place the tube on a magnetic stand, let it stand for 2 minutes until the solution is clear, and remove the supernatant;

(13) Add 200μL of freshly prepared 80% ethanol to the tube, incubate at room temperature for 30s, and remove the supernatant;

(14) Repeat step 13 once

(15) Place the tube on a magnetic stand, dry at room temperature for 3-5 minutes, add 25μL of elution buffer to the tube, resuspend the magnetic beads, and incubate at room temperature for 2 minutes;

(16) Place the tube on the magnetic stand and let it stand for 2 minutes until the solution is clear. Transfer 20 μL of supernatant to a new 200 μL tube for use.

Step 4: Library amplification

(1) Add 25 μL of amplification buffer and 5 μL of primer mixture to the 20 μL of nucleic acid solution prepared in step 3 above, and mix well;

(2) Place the reaction tube in the PCR machine and proceed with the reaction according to the following procedure

98℃, 45s, 1 cycle; 98℃, 15s→60℃, 30s→72℃, 1min, 8-10 cycles; 72℃, 1min, 1 cycle, 4℃, hold

(3) After the reaction is over, add 50 μL (1×) KAPA purified magnetic beads to the reaction tube, vortex to mix, and place at room temperature for 5 minutes;

(4) Place the reaction tube on the magnetic plate and let it stand for 3 minutes until the solution is clear, then remove the supernatant;

(5) Add 200μL of freshly prepared 80% ethanol to the tube, incubate at room temperature for 30s, and remove the supernatant;

(6) Repeat step 5 once;

(7) Dry at room temperature for 3-5 minutes, and remove the tube from the magnetic plate;

(8) Add 30μL of elution buffer, mix well, and incubate for 2min at room temperature;

(9) Place the tube on a magnetic stand, let it stand for 2 minutes until the solution becomes clear, transfer the supernatant to a 200 μL PCR tube, and perform Qubit quantification and Agilent 2100 library quality identification.

3. On-machine sequencing

The MiSeq sequencer is used for sequencing, and the sequencing process is completed by Beijing Boao Jingdian Biotechnology Co., Ltd.

4. Bioinformatics analysis

The analysis method and process are the same as the bioinformatics analysis process of Example 1.

Example 3. Clinical application of the method for detecting free nucleic acid of alveolar hydatid in peripheral blood based on the Ion Torrent platform

In this embodiment, the detection method provided in embodiment 1 is used to detect clinical samples. A total of 119 clinical samples were tested, including 38 negative samples and 81 samples from patients diagnosed as AE-positive (including 60 preoperative samples and 21 postoperative samples). The test results of the present invention were used with clinicians to pass imaging and serum The results of the diagnostic tests are compared, and the results are shown in Table 1.

Table 1

Comparing 98 samples before surgery, the results are shown in Table 2. The clinical diagnosis results are used as the gold standard. According to statistics, the positive coincidence rate of this method is 100%, and the negative coincidence rate is 94.7%. There are 2 samples The clinical diagnosis was negative, and the test result by this method was weakly positive. The two patients were re-diagnosed clinically through various testing techniques, and the final diagnosis was early AE, indicating that this test method can assist in the early diagnosis of AE, and also that the test The method has high sensitivity and high specificity.

Table 2

The AE-cfDNA abundance detected by this method was compared with the clinical diagnosis results, and the statistical results are shown in Table 3. It can be clearly found from the comparison results that the test results indicate that the abundance of AE-cfDNA in peripheral blood is closely related to the prevalence of AE, which further shows that this method can assist in AE diagnosis and postoperative non-invasive monitoring.

table 3

Example 4.

In the process of performing bioinformatics analysis on the obtained sequencing data described in Example 1, the AE sequence obtained in step 3) is again compared with the sequence in the NCBI nt database to remove matches with other species other than AE The remaining sequence is the AE-specific sequence. As a comparison, the present inventors also used 20 negative samples and 30 positive samples to repeat the experiment described in Example 1. The difference is that step (3) was not performed, that is, the amount of effective sequencing data removed from the host sequence was directly recorded as For AE sequence b, calculate the abundance of AE-cfDNA according to the same formula, and the results obtained are as follows. The following table shows the results of the secondary comparison and the non-secondary comparison. Among the 20 samples diagnosed as negative by the hospital, the results of the secondary comparison analysis were all negative, and the secondary comparison analysis was not performed. In the results, 6 samples were false positives. Among the 30 positive samples, the final analysis results were consistent with and without the second comparison.

The following table shows the results of the secondary comparison and the non-secondary comparison.

Due to the extremely low content of AE sequences in cfDNA, it is necessary to increase sensitivity to detect them. However, as the sensitivity increases, the specificity becomes worse. Some samples contain low AE-cfDNA content or do not contain AE-cfDNA specific sequences. If the secondary comparison is not performed, it may be misjudged as positive, resulting in false positives. The method of the present invention solves this problem to a large extent, and can maintain extremely high specificity while ensuring the maximum detection sensitivity. From the data point of view, the occurrence of false positives is basically eliminated.

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been published, and these changes are within the protection scope of the present invention . All of the present invention is given by the appended claims and any equivalents thereof.

Claims (9)

1. A method for detecting AE-cfDNA in a sample of a subject, which includes the following steps:

   a) Extract and enrich free DNA in the subject's sample;

   b) Library construction and sequencing of the free DNA obtained in step a); and

   c) Perform bioinformatics analysis on the sequencing data of step b).
2. The method according to claim 1, wherein step c) said bioinformatics analysis comprises the following steps:

   1) Remove the linker sequence, repetitive sequence, and sequence less than 60bp, use the remaining sequence as data for AE sequence analysis, and record the number as a;

   2) Align the sequence obtained in step 1) with the host reference genome sequence to remove the sequence matching the host genome;

   3) Compare the sequence obtained in step 2) with the AE reference genome to obtain the AE sequence;

   4) Align the AE sequence obtained in step 3) with the sequence in the NCBI nucleic acid database again, remove the sequence that can match with other species other than AE, and obtain the AE-specific sequence, the number of which is recorded as b;

   5) Calculate the AE-cfDNA abundance according to the following formula, which means the content of AE-cfDNA in every tens of millions of free nucleic acid sequences in peripheral blood;

   Abundance _(AE-cfDNA) = b/a×10 ⁸ .
3. The method according to claim 2, further comprising the following steps:

   6) Judging the test results through the results obtained in step 5):

   i) Abundance _(AE-cfDNA) = 0, indicating that the subject sample is negative for AE-cfDNA;

   ii) 0 <Abundance _(AE-cfDNA) ≤ 5, indicating that the subject sample is weakly positive for AE-cfDNA;

   iii) Abundance _(AE-cfDNA) >5, indicating that the subject sample is positive for AE-cfDNA.
4. The method according to any one of claims 1 to 3, wherein the sample is a blood sample, in particular a plasma sample, preferably, the sample is a plasma sample from a human infected with Echinococcus.
5. Use of a reagent for detecting AE-cfDNA in a sample of a subject in preparing a kit for diagnosing whether a subject is suffering from echinococcosis infection, wherein the diagnosis includes the following steps:

   a) Extract and enrich free DNA in the subject's sample;

   b) Library construction and sequencing of the free DNA obtained in step a); and

   c) Perform bioinformatics analysis on the sequencing data of step b) through the following steps:

   1) Remove the linker sequence, repetitive sequence, and sequence less than 60bp, use the remaining sequence as data for AE sequence analysis, and record the number as a;

   2) Align the sequence obtained in step 1) with the host reference genome sequence to remove the sequence matching the host genome;

   3) Compare the sequence obtained in step 2) with the AE reference genome to obtain the AE sequence;

   4) Align the AE sequence obtained in step 3) with the sequence in the NCBI nucleic acid database again, remove the sequence that can match with other species other than AE, and obtain the AE-specific sequence, the number of which is recorded as b;

   5) Calculate the AE-cfDNA abundance according to the following formula, which means the content of AE-cfDNA in every tens of millions of free nucleic acid sequences in peripheral blood;

   Abundance _(AE-cfDNA) = b/a×10 ⁸ ,

   6) Judging the test results through the results obtained in step 5):

   i) Abundance _(AE-cfDNA) = 0, indicating that the subject sample is negative for AE-cfDNA;

   ii) 0 <Abundance _(AE-cfDNA) ≤ 5, indicating that the subject sample is weakly positive for AE-cfDNA;

   iii) Abundance _(AE-cfDNA) >5, indicating that the subject sample is positive for AE-cfDNA.
6. The use according to claim 5, wherein the kit further comprises at least one reagent selected from the group consisting of: a reagent for extracting and enriching free DNA in a subject sample, a reagent for constructing a DNA library, Reagents for high-throughput sequencing, and instructions for use.
7. A system for determining whether a subject has a vesicular hydatid infection, which includes:

   A) A peripheral blood sample obtained from the subject;

   B) Tools for extracting and enriching free DNA in subject samples;

   C) Tools for library construction and sequencing of free DNA; and

   D) A computer-readable medium with multiple instructions for performing the following steps:

   1) Remove the linker sequence, repetitive sequence, and sequence less than 60bp, use the remaining sequence as data for AE sequence analysis, and record the number as a;

   2) Align the sequence obtained in step 1) with the host reference genome sequence to remove the sequence matching the host genome;

   3) Compare the sequence obtained in step 2) with the AE reference genome to obtain the AE sequence;

   4) Align the AE sequence obtained in step 3) with the sequence in the NCBI nucleic acid database again, remove the sequence that can match with other species other than AE, and obtain the AE-specific sequence, the number of which is recorded as b;

   5) Calculate the AE-cfDNA abundance according to the following formula, which means the content of AE-cfDNA in every tens of millions of free nucleic acid sequences in peripheral blood;

   Abundance _(AE-cfDNA) = b/a×10 ⁸

   6) Judging the test results through the results obtained in step 5):

   i) Abundance _(AE-cfDNA) = 0, indicating that the sample of the subject does not contain AE-cfDNA, which is negative for AE-cfDNA;

   ii) 0 <Abundance _(AE-cfDNA) ≤ 5, indicating that the subject sample may contain AE-cfDNA, which is weakly positive for AE-cfDNA;

   iii) Abundance _(AE-cfDNA) >5, indicating that the subject sample contains AE-cfDNA and is AE-cfDNA positive.
8. A kit for diagnosing whether a subject is suffering from alveolar hydatid infection, which comprises a reagent for detecting alveolar hydatid free nucleic acid (AE-cfDNA) in a sample of the subject and at least one selected from the following Species of reagents: reagents for extracting and enriching free DNA in subject samples, reagents for DNA library construction, reagents for high-throughput sequencing, and instructions for use.
9. A method for diagnosing whether a subject is suffering from vesicular hydatid infection, including the following steps:

   a) Extract and enrich free DNA in the subject's sample;

   b) Library construction and sequencing of the free DNA obtained in step a); and

   c) Perform bioinformatics analysis on the sequencing data of step b) through the following steps:

   1) Remove the linker sequence, repetitive sequence, and sequence less than 60bp, use the remaining sequence as data for AE sequence analysis, and record the number as a;

   2) Align the sequence obtained in step 1) with the host reference genome sequence to remove the sequence matching the host genome;

   3) Compare the sequence obtained in step 2) with the AE reference genome to obtain the AE sequence;

   4) Align the AE sequence obtained in step 3) with the sequence in the NCBI nucleic acid database again, remove the sequence that can match with other species other than AE, and obtain the AE-specific sequence, the number of which is recorded as b;

   5) Calculate the AE-cfDNA abundance according to the following formula, which means the content of AE-cfDNA in every tens of millions of free nucleic acid sequences in peripheral blood;

   Abundance _(AE-cfDNA) = b/a×10 ⁸ ,

   6) Judging the test results through the results obtained in step 5):

   i) Abundance _(AE-cfDNA) = 0, indicating that the subject sample is negative for AE-cfDNA;

   ii) 0 <Abundance _(AE-cfDNA) ≤ 5, indicating that the subject sample is weakly positive for AE-cfDNA;

   iii) Abundance _(AE-cfDNA) > 5, indicating that the subject sample is positive for AE-cfDNA;

   d) If the subject is suffering from alveolar hydatid infection, treatment for alveolar hydatid infection is performed on the subject.