Automatic body fluid sample pathogen and host genome nucleic acid synchronous extraction method

Abstract

The invention discloses an automatic body fluid sample pathogen and host genome nucleic acid synchronous extraction method. The method comprises the following steps: S1, mixing a large-volume body fluid sample with a lysis buffer solution Buffer LA containing chaotropic salt and protease K, and carrying out lysis to release nucleic acid; s2, amino-modified magnetic beads are added, nucleic acid is captured through electrostatic interaction, after room-temperature combination, magnetic separation is conducted, supernatant is abandoned, and the amino-modified magnetic beads are reserved; s3, eluting nucleic acid on the amino-modified magnetic beads, and performing magnetic separation to retain eluent; s4, adding a protective agent Buffer GTN, isopropanol and ion exchange magnetic beads into the eluent, uniformly mixing and combining, performing magnetic separation, discarding supernatant, and retaining the ion exchange magnetic beads; s5, washing the ion exchange magnetic beads, drying, and eluting with Buffer EB1 to obtain whole nucleic acid; according to the method, large-volume, high-efficiency and high-purity nucleic acid synchronous extraction is realized through a double-magnetic-bead staged capturing and cracking-protective agent combination technology.

Description

Technical Field

[0001] This invention relates to an automated method for simultaneous extraction of pathogens and host genomic nucleic acids from bodily fluid samples, belonging to the field of biotechnology. Background Technology

[0002] Nucleic acid extraction is a crucial step in molecular biology research and clinical diagnosis, especially in infectious diseases and tumor gene detection. High-quality nucleic acids are fundamental to ensuring the accuracy of downstream detection (such as PCR and sequencing). However, traditional nucleic acid extraction methods have the following main problems when processing large volumes of body fluid samples (such as bronchoalveolar lavage fluid, cerebrospinal fluid, pleural effusion, and ascites): 1. Limitations of the single magnetic bead method: Currently, common nucleic acid extraction methods on the market (such as Qiagen) The main approach relies on single-type magnetic beads (such as silica beads) to capture nucleic acids through electrostatic adsorption or hydrophobic interactions. However, this method cannot effectively lyse pathogen cell walls. Many pathogens (such as bacteria and fungi) have tough cell walls or membranes, which are difficult to completely destroy using conventional lysis buffers (such as proteinase K+ ionizing salts), resulting in insufficient release of the pathogen genome and low nucleic acid recovery rates. Furthermore, existing nucleic acid extraction methods only capture free nucleic acids; host cell-free DNA (cfDNA) and some RNA can be effectively adsorbed, but the pathogen genome is lost due to insufficient release, affecting the sensitivity of infectious disease detection. 2. Efficiency issues with stepwise extraction methods: Existing nucleic acid extraction methods employ a stepwise DNA and RNA extraction strategy, extracting DNA first and then RNA. This requires processing the same sample in two parts, increasing sample volume requirements, especially unsuitable for large volumes of body fluids. Multiple centrifugation and liquid changes are necessary, increasing the risk of experimental errors. Automation is difficult, and the operation is cumbersome. Additionally, stepwise operations may lead to nucleic acid degradation during repeated processing, affecting downstream sequencing or qPCR results. 3. Residual inhibitors affect downstream detection: Large-volume body fluid samples often contain a large number of inhibitors, such as mucin, hemoglobin, and bile salts, which can interfere with nucleic acid extraction and subsequent amplification. 4. Existing nucleic acid extraction methods (such as a method for extracting whole nucleic acids from bronchoalveolar lavage fluid disclosed in the applicant's previous patent application 202110918528.7) cannot guarantee pathogen extraction, and the extracted samples cannot be guaranteed to be used simultaneously for pathogen and tumor detection. Therefore, there is an urgent need for a nucleic acid extraction method that is sample-saving, highly automated and compatible, can simultaneously extract host and pathogen nucleic acids, and has high recovery and high purity. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing an automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples. This method utilizes a dual-magnetic-bead staged capture technique (enriching whole nucleic acids with amino-modified magnetic beads and specifically purifying them with ion-exchange magnetic beads) and a lysis-protection agent co-process to achieve simultaneous extraction of large-volume, efficient, and high-purity nucleic acids. The method is simple to operate and suitable for tumor gene detection, etc., and has advantages such as high recovery rate, low inhibitor residue, and low sample consumption.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] This invention provides an automated method for simultaneous extraction of pathogens and host genomic nucleic acids from bodily fluid samples, comprising the following steps:

[0006] S1. Mix the body fluid sample with lysis buffer LA containing dissociation salt and proteinase K to lyse and release nucleic acids;

[0007] S2. Add amino-modified magnetic beads to capture nucleic acids through electrostatic interaction. After binding at room temperature, magnetic separation is performed to discard the supernatant and retain the amino-modified magnetic beads.

[0008] S3. Add high-salt elution buffer EB2 to elute the nucleic acids on the amino-modified magnetic beads, and magnetically separate to retain the elution buffer; the high-salt elution buffer EB2 is a NaCl solution;

[0009] S4. Add the protective agent Buffer GTN, isopropanol and ion exchange magnetic beads to the eluent, mix well and bind, then magnetically separate and discard the supernatant, retaining the ion exchange magnetic beads.

[0010] S5. Wash the ion exchange magnetic beads sequentially with Buffer W1 and Buffer W2, dry them, and then elute with low-salt elution buffer EB1 to obtain whole nucleic acids; the buffer EB1 is DEPC water.

[0011] Preferably, the body fluid sample in step S1 is a large-volume body fluid sample, which is bronchoalveolar lavage fluid.

[0012] Preferably, in step S1: the Buffer LA contains GuSCN, KCl, Triton X-100 and EDTA, the concentration of the proteinase K is 20 mg / mL; the lysis temperature is 60°C and the time is 30-60 minutes.

[0013] Preferably, in step S1, the amount of Buffer LA added is 1.2 ml Buffer LA / 4 ml bronchoalveolar lavage fluid; the amount of proteinase K added is 200 μl proteinase K / 4 ml bronchoalveolar lavage fluid.

[0014] Preferably, in step S2: the amino-modified magnetic beads have an amino density ≥ 5 μmol / mg and a particle size of 500 nm.

[0015] Preferably, in step S2, the amount of amino-modified magnetic beads added is: 9.6 mg amino-modified magnetic beads / 4 ml bronchoalveolar lavage fluid.

[0016] Preferably, in step S3, the amount of Buffer EB2 added is: 400 μL Buffer EB2 / 4 ml bronchoalveolar lavage fluid, based on the initial amount of bronchoalveolar lavage fluid; the Buffer EB2 is a 1.4-1.5 M NaCl solution.

[0017] Preferably, the ion-exchange magnetic beads in step S4 are hydroxyl magnetic beads.

[0018] Preferably, in step S4, the Buffer GTN is a buffer solution with pH 8.5 containing SDS, tetraethylammonium bromide, and Tris-HCl; the Buffer GTN is a nucleic acid binding enhancer, and the amount of Buffer GTN added is: 200 μl Buffer GTN / 4 ml bronchoalveolar lavage fluid based on the initial volume of bronchoalveolar lavage fluid; the amount of isopropanol added is: 700 μL 100% isopropanol / 4 ml bronchoalveolar lavage fluid based on the initial volume of bronchoalveolar lavage fluid; and the amount of ion exchange magnetic beads added is: 500 μL ion exchange magnetic beads / 4 ml bronchoalveolar lavage fluid based on the initial volume of bronchoalveolar lavage fluid.

[0019] Preferably, in step S5, Buffer W1 is a solution containing 50% v / v ethanol and 4M GuHCl; Buffer W2 is an 80% v / v ethanol solution; the amount of Buffer W1 added is: 1000 μL Buffer W1 / 4 ml bronchoalveolar lavage fluid, based on the initial amount of bronchoalveolar lavage fluid; the amount of Buffer W2 added is: 1000 μL Buffer W2 / 4 ml bronchoalveolar lavage fluid, based on the initial amount of bronchoalveolar lavage fluid; and the amount of Buffer EB1 added is: 100 μL Buffer EB1 / 4 ml bronchoalveolar lavage fluid, based on the initial amount of bronchoalveolar lavage fluid.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention achieves efficient and high-purity simultaneous nucleic acid extraction through a dual-magnetic-bead staged capture technique (enrichment of whole nucleic acids with amino-modified magnetic beads and specific purification with ion-exchange magnetic beads) and a lysis-protection agent coupling technology. The operation is simple and suitable for tumor gene detection and metagenomic sequencing detection of infectious pathogens, offering advantages such as high recovery rate, low inhibitor residue, and low sample consumption. Specifically, this invention has the following advantages: 1) Efficient simultaneous extraction: Through dual-magnetic-bead staged capture, i.e., enrichment of whole nucleic acids with amino-modified magnetic beads and specific purification with ion-exchange magnetic beads, simultaneous extraction of host and pathogen nucleic acids is achieved, with a recovery rate ≥80%. 2) Simple operation: A single processing step is sufficient, reducing the operation time to 110 minutes, making it suitable for automated equipment. 3) High-purity nucleic acid extraction: The lysis-protection agent coupling technology reduces inhibitor residue and improves downstream detection efficiency (such as PCR and sequencing). 4) Sample saving: Avoiding step-by-step extraction significantly reduces sample consumption. Attached Figure Description

[0022] Figure 1 This is an Agilent 4200D1000 film image of the present invention. Detailed Implementation

[0023] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0024] In the following examples, Buffer LA, amino-modified magnetic beads, ion-exchange magnetic beads, Buffer EB1, Buffer EB2, Buffer W1, Buffer W2, and Buffer GTN were all purchased from Kaishuo Biotechnology (Xiamen) Co., Ltd.

[0025] The components of Buffer LA include:

[0026] 20~40mM GuSCN, 100mM KCl, 5mM EDTA, 5% TritonX-100, pH 5.5;

[0027] The components of Buffer GTN include:

[0028] 10 mM EDTA, 300 mM TEAB, 30 mM Tris-HCl, 1% v / v SDS (20 wt% sodium dodecyl sulfate solution), pH 8.5;

[0029] Buffer W1 consists of: 2.4M GuHCl, 70% v / v EtOH;

[0030] The composition of Buffer W2 is: 10 mM Tris-HCl, 80% v / v EtOH;

[0031] Buffer EB1: DEPC water (Diethyl Pyrocarbonate-treated Water);

[0032] Buffer EB2: 1.44M NaCl;

[0033] The ion-exchange magnetic beads are hydroxyl magnetic beads (Mag-OH) with a large number of silanol groups (hydroxyl groups) modified on their surface.

[0034] An automated method for simultaneous extraction of pathogens and host genomic nucleic acids from bodily fluid samples includes the following steps:

[0035] S1. Mix a large volume of body fluid sample with lysis buffer LA containing dissociation salt and proteinase K, and lyse at 60°C for 15-30 minutes to release nucleic acids;

[0036] In step S1, the large-volume body fluid sample is bronchoalveolar lavage fluid;

[0037] In step S1, the Buffer LA contains GuSCN, KCl, Triton X-100 and EDTA, and the proteinase K concentration is 20 mg / mL;

[0038] In step S1, the amount of Buffer LA added is 1.2 ml Buffer LA / 4 ml bronchoalveolar lavage fluid; the amount of Proteinase K added is 200 μl Proteinase K / 4 ml bronchoalveolar lavage fluid.

[0039] S2. Add amino-modified magnetic beads to capture nucleic acids through electrostatic interaction. After binding at room temperature, magnetic separation is performed to discard the supernatant and retain the amino-modified magnetic beads.

[0040] In step S2, the amino-modified magnetic beads have an amino density ≥ 5 μmol / mg and a particle size of 500 nm;

[0041] In step S2, the amount of amino-modified magnetic beads added is: 9.6 mg amino-modified magnetic beads / 4 ml bronchoalveolar lavage fluid;

[0042] S3. Add high-salt elution buffer EB2 to elute the nucleic acids on the amino-modified magnetic beads, and magnetically separate to retain the elution buffer;

[0043] In step S3, the amount of Buffer EB2 added is: 400 μL Buffer EB2 / 4 ml bronchoalveolar lavage fluid, based on the initial volume of bronchoalveolar lavage fluid.

[0044] S4. Add the protective agent Buffer GTN, isopropanol and ion exchange magnetic beads to the eluent, mix well and bind for 10 minutes, then magnetically separate and discard the supernatant, keeping the ion exchange magnetic beads.

[0045] In step S4, the Buffer GTN is a pH 8.5 buffer containing SDS, tetraethylammonium bromide, and Tris-HCl; the Buffer GTN is a nucleic acid binding enhancer, and the amount of Buffer GTN added is: 200 μl Buffer GTN / 4 ml bronchoalveolar lavage fluid based on the initial volume of bronchoalveolar lavage fluid; the amount of isopropanol added is: 700 μl 100% isopropanol / 4 ml bronchoalveolar lavage fluid based on the initial volume of bronchoalveolar lavage fluid; the amount of ion exchange magnetic beads added is: 500 μl ion exchange magnetic beads / 4 ml bronchoalveolar lavage fluid based on the initial volume of bronchoalveolar lavage fluid.

[0046] S5. Wash the ion exchange magnetic beads sequentially with Buffer W1 and Buffer W2, dry them, and then elute with low-salt elution buffer EB1 to obtain whole nucleic acids;

[0047] In step S5, Buffer W1 is a solution containing 50% ethanol and 4M GuHCl (guanidine hydrochloride); Buffer W2 is an 80% ethanol solution; the amount of Buffer W1 added is 1000 μL Buffer W1 / 4 ml bronchoalveolar lavage fluid, based on the initial volume of bronchoalveolar lavage fluid; the amount of Buffer W2 added is 1000 μL Buffer W2 / 4 ml bronchoalveolar lavage fluid, based on the initial volume of bronchoalveolar lavage fluid; and the amount of Buffer EB1 added is 100 μL Buffer EB1 / 4 ml bronchoalveolar lavage fluid, based on the initial volume of bronchoalveolar lavage fluid.

[0048] Using the aforementioned automated method for simultaneous extraction of pathogens and host genomic nucleic acids from bodily fluid samples, nucleic acid extraction was performed on samples B1, C1, D1, E1, F1, and G1, respectively. The results of the extracted nucleic acid Qubit concentration tests are shown in Table 1. Agilent 4200D1000 gel images are shown below. Figure 1 As shown.

[0049] Table 1 shows the results of nucleic acid Qubit concentration testing.

[0050]

[0051]

[0052] Using the aforementioned automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples, NGS testing was performed on bronchoalveolar lavage fluid (BALF). The test results for microorganisms are shown in Table 2, and the test results for tumor genes are shown in Table 3.

[0053] Table 2 shows the NGS test results of microorganisms in bronchoalveolar lavage fluid (BALF).

[0054]

[0055] Table 3 shows the NGS test results of tumor genes in bronchoalveolar lavage fluid (BALF).

[0056]

[0057] The automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples described in this invention can be applied to whole nucleic acid samples extracted by automated nucleic acid extraction equipment and can be used for downstream PCR, sequencing, or gene detection.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. An automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples, characterized in that, Includes the following steps: S1. Mix the body fluid sample with lysis buffer LA containing dissociation salt and proteinase K to lyse and release nucleic acids; S2. Add amino-modified magnetic beads to capture nucleic acids through electrostatic interaction. After binding at room temperature, magnetic separation is performed to discard the supernatant and retain the amino-modified magnetic beads. S3. Add high-salt elution buffer EB2 to elute the nucleic acids on the amino-modified magnetic beads, and magnetically separate to retain the elution buffer; the high-salt elution buffer EB2 is a NaCl solution; S4. Add the protective agent Buffer GTN, isopropanol and ion exchange magnetic beads to the eluent, mix well and bind, then magnetically separate and discard the supernatant, retaining the ion exchange magnetic beads. S5. Wash the ion exchange magnetic beads sequentially with Buffer W1 and Buffer W2, dry them, and then elute with low-salt elution buffer EB1 to obtain whole nucleic acids; the low-salt elution buffer EB1 is DEPC water.

2. The automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples as described in claim 1, characterized in that, The body fluid sample in step S1 is a large-volume body fluid sample, which is bronchoalveolar lavage fluid.

3. The automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples as described in claim 2, characterized in that, In step S1: the Buffer LA contains GuSCN, KCl, Triton X-100 and EDTA, the concentration of proteinase K is 20 mg / mL; the lysis temperature is 60°C and the time is 30-60 minutes.

4. The automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples as described in claim 3, characterized in that, In step S1, the amount of Buffer LA added is 1.2 ml Buffer LA / 4 ml bronchoalveolar lavage fluid; the amount of proteinase K added is 200 μl proteinase K / 4 ml bronchoalveolar lavage fluid.

5. The automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples as described in claim 1, characterized in that, In step S2: the amino-modified magnetic beads have an amino density ≥ 5 μmol / mg and a particle size of 500 nm.

6. The automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples as described in claim 2, characterized in that, In step S2, the amount of amino-modified magnetic beads added is: 9.6 mg amino-modified magnetic beads / 4 ml bronchoalveolar lavage fluid.

7. The automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples as described in claim 2, characterized in that, In step S3, the amount of Buffer EB2 added is: 400 μL Buffer EB2 / 4 ml bronchoalveolar lavage fluid, based on the initial volume of bronchoalveolar lavage fluid; the Buffer EB2 is a 1.4-1.5 M NaCl solution.

8. The automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples as described in claim 1, characterized in that, The ion-exchange magnetic beads in step S4 are hydroxyl magnetic beads.

9. The automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples as described in claim 2, characterized in that, In step S4, the Buffer GTN is a pH 8.5 buffer containing SDS, tetraethylammonium bromide, and Tris-HCl; the Buffer GTN is a nucleic acid binding enhancer, and the amount of Buffer GTN added is: 200 μl Buffer GTN / 4 ml bronchoalveolar lavage fluid based on the initial volume of bronchoalveolar lavage fluid; the amount of isopropanol added is: 700 μl 100% isopropanol / 4 ml bronchoalveolar lavage fluid based on the initial volume of bronchoalveolar lavage fluid; the amount of ion exchange magnetic beads added is: 500 μl ion exchange magnetic beads / 4 ml bronchoalveolar lavage fluid based on the initial volume of bronchoalveolar lavage fluid.

10. The automated method for simultaneous extraction of pathogens and host genomic nucleic acids from body fluid samples as described in claim 2, characterized in that, In step S5, Buffer W1 is a solution containing 50% v / v ethanol and 4M GuHCl; Buffer W2 is an 80% v / v ethanol solution; the amount of Buffer W1 added is 1000 μL Buffer W1 / 4 ml bronchoalveolar lavage fluid, based on the initial amount of bronchoalveolar lavage fluid; the amount of Buffer W2 added is 1000 μL Buffer W2 / 4 ml bronchoalveolar lavage fluid, based on the initial amount of bronchoalveolar lavage fluid; and the amount of Buffer EB1 added is 100 μL Buffer EB1 / 4 ml bronchoalveolar lavage fluid, based on the initial amount of bronchoalveolar lavage fluid.

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