CN118510910A - Method for processing nucleic acid containing samples - Google Patents

Abstract

The present invention provides a method for treating a nucleic acid-containing sample, the method comprising the following steps: a) contacting the nucleic acid-containing sample with an ion exchange resin, a protease and an albumin; b) heating at a temperature of 60° C. to 120° C.; c) allowing the formation of a sediment and a supernatant; and d) recovering the supernatant containing the nucleic acid. The present invention also provides a composition comprising an ion exchange resin, a protease and an albumin; a kit; use of the composition for treating a nucleic acid-containing sample; a method for amplifying nucleic acid, and a treated nucleic acid-containing sample obtainable by the method.

Description

Method for processing nucleic acid containing samples

This application claims the priority benefit of European patent application EP22382008 filed on January 10, 2022.

Technical Field

The present invention belongs to the field of biological sample processing, in particular the processing of samples containing nucleic acids. The method of the present invention is particularly suitable for preparing biological samples for diagnosing infectious diseases by genetic analysis.

Background Art

The role of molecular diagnostics is crucial in today’s global healthcare environment. In developing countries, most deaths are due to lack of proper diagnosis and subsequent treatment of infectious diseases such as acute respiratory infections, malaria, HIV/AIDS, and tuberculosis. Furthermore, the recent SARS-CoV-2 pandemic has highlighted the need for tools to effectively detect and control infectious diseases anywhere in the world.

Molecular diagnostic procedures often require the detection of specific nucleic acid sequences present in a biological sample. Before nucleic acid molecules can be detected or quantified, the sample must be made usable by processing it. In fact, sample preparation is a major cost and time component of genetic testing.

Typically, the target nucleic acid is contained within a viral particle, a bacterial cell, a fungal cell, or a cell of a more complex organism, such as a human leukocyte.

Therefore, sample preparation methods for nucleic acid analysis usually require a first lysis step to release the nucleic acids into solution, followed by a nucleic acid purification step.

Traditional sample preparation methods involve laborious extraction procedures including several washing and centrifugation steps, making them cumbersome, labor-intensive, and slow. Furthermore, organic extraction methods often involve the use of chaotropic agents such as ammonium thiocyanate and organic solvents to lyse cells and denature proteins, which makes them hazardous and highly contaminating. Furthermore, these substances remaining in the eluted sample can interfere with subsequent enzymatic processing of the isolated nucleic acids, such as nucleic acid sequencing or amplification. Therefore, these methods have limitations as they can generate amplification problems due to the inability to remove inhibitors, undesirable interference of reagents, or simply because the method is not efficient enough.

In addition, several inorganic methods have been developed, including the use of high salt concentrations, glass powders, or silica gel suspensions, but these methods are time-consuming and require several steps, washing and desalting procedures or multiple transfers of the sample to different containers.

Nucleic acid extraction kits are relatively expensive and almost unavailable in resource-poor areas. In addition, the clinical application of genetic testing has increased dramatically due to the COVID-19 pandemic, leading to a global shortage of reagents and kits, which is also a major bottleneck for disease diagnosis in developed countries.

For all these reasons, many research groups around the world have been working to simplify or eliminate the nucleic acid extraction step for genetic testing. However, methods disclosed in the prior art are either too slow and cumbersome, or fail to effectively remove enough contaminants from the sample, rendering the subsequent genetic analysis results unreliable.

Furthermore, methods that allow rapid and very simple extraction of nucleic acids remain necessary in developing countries, where poverty and poor sanitary conditions spread certain diseases that could lead to emergent epidemic situations.

Therefore, despite the progress achieved to date, there is still a need for rapid, efficient and safe methods to process samples that allow direct detection of nucleic acids.

SUMMARY OF THE INVENTION

The present inventors have developed a method for processing nucleic acid-containing samples that enables direct detection of DNA or RNA without the need for cumbersome nucleic acid extraction steps or the use of hazardous chemicals.

As shown in the following examples, through a large number of experiments, the present inventors unexpectedly discovered that by mixing a sample with a ternary combination comprising an ion exchange resin, a protease and an albumin, followed by a brief heat treatment, a supernatant containing nucleic acids suitable for genetic analysis techniques such as PCR can be obtained quickly and safely without the need for a complicated purification step. Therefore, anyone can obtain nucleic acids from a sample without exposure to hazardous chemicals.

Advantageously, the inventors have found that the addition of albumin to the mixture synergistically enhances the removal of inhibitory or degradative enzymes and other contaminants, which allows obtaining samples that can be directly used for detection analysis without the need for any further purification steps (see Tables 2 and 5). It is worth noting that this effect was not obtained when other reagents were added to the mixture (see Table 3).

The present invention provides unprecedented quick, simple and effective method to provide nucleic acid that can be used as diagnostic analysis reagent immediately.Compared with conventional extraction method, the method provided herein provides equivalent genetic results, has the advantages of shortening analysis time and reducing cost.In fact, the method of the present invention can be carried out in less than 20 minutes.

The extraction-free method presented here is very versatile because it can process a variety of samples, from tissue swabs to saliva samples, and it can be used in conjunction with a variety of nucleic acid amplification and detection kits.

In situations of reagent shortages, such as those caused by the COVID-19 pandemic, the rapid identification of infected patients enabled by the methods of the present invention may be particularly important.

In summary, the results presented herein demonstrate that the present invention provides an effective method that allows bypassing traditional nucleic acid extraction procedures and can help improve detection capabilities, especially in health emergencies or in countries with limited resources.

Therefore, in a first aspect, the present invention provides a method for treating a nucleic acid-containing sample, the method comprising the following steps: a) contacting the nucleic acid-containing sample with an ion exchange resin, a protease and an albumin; b) heating at a temperature of 60°C to 120°C; c) allowing a sediment (i.e., a precipitate) and a supernatant to form; d) recovering the supernatant containing the nucleic acid.

In a second aspect, the present invention provides a composition comprising an ion exchange resin, a protease and an albumin; specifically, a composition for treating a nucleic acid-containing sample comprising an ion exchange resin, a protease and an albumin.

In a third aspect, the present invention provides a kit comprising an ion exchange resin, a protease, an albumin, optionally instructions for use thereof, and optionally a PCR buffer; in particular wherein the kit is for processing a sample containing nucleic acids.

In a fourth aspect, the present invention provides the use of an ion exchange resin, a protease and an albumin for treating a sample containing nucleic acid.

In a fifth aspect, the present invention provides a method for amplifying nucleic acids from a nucleic acid-containing sample, the method comprising the following steps: a) treating the sample using the method defined in the first aspect to obtain a supernatant containing nucleic acids; and b) amplifying the nucleic acids contained in the supernatant.

In a sixth aspect, the present invention provides a processed nucleic acid-containing sample obtained by the method defined in the first aspect.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, all terms used in this application should be understood as having ordinary meanings known in the art. Other more specific definitions of certain terms used in this application are described below and are intended to be uniformly applied throughout the specification and claims, unless otherwise explicitly listed definitions provide a broader definition.

For the purposes of the present invention, any range given includes the lower and upper limits of the range. Given ranges, such as concentrations, etc., should be considered approximate unless otherwise specified.

As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more than one". Unless otherwise indicated, definite articles such as "the" used herein also include the plural form of the noun. As used herein, the term "about" refers to a numerical range that is 15% greater or less than a fixed value.

As described above, the present invention provides in the first aspect a method for processing a nucleic acid-containing sample based on the synergistic action of an ion exchange resin, a protease and an albumin. Those skilled in the art will appreciate that the method does not exclude the insertion of additional steps before, after or between the steps shown. In addition, those skilled in the art will appreciate that the components of the composition mixed with the sample can be provided together or separately. The expression "processing a nucleic acid-containing sample" used herein refers to a process for removing or inactivating compounds (such as inhibitory or degradative enzymes and other contaminants) that hinder nucleic acid detection, thereby producing a processed sample that can be directly used for detection analysis without any further purification steps. In particular, it is meant to include "extracting nucleic acids from a sample containing nucleic acids", but does not include subsequent detection steps such as PCR amplification.

In the method of the present invention, a sample containing nucleic acid is mixed with the ternary combination, which is then heated to lyse the sample and inactivate the protease. The resin is then allowed to sediment, and the supernatant containing nucleic acid now appears to be substantially free of contaminants. Thus, the method of the present invention produces an extract containing nucleic acid.

In a specific embodiment of the method of the first aspect, optionally in combination with any embodiment provided above or below, the method comprises the following steps in order: a) contacting the nucleic acid-containing sample with an ion exchange resin, a protease and an albumin; b) heating at a temperature of 60°C to 120°C; c) allowing a precipitate and a supernatant to form; d) recovering the supernatant.

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, the method comprises the following steps: a) contacting a nucleic acid-containing sample with an ion exchange resin, a protease and an albumin; b) heating the mixture obtained in step (a) at a temperature of 60°C to 120°C; c) allowing a precipitate or sediment and a supernatant to form in the heated mixture obtained in step (b); d) recovering the supernatant obtained in step (c).

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, the method consists of the following steps: a) contacting a sample containing nucleic acid with an ion exchange resin, a protease and an albumin; b) heating at a temperature of 60°C to 120°C; c) allowing the formation of a precipitate and a supernatant; d) recovering the supernatant.

In a specific embodiment of the method of the first aspect, the method is used to extract nucleic acid from a sample containing nucleic acid.

In a specific embodiment of the method of the first aspect, the sample is selected from whole blood, plasma, serum, urine, saliva, sputum, nasal mucus, respiratory lavage fluid, tears, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid, feces, prostatic fluid, semen, lymph fluid, bile, sweat, breast milk, breast fluid, non-human embryonic cells, non-human fetal cells, human or non-human stem cells, cultured cells, cultured bacteria, cultured yeast, tissue biopsy (such as colorectal biopsy, epithelial biopsy, skin biopsy), tissue swab or smear (such as nasopharyngeal swab, oropharyngeal swab, vaginal swab, urethral swab or cervical swab), cervical cytology, synovial fluid, food, feed, soil, agricultural products, water (such as drinking water, domestic water system, water storage or water tank, food processing water or food production water, recreational water, irrigation water, agricultural water system or agricultural water, water distribution system, water from wells, rivers, lakes, swimming and bathing areas), surface swabs, surface washes, and combinations thereof.

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, the sample is a biological sample.As used herein, "biological sample" refers to any sample comprising biological material in which nucleic acids are present.

In a more specific embodiment, the sample is a liquid sample or a solid or semi-solid diluted sample, in particular a liquid biological sample. In a more specific embodiment, the biological sample comprises a pathogen, such as a virus, a virosome or a viral particle, a prion, a bacterium, a fungus, a yeast, a protozoa, a worm, a parasite, a pinworm, a trematode, a tissue cell, or a combination thereof.

Non-limiting examples of pathogens and/or diseases caused by pathogens encompassed by the methods of the present invention include herpes simplex, varicella-zoster virus (VZV), respiratory syncytial virus (RSV), Epstein-Barr virus, cytomegalovirus (CMV), human polyomavirus 8, human herpesvirus 8, Legionella pneumophila, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Aspergillus, coccal pneumonia, microsporidia, rotavirus, hepatitis, genital warts (human papillomavirus or HPV, influenza (flu) and BK virus, cholera (caused by Vibrio cholerae), diarrhea, colitis and severe intestinal disease (caused by Clostridium difficile), bacillary dysentery (caused by Shigella), salmonellosis (caused by Salmonella typhi, Salmonella paratyphi or Salmonella enteritidis), gastrointestinal disease caused by Vibrio parahaemolyticus), gastroenteritis, urinary tract infection, neonatal meningitis, hemorrhagic colitis, Crohn's disease, Gram-negative pneumonia (caused by E. coli, enterotoxigenic E. coli), Campylobacter infection (caused by C. jejuni, C. hyosintestinalis, C. seagull, C. rectum, C. Uppsala, or C. coli), candidiasis (caused by Candida albicans), hepatitis (caused by hepatitis A virus or hepatitis E virus), common cold, hand, foot and mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis (caused by enterovirus A-L, poliovirus, coxsackievirus, echovirus), vomiting virus and norovirus acute gastroenteritis (caused by norovirus or norwalk virus), poliomyelitis (caused by poliovirus 1, poliovirus 2, poliovirus 3), respiratory diseases, including severe acute respiratory syndrome (caused by coronaviruses such as 229E, OC43, SARS-CoV, HCoV NL63, HCoV HKU1, MERS-CoV, and SARS-CoV-2), stomach flu and gastroenteritis (caused by rotavirus A-J), mild cold or flu-like illness (caused by adenovirus), amebiasis (caused by Entamoeba histolytica), giardiasis (caused by Giardia lamblia), cryptosporidiosis (caused by Cryptosporidium), toxoplasmosis (caused by Toxoplasma gondii), taeniasis (caused by tapeworms), helminthiasis, trichomoniasis or whipworms, and soil-transmitted helminthiasis (caused by Ancylostoma americanum, Hookworm, and Trichocephalus) foodborne trematodes (caused by several parasites, such as Clonorchis sinensis, Fasciola, Opisthorchis, and Paragonimus), malaria (caused by Plasmodium), tuberculosis (caused by Mycobacterium tuberculosis), Chagas disease (caused by Trypanosoma cruzi), schistosomiasis (caused by flatworms or schistosomes), Zika fever (caused by Zika virus), dengue fever (caused by dengue virus DENV), Japanese encephalitis (caused by Japanese encephalitis virus), West Nile fever (caused by West Nile virus), ascariasis (caused by Ascaris lumbricoides ), leishmaniasis (caused by Leishmania), mycetoma (actinomycetoma caused by Nocardia brasiliensis, Streptomyces somaliensis, Actinomycetes maduraensis and Actinomyces balaensis, or fungal bone abscess caused by Mycomadura mycoides), Buruli ulcer (caused by Mycobacterium ulcerans), leprosy or Hansen's disease (caused by Mycobacterium leprae), chromoblastomycosis (caused by Chromosporium personi, Cladosporium fasciatum, Cladosporium carinii, Chromosporium compactum), paracoccidioidomycosis (caused by Paracoccidioidomycosis), trachoma (caused by Chlamydia trachomatis), yaws, syphilis or non-venereal syphilis (caused by Treponema pallidum), dracunculiasis or guinea worm disease (caused by Guinea worm), echinococcosis (caused by Echinococcus granulosus, Echinococcus multilocularis, Echinococcus minor or Echinococcus fowleri), lymphatic filariasis (caused by filariae such as Wuchereria bancrofti, Brugia malayi and Brugia timorensis), onchocerciasis (caused by Onchocerca volvulus), rabies (caused by lyssaviruses such as lyssavirus and Australian bat lyssavirus).

In a more specific embodiment, the pathogen causes a disease selected from the group consisting of: COVID-19 (caused by severe acute respiratory syndrome coronavirus-2, SARS-CoV-2), aspergillosis (caused by Aspergillus fumigatus), acquired immunodeficiency syndrome (AIDS, caused by human immunodeficiency virus, HIV), chlamydia, gonorrhea, syphilis, trichomoniasis, cold sores, chickenpox, measles, influenza, certain types of cancer, etc.

In a more specific embodiment, the biological sample is a nasopharyngeal or oropharyngeal swab diluted in a viral transport medium (VTM). As used herein, "viral transport medium" or "VTM" is any medium suitable for non-reproductive transport of viruses. In another specific embodiment, the sample can be diluted in any transport medium, such as a universal transport medium (UTM).

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, the sample comprises a polymerase chain reaction inhibitor, and the supernatant recovered in step (d) is substantially free of polymerase chain reaction inhibitors, particularly cations. As used herein, a "polymerase chain reaction inhibitor" is an agent that prevents nucleic acid from being amplified by the polymerase chain reaction; a "supernatant substantially free of polymerase chain reaction inhibitors" refers to a supernatant containing a polymerase chain reaction inhibitor whose concentration is sufficiently low so as not to significantly hinder the polymerase chain reaction.

In a specific embodiment of the method of the first aspect, optionally in combination with any embodiment provided above or below, the nucleic acid is selected from DNA, RNA and combinations thereof. In a more specific embodiment, the nucleic acid is genomic DNA, plasmid DNA, viral DNA, DNA obtained from a DNA amplification reaction, RNA, viral RNA, RNA obtained from an RNA amplification reaction, DNA (i.e., cDNA) reverse transcribed from an RNA sample and combinations thereof.

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, step (a) comprises mixing the nucleic acid-containing sample with an ion exchange resin, a protease and an albumin.

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, step (b) is performed directly after step (a) or (a'); and/or step (c) is performed directly after step (b) or (b').

In specific embodiments of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, the temperature in step (b) is 70°C to 110°C, 80°C to 105°C, 90°C to 100°C or about 100°C.

In the specific embodiment of the method for the first aspect, optionally in combination with any embodiment provided above or below, step (b) is carried out until the protease is inactivated and/or the sample is cracked. Those skilled in the art can perform routine tests to calculate the suitable incubation time for deactivating the protease and the cracked sample. Those skilled in the art can adjust the technical parameters to obtain the best results. For illustrative purposes, the inactivation of the protease can be measured by incubating the protease with the protein-containing sample under the conditions suitable for protease activity, then by Western blotting analysis of the reaction product.

In the specific embodiment of the method for the first aspect, optionally in combination with any embodiment provided above or below, step (b) is carried out for at least 1 minute, at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes or at least 20 minutes. In another specific embodiment, step (b) is carried out for 1 minute to 30 minutes, 5 minutes to 20 minutes, 10 minutes to 15 minutes, or about 10 minutes.

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, step (C) is carried out at a temperature of 15°C to 25°C, in particular at about room temperature.

In a specific embodiment of the method of the first aspect, optionally in combination with any embodiment provided above or below, step (c) comprises allowing the heated mixture to stand until a sediment and a supernatant are formed; or centrifuging the heated mixture until a sediment and a supernatant are formed. The higher density of the ion exchange resin will cause it to settle or settle, which can be accelerated by centrifuging the mixture. Those skilled in the art can know when the sediment and supernatant are formed by simple visual inspection.

In a specific embodiment of the method of the first aspect, optionally in combination with any embodiment provided above or below, step (c) comprises allowing to form a sediment or a sediment comprising an ion exchange resin and a supernatant comprising nucleic acids, albumin and inactivated proteases. In a more specific embodiment, step (c) comprises allowing to form a sediment or a sediment and a supernatant, wherein the supernatant is substantially free of polymerase chain reaction inhibitors.

The step (d) of reclaiming the supernatant can be carried out by any conventional method known in the art, such as decantation, filtration or pipetting. In a specific embodiment of the method of the first aspect, optionally in combination with any embodiment provided above or below, step (d) comprises reclaiming the supernatant by decantation.

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, the method further comprises a step (a') of vortexing the mixture obtained in step (a). In a more specific embodiment, the vortexing of step (a') is performed for at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds or at least 5 seconds. In a more specific embodiment, the vortexing of step (a') is performed for 1 second to 10 seconds, 2 seconds to 8 seconds or about 5 seconds.

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, the method further comprises a step (b') of vortexing the mixture obtained in step (b). In a more specific embodiment, the vortexing of step (b') is performed for at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds or at least 5 seconds. In a more specific embodiment, the vortexing of step (b') is performed for 1 second to 10 seconds, 2 seconds to 8 seconds or about 5 seconds.

In a specific embodiment of the method of the first aspect, optionally in combination with any embodiment provided above or below, the method comprises or consists of the following steps: a) contacting a nucleic acid-containing sample with an ion exchange resin, a protease and an albumin; a') vortexing the mixture obtained in step (a); b) heating at a temperature of 60°C to 120°C; b') vortexing the mixture obtained in step (b); c) allowing a sediment and a supernatant to form; and d) recovering the supernatant containing the nucleic acid.

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, the method further comprises a step (c') of purifying or isolating the nucleic acid after step (c). Any nucleic acid purification or separation method known to those skilled in the art can be used for step (c').

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, step (a) comprises contacting the nucleic acid-containing sample with a composition comprising an ion exchange resin, a protease and an albumin, and the volume ratio of the contact (nucleic acid-containing sample:composition) is 1:0.1 to 1:10, 1:0.2 to 1:8, 1:0.3 to 1:7, 1:0.4 to 1:6, 1:0.5 to 1:5, 1:0.6 to 1:4, 1:0.7 to 1:3, 1:0.8 to 1:2 or a volume ratio of about 1:1.

In a specific embodiment of the method of the first aspect, optionally in combination with any embodiment provided above or below, the method has the proviso that no nucleic acid separation or purification step is performed. In a more specific embodiment, the method has the proviso that the method does not comprise an organic solvent extraction step, particularly a phenol-chloroform extraction step or a phenol-chloroform-isoamyl alcohol extraction step.

In a specific embodiment of the method of the first aspect, optionally in combination with any of the embodiments provided above or below, the method comprises the following steps: a) contacting a nucleic acid-containing sample with a chelating resin, in particular Chelex-100; a protease, in particular proteinase K; and an albumin, in particular bovine serum albumin; b) heating at a temperature of 60°C to 120°C; c) allowing a precipitate and a supernatant to form; and d) recovering the supernatant containing the nucleic acid.

As described above, in a second aspect, the present invention provides a composition for treating a nucleic acid-containing sample, which comprises an ion exchange resin, a protease and an albumin.

In a specific embodiment of the composition of the second aspect, optionally in combination with any of the embodiments provided above or below, the composition is lyophilized.

In a specific embodiment of the composition of the second aspect, optionally in combination with any of the embodiments provided above or below, the composition further comprises a polar solvent. "Polar solvent" as used herein refers to a solvent that is miscible with water and other polar solvents. Polar solvents are well known and can be evaluated by measuring any parameter known to those skilled in the art, including dielectric constant, polarity index and dipole moment. Considering that polar solvents must be suitable for loading ion exchange resins, proteases, albumins and nucleic acids, those skilled in the art will know which polar solvent to select for the composition of the present invention.

In a specific embodiment of the composition of the second aspect, optionally in combination with any of the embodiments provided above or below, the polar solvent is water or a tampon buffer. In a more specific embodiment, the polar solvent is milli-Q water and the tampon buffer is TE buffer or CAPS. As used herein, "TE buffer" is a buffer solution commonly used in molecular biology, especially in procedures involving DNA, cDNA or RNA, which contains Tris and EDTA.

In a specific embodiment of the composition of the second aspect, optionally in combination with any of the embodiments provided above or below, the composition comprises at least one of:

- an ion exchange resin in a concentration of 4 to 99 wt %, 10 to 80 wt %, 20 to 60 wt %, 30 to 50 wt % or about 40 wt %.

- Protease in a concentration of 0.05% to 5%, 0.1% to 1%, 0.2% to 0.8%, 0.3% to 0.7% or about 0.5% by weight.

- albumin in a concentration of 0.2% to 20% by weight, 0.5% to 10% by weight, 1% to 4% by weight, 1.5% to 3% by weight or about 2% by weight,

The sum of the ingredients reaches 100%.

In a specific embodiment of the composition of the second aspect, optionally in combination with any of the embodiments provided above or below, the concentration of the ion exchange resin is from 10 wt% to 80 wt%, in particular about 40 wt%; the concentration of the protease is from 0.1 wt% to 1 wt%, in particular about 0.5 wt%; and/or the concentration of the albumin is from 0.5 wt% to 4 wt%, in particular about 2 wt%; the sum of the ingredients reaches 100%.

As used herein, the term "weight %" or "weight percent" of a component refers to the amount of a single component relative to the total weight of the composition, or if specifically mentioned, relative to the total weight of another component.

As described above, in a third aspect, the present invention provides a kit comprising an ion exchange resin; a protease; an albumin, optionally, instructions for use thereof; and optionally a PCR buffer. As used herein, "PCR buffer" refers to any buffer solution that allows DNA polymerase activity and thus produces DNA amplicons. In a more specific embodiment, the PCR buffer comprises ammonium sulfate, Tris/HCl, MgCl ₂ , Tween 20 and optionally PEG-200; in particular 26.67 mM ammonium sulfate, 111.6 mM Tris/HCl pH 8.8, 5 mM MgCl ₂ and 0.33% Tween 20.

As described above, in a fourth aspect, the present invention provides the use of ion exchange resin, protease and albumin for treating nucleic acid-containing samples. The above aspects can also be expressed as using ion exchange resin, protease and albumin to extract nucleic acid from nucleic acid-containing samples. The present invention also provides the use of the composition defined in the second aspect or the kit defined in the third aspect for treating nucleic acid-containing samples, particularly in the method defined in claim 1.

In a specific embodiment of the use provided by the fourth aspect, the sample is contacted with the ion exchange resin, the protease and the albumin simultaneously.

In specific embodiments of any of the preceding aspects, optionally in combination with any of the embodiments provided above or below, the weight ratio (weight/weight/weight) of ion exchange resin, protease and albumin (cation exchange resin/protease/albumin) is 1:0.25:2 to 1:25:200; 1:1:5 to 1:10:80; 1:2:10 to 1:5:40, or about 1:2.5:20.

In the specific embodiments of any of the aforementioned aspects, optionally in combination with any embodiment provided above or below, the ion exchange resin is a chelating resin. In even more specific embodiments, the chelating resin is selected from Chelex-100, Sepharose 2B, SP Sepharose FF, Ni Sepharose 6FF, DEAE Sepharose FF, AG 50W, AG MP-50, Bio-Rex 70, Lewatit TP 260, Diaion CR11, Ambersep M4195 and combinations thereof. In more specific embodiments, the ion exchange resin is a cation exchange resin. In more specific embodiments, the cation exchange resin is a styrene-divinylbenzene copolymer containing iminodiacetic acid groups. In more specific embodiments, the cation exchange resin is Chelex-100, particularly Chelex-100 50-100 mesh or Chelex-100 200-400 mesh. As used herein, "ion exchange resin" is a resin capable of exchanging ions, and "cation exchange resin" is a resin capable of exchanging cations. As used herein, "chelating resins" are ion exchange resins comprising a chelating agent covalently attached to a polymer matrix. These resins can be obtained from commercial sources (e.g., Chelex-100 can be obtained from BioRad or Merck, CAS 11139-85-8).

In a specific embodiment of any of the foregoing aspects, optionally in combination with any embodiment provided above or below, the protease is a proteolytic enzyme. In a more specific embodiment, the protease is selected from lysozyme, proteolytic enzyme K, trypsin and pepsin. As used herein, "protease" refers to an enzyme that degrades proteins by hydrolysis of peptide bonds. The term "proteolytic enzyme" or "endopeptidase" refers to a specific type of protease that hydrolyzes internal peptide bonds. Protease, particularly proteolytic enzyme K, can be obtained from commercial sources (e.g., proteolytic enzyme K from Merk#70663) or produced according to standard methods in molecular biology.

In a specific embodiment of any of the preceding aspects, optionally in combination with any of the embodiments provided above or below, the albumin is a serum albumin, in particular a serum albumin selected from bovine serum albumin (BSA), human serum albumin (HSA) and mouse serum albumin (MSA). In another specific embodiment, the albumin is in the form of powdered skim milk powder. Albumin, in particular BSA, HSA and MSA can be obtained from commercial sources or produced according to standard methods in molecular biology.

In specific embodiments of any of the aforementioned aspects, optionally in combination with any of the embodiments provided above or below, the ion exchange resin is a chelating resin, particularly Chelex-100; the protease is a protease, particularly protease K; and the albumin is serum albumin, particularly BSA.

In a specific embodiment of any of the foregoing aspects, optionally in combination with any of the embodiments above or below, the kit further comprises polyethylene glycol (PEG), N-acetylcysteine and/or ammonium sulfate. In another specific embodiment of any of the foregoing aspects, optionally in combination with any of the embodiments provided above or below, step (a) of the method comprises contacting the nucleic acid-containing sample with a cation exchange resin, a protease, albumin and polyethylene glycol (PEG); or treating the nucleic acid-containing sample with a cation exchange resin, a protease, albumin and ammonium sulfate; or treating the nucleic acid-containing sample with a cation exchange resin, a protease, albumin and N-acetylcysteine.

As described above, in the fifth aspect, the present invention provides a method for amplifying nucleic acid contained in a sample, comprising treating the sample using the method defined in the first aspect.

In a specific embodiment of the fifth aspect, optionally in combination with any of the embodiments provided above or below, the nucleic acid amplification is RT-PCR, PCR, qPCR, RT-qPCR, SDA, iSDA, HDA, RPA, NASBA, LAMP or EXPAR.

In a specific embodiment of the fifth aspect, optionally in combination with any of the embodiments provided above or below, the nucleic acid is RNA and the amplification reaction comprises the following steps: (i) reverse transcribing the RNA into cDNA using a reverse transcriptase, and (ii) amplifying the cDNA by PCR, qPCR, LAMP or EXPAR.

In a specific embodiment of the fifth aspect, optionally in combination with any of the embodiments provided above or below, the method further comprises a step (c) of detecting or quantifying the amplified nucleic acid.

In a sixth aspect, the present invention provides a processed nucleic acid-containing sample obtainable by the method defined in the first aspect. The nucleic acid-containing sample processed according to the method of the present invention is particularly suitable for direct nucleic acid amplification, as demonstrated by the lower Cq values shown in the following examples. The processed samples prepared according to the present invention are substantially free of nuclease activity and polymerase chain reaction inhibitors, and therefore they are excellent substrates for polymerases.

The processed nucleic acid-containing sample "obtainable" by the method as defined above is used herein to define the sample by its preparation method and relates to a sample obtained by a method comprising the above steps a), b) and c). For the purposes of the present invention, the expressions "obtainable", "obtained" and equivalent expressions are used interchangeably, and in any case the expression "obtainable" includes the expression "obtained".

In a specific embodiment of the sixth aspect, optionally in combination with any embodiment provided above or below, the treated nucleic acid-containing sample further comprises a PCR buffer. In a more specific embodiment, the PCR buffer comprises ammonium sulfate, PEG-200, Tris/HCl, MgCl ₂ and Tween 20, particularly 26.67 mM ammonium sulfate, 111.6 mM Tris/HCl pH8.8, 5 mM MgCl ₂ and 0.33% Tween 20.

All embodiments disclosed above in relation to the method of the first aspect of the invention are also intended to apply to the treated nucleic acid-containing sample of the sixth aspect of the invention.

Throughout the specification and claims, the word "comprise" and its variants are not intended to exclude other technical features, additives, ingredients or steps. In addition, the word "comprise" includes the situation of "consisting of ...". Other objects, advantages and features of the present invention will become apparent to those skilled in the art after reading the specification, or can be understood by the practice of the present invention. The following examples are provided by way of illustration, and they are not intended to limit the present invention. In addition, the present invention covers all possible combinations of the specific and preferred embodiments described herein.

Example

Example 1

200 μl of saliva sample or VTM sample (TM011, Vircell) spiked with heat-inactivated SARS-CoV-2 (ATCC, VR-1986HK) was added to a vial with 200 μl of treatment mixture containing 40 wt% Chelex 200-400 mesh (95621-100G-F Merck) and 5 mg/ml protease K (A3830.0500 Panreac Applichem) in milli-Q water with or without 2% BSA (1010-30-100ML Seqens)

The vial was vortexed for 5 seconds, then heated at 100°C for 10 minutes and vortexed again for 5 seconds. The vial was then allowed to stand until the contents were separated by gravity (i.e., the resin was allowed to settle). The supernatant containing the processed sample (i.e., nucleic acid) was recovered by decantation. The supernatant was used directly in a kit suitable for detecting infection by PCR without further purification.

As a control, the automated extraction platform ( 12gC instrument, use Dx SV kit, called MagLEAD onwards) to extract equivalent nucleic acid-containing samples.

RT-qPCR was performed using the VIASURE SARS-CoV-2 Real-Time PCR Detection Kit (Certest Biotec) according to the manufacturer's instructions. Specifically, the freeze-dried qPCR vials of the kit were rehydrated with 12 μl of rehydration buffer containing 26.67 mM ammonium sulfate, 111.6 mM Tris/HCl pH 8.8, 5 mM MgCl ₂ , and 0.33% Tween 20. Then, 8 μl of the treated sample was added to the qPCR vial. The thermal cyclers used were the CFX96 ^TM Real-Time PCR Detection System (Bio-Rad) and the AriaMx Real-Time PCR System (Agilent Technologies).

For the detection of Sars-CoV-2, genes ORF1ab and N were targeted. The primers and probes used were as follows (primers and probes for the detection of 2019-nCoV from the Chinese Center for Disease Control and Prevention, targeting 1 and 2, published on January 24, 2020):

-ORF1ab gene: Fw CCCTGTGGGTTTTACACTTAA (SEQ ID NO: 1); RvACGATTGTGCATCAGCTGA (SEQ ID NO: 2), Pr CCGTCTGCGGTATGTGGAAAGGTTATGG (SEQ ID NO: 3).

-N gene: Fw GGGGAACTTCTCCTGCTAGAAT (SEQ ID NO: 4); RVCAGACATTTTGCTCTCAAGCTG (SEQ ID NO: 5); Pr TTGCTGCTGCTTGACAGATT (SEQ ID NO: 6).

The qPCR thermal protocol used is shown in Table 1:

Table 1

FAM ^TM , ROX and Hex were used as dyes for qPCR. The target gene ORF1ab was detected in the FAM channel, the N gene was detected in the ROX channel, and the internal control was detected in the Hex channel. The qPCR results were analyzed using standard procedures with the thermal cycler software. In particular, the threshold was manually adjusted. The threshold was calculated as 10 times the standard deviation of the average baseline fluorescence signal between 3 and 15 cycles. The fluorescence signal detected above the threshold was considered to be a true signal and can be used to define the quantitative cycle (Cq) value of the sample. The threshold was manually changed in each experiment to find the exponential amplification region of all existing curves.

The Cq value is the most important parameter used in the experiment to evaluate the best reagent combination for sample treatment. Lower Cq values indicate better sample treatment (i.e., lower concentrations of contaminants or inhibitory enzymes for qPCR). Endpoint fluorescence is used in the experiment as a measure of qPCR inhibition problems. A significant drop in fluorescence can cause problems with sample detection within the limit of detection (LOD). The ΔCq value is calculated by subtracting the Cq value obtained in the reference from the Cq value obtained in the sample treated by the method of the invention (ΔCq = method of the invention - reference control).

As shown in Table 2 below, the addition of BSA to the processing buffer significantly improved the qPCR results, reducing the Cq value by 1 full cycle (Cq = 29 vs. Cq = 30 for FAM) and provided better results than samples extracted with MagLEAD (Cq = 29 vs. Cq = 29.5 for FAM).

Table 2

Concentration values given are given after adding samples to treatment buffer at a volume ratio of 1:1; *ΔCq (condition - MagLEAD); RFU refers to relative fluorescence units.

These results clearly demonstrate that BSA synergistically enhances the action of the resin and protease to remove inhibitory or degradative enzymes and other contaminants, resulting in samples that are more suitable for nucleic acid detection analysis than those obtained through laborious purification steps in expensive equipment (MagLEAD).

Example 2

The composition of the treatment buffer was varied, and the samples were treated and analyzed as described in Example 1. In particular, BSA was replaced by spermidine (S0266-1G), sodium lauryl sarcosinate (L7414-50ML), or PEG (BioUltra, 200, 88440-250ML-F). As shown in Table 3, only BSA enhanced the effect of the resin and protease. The addition of other reagents failed to improve the effect of the resin and protease, as shown by the decrease in RFU and the increase in Cq values:

Table 3

Concentration values given are given after adding samples to treatment buffer at a 1:1 volume ratio; *ΔCq (condition - MagLEAD).

Example 3

To forgo the improved qPCR results obtained by adding BSA as a result of the action of BSA during the qPCR reaction rather than during sample processing, samples were processed and analyzed as described in Example 1 with the addition of BSA to the processing buffer or qPCR buffer (ie, after processing).

As shown in Table 4 below, only the addition of BSA to the treatment buffer (row 6) significantly improved the qPCR results (ie, Cq = 29 vs. Cq = 30.56). This effect was not observed when BSA was added to the qPCR buffer.

Table 4

The concentration values given are given after adding the samples to the treatment buffer at a volume ratio of 1:1; *ΔCq (condition - control (rehydration buffer)). DILBM refers to 16.16 mM ammonium sulfate, 67.63 mM Tris/HCl pH 8.8, 3 mM MgCl ₂ and 0.2% Tween 20.

These results clearly demonstrate that BSA acts synergistically during sample processing to produce a nucleic acid-containing supernatant that is highly suitable for further nucleic acid detection.

Example 4

The same procedure described in Example 1 was repeated at VTM with authentic SARS-CoV-2 nasopharyngeal swabs. The primers and probes used for amplification were the same as in Example 1. The qPCR results obtained with samples treated according to the method of the present invention and control samples extracted with MagLEAD are shown in Table 5:

Table 5

Concentration values given are given after adding samples to treatment buffer at a 1:1 volume ratio; *ΔCq (condition - MagLEAD).

These results demonstrate that real clinical samples contaminated with SARS-CoV-2 treated with the methods of the present invention provide significantly better viral detection results compared to samples treated with buffer without albumin, and notably, provide better detection results than samples treated with MagLEAD.

Example 5

The method of the present invention was used to process various types of samples contaminated with different microorganisms, including viruses and bacteria, and provided qPCR results that were equivalent to or even better than those of MagLEAD.

Example 6

The method of the present invention is further used to process biological samples contained in different buffers, including several commercial buffers commonly used in clinical practice. Table 6 below shows successful nucleic acid detection in samples processed by the method of the present invention, without relying on the buffer used for sample collection, according to the method described in Example 1 above.

Table 6

*ΔCq (condition-MagLEAD).

Example 7

As described in Example 1, the method of the invention was performed using different proteases, ion exchange resins and albumin.

As shown in Table 7 below, the same results were obtained when mouse albumin or skimmed milk powder was used instead of BSA. Similarly, the proteases lysozyme and trypsin and the chelating resin Ni Sepharose 6FF also provided good results.

Table 7

The given concentration values were obtained after adding the samples to the treatment buffer in a 1:1 volume ratio.

In summary, these results demonstrate that the method of the present invention can be successfully performed with different ion exchange resins, proteases and albumin.

Example 8

The method of the present invention was further used to treat different food samples spiked with Listeria monocytogenes or Salmonella according to the protocol described above in Example 1. Table 8 below shows that the method of the present invention can also be used to treat food samples and that BSA enhances the action of resins and proteases in food samples regardless of the type of food sample and pathogen detected.

Table 8

Example 9

The method of the present invention was also used to process various types of clinical samples as described in Example 1. Table 9 below shows the successful detection of all samples processed by the method of the present invention.

Table 9

Example 10

The samples (saliva samples spiked with heat-inactivated SARS-CoV-2) were processed and analyzed as described in Example 1, but the composition of the processing buffer was changed. The results in Table 10 below clearly show that BSA and protease K synergistically enhance the effect of the exchange resin (column 1 vs. column 2 to column 4), which results in unexpected improvements in subsequent nucleic acid detection.

Table 10

Claims (15)

1. A method for processing a nucleic acid-containing sample, comprising the steps of:

a) Contacting the nucleic acid-containing sample with an ion exchange resin, a protease, and albumin;

b) Heating at a temperature of 60 ℃ to 120 ℃;

c) Allowing sediment and supernatant to form; and

D) Recovering the supernatant containing the nucleic acid.

2. The method of claim 1, wherein the sample is selected from the group consisting of whole blood, plasma, serum, urine, saliva, sputum, nasal mucus, respiratory lavage, tears, chorionic villus, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid, stool, prostatic fluid, semen, lymph fluid, bile, sweat, breast milk, breast fluid, non-human embryonic cells, non-human fetal cells, human or non-human stem cells, cultured bacteria, cultured yeast, tissue biopsies, tissue swabs, cervical cytology, synovial fluid, food, feed, soil, agricultural products, water, surface swabs, surface washes, and combinations thereof.

3. The method of any one of claims 1-2, wherein the nucleic acid is DNA, RNA, or a combination thereof.

4. A method according to any one of claims 1 to 3, wherein:

-the temperature in step (b) is from 70 ℃ to 110 ℃, in particular from 80 ℃ to 105 ℃;

-the heating in step (b) is carried out for 1 to 30 minutes, in particular 5 to 20 minutes; and/or

-Step (c) comprises allowing the heated mixture to stand until a sediment and a supernatant are formed; or centrifuging the heated mixture until a sediment and supernatant are formed.

5. The method of any one of claims 1 to 4, further comprising:

-a step (a') of swirling the mixture obtained in step (a); and/or

-A step (b') of swirling the heated mixture obtained in step (b).

6. A composition for treating a nucleic acid-containing sample comprising an ion exchange resin, a protease, and albumin.

7. The composition of claim 6, which is lyophilized.

8. The composition according to claim 6, further comprising a polar solvent, in particular water.

9. The composition according to any one of claims 6 to 8, wherein:

The concentration of the ion exchange resin is from 10% to 80% by weight, in particular about 40% by weight;

the concentration of protease is from 0.1% to 1% by weight, in particular about 0.5% by weight; and/or

The concentration of albumin is from 0.5% to 4% by weight, in particular about 2% by weight.

10. A kit, comprising:

-an ion exchange resin;

-a protease;

-an albumin protein, which is present in the mixture,

-Optionally, instructions for use thereof; and

-Optionally, PCR buffer.

11. Use of ion exchange resins, proteases and albumin for the treatment of nucleic acid containing samples.

12. The method as defined in any one of claims 1 to 5, the composition as defined in any one of claims 6 to 9, the kit as defined in claim 10 or the use as defined in claim 11, wherein the weight ratio (weight/weight) of ion exchange resin, protease and albumin is from 1:1:5 to 1:10:80; in particular 1:2:10 to 1:5:40.

13. The method as defined in any one of claims 1 to 5 and 12, the composition as defined in any one of claims 6 to 9 and 12, the kit as defined in any one of claims 10 and 12 or the use as defined in any one of claims 11 to 12, wherein:

the ion exchange resin is a chelating resin, in particular Chelex-100;

-the protease is a proteolytic enzyme, in particular proteinase K; and/or

-Albumin is selected from Bovine Serum Albumin (BSA), human Serum Albumin (HSA) and Mouse Serum Albumin (MSA).

14. A method for amplifying nucleic acid from a nucleic acid-containing sample, comprising the steps of:

a) Treating the sample with a method as defined in any one of claims 1 to 5, thereby obtaining a supernatant containing nucleic acids; and

B) Amplifying the nucleic acid contained in the supernatant.

15. The method of claim 14, wherein the amplification in step (b) is selected from RT-PCR, PCR, qPCR, RT-qPCR, SDA, iSDA, HDA, RPA, NASBA, LAMP and EXPAR.