WO2022029219A1

WO2022029219A1 - Methods for rapid and sensitive detection of nucleic acids

Info

Publication number: WO2022029219A1
Application number: PCT/EP2021/071854
Authority: WO
Inventors: Damien Yann Marie-Joseph Thomas; Sébastien LHOSPICE; Younes LAZRAK
Original assignee: C4Diagnostics
Priority date: 2020-08-05
Filing date: 2021-08-05
Publication date: 2022-02-10

Abstract

The present invention relates to a novel in vitro method for rapid detection of nucleic acids. More particularly, the present invention relates to an improved loop-mediated isothermal amplification method, which is particularly useful for the fast and effective detection of a specific micro-organism in a sample.

Description

METHODS FOR RAPID AND SENSITIVE DETECTION OF NUCLEIC ACIDS

INTRODUCTION

Three coronaviruses have crossed the species barrier to cause deadly pneumonia in humans since the beginning of the 21st century: severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV- 2. SARS-CoV-2, was discovered in December 2019 in Wuhan, Hubei province of China, and its genome has been fully sequenced in early January 2020. The SARS-CoV-2 is a coronavirus responsible for the emergence of a new respiratory infectious disease called Covid- 19 that has caused a global pandemic, which led the World Health Organization to declare this disease a public health emergency of international concern in early January 2020. Without being bound by theory, Covid- 19 is thought to be mainly transmitted by air, with a high reproduction rate of between 2 and 3 in the absence of control and prevention measures. The severity of the clinical signs of the disease varies greatly from one individual to another, with a preponderance of severe forms in elderly individuals or those suffering from other diseases. The scientific data available to date estimate that about 30% to 60% of infected subjects are asymptomatic but can transmit the disease. In this context, it is essential to be able to carry out diagnostic tests on a massive scale in order to limit the spread of the virus.

In view of this public health emergency, the scientific community developed SARS-CoV- 2 diagnostic tests that are essentially based on semi -quantitative detection of circulating SARS- CoV-2 RNA by conventional real-time reverse transcription-polymerase chain reaction assay (RT-PCR). These laboratory -based molecular diagnostic tests are still routinely used to this day but are resource-intensive, and it may take 24 hours for a patient to know the results of the test, making these tests less readily available for the masses.

In light of the need for rapid and ultrasensitive assay, some scientists have more recently investigated an alternative to the standard PCR tests: the loop-mediated isothermal amplification (LAMP). Loop-mediated isothermal amplification (LAMP) is a single-tube method of specific isothermal amplification of a nucleotide sequence with incorporation of a fluorescent nucleic acid stain during amplification (Li & Macdonald, Biosensors and Bioelectronics, 2015, Vol. 64, pp. 196-211; Nagamine et al., Molecular and Cellular Probes, 2002, 16(3), 223-229; Notomi et al., Nucleic Acids Research, 2000, Vol. 28, Issue 12; Yan et al., Molecular BioSystems, Royal Society of Chemistry, 2014, Vol. 10, Issue 5, pp. 970-1003). Compared to traditional detection methods such as Polymerase Chain Reaction (PCR), LAMP has the particularity to amplify a target sequence with two to three sets of primers at a constant temperature of 65°C (no temperature cycling), and is more specific and faster (Aebischer et al., Journal of Clinical Microbiology, 2014, 52(6), 1883-1892). LAMP is also advantageous as it produces more amplicons than traditional PCR and is less sensitive to polymerase inhibitors that are naturally present in biological matrices (Chander et al., Frontiers in Microbiology, 2014, https://doi.org/10.3389/fmicb.2014.00395; Francois et al., FEMS Immunology and Medical Microbiology, 2011, 62(1), 41-48; Nie et al., PLoS ONE, 2012, 7(12), https://doi.org/10.1371/journal.pone.0052486 2012). In short, LAMP can efficiently produce a high yield of DNA, in a very short time period (less than 30 minutes, while PCR typically requires more than Ih).

Because of its unique properties, LAMP is a promising technology for the detection of pathogenic micro-organisms that are highly infectious, such as SARS-CoV-2, and that require a fast identification of infected subjects and/or contaminated environments so as to prevent further spread and future infectious waves. In such context, LAMP can provide a rapid and robust diagnostic test, conductible in the field and at local point-of-care (POC) centers, without the requirement of specialized equipment and/or highly trained professionals to interpret the results.

However, the few SARS-CoV-2 LAMP tests developed to this day typically require the extraction and purification of the nucleic acid material from the test samples prior to the amplification, thereby leading to a loss of genomic material and thus to potential false negative results. One such test that recently reached the market requires a pre-treatment of the sample by (i) an extraction of viral RNA with chemical lysis and precipitation by chaotropic agents (RNA extraction), followed by (ii) a neutralization of the amplification inhibitors that may be present in the lysate, and (iii) a subsequent filtration of the lysate to remove aggregates of cell debris and reagents precipitated during neutralization, prior to (iv) performing the RT-LAMP amplification. The present Inventors have developed a fast-tracked LAMP method that can be performed from a “dirty” swab environmental or biological sample that is minimally processed, and thus does not require to fully isolate and purify the nucleic acid prior to amplification. This method proved to be not only faster, but also better in limit of detection compared to the methods performed on a manually purified nucleic acid sample. This novel method can be used to detect any micro-organism at point of care, such as SARS-CoV-2.

SUMMARY OF THE INVENTION

The present invention relates to an in vitro method of detecting the presence of a microorganism in a sample, comprising or consisting of: a) contacting a raw sample with a basic buffer, a denaturing detergent, a chaotropic agent or an amplification enhancer, or any combination thereof; and/or pre-heating the raw sample; b) selectively amplifying the DNA or RNA of the micro-organism from the pre-treated sample of step a) by a loop-mediated isothermal amplification (LAMP) or a reversetranscription loop-mediated isothermal amplification (RT-LAMP); and c) detecting the amplification product of step b), thereby detecting the presence of the micro-organism in the sample.

In a preferred embodiment, no purification step that is directed at purifying DNA or RNA is carried out, especially prior to step b).

In a preferred embodiment, the basic buffer is used under conditions sufficient for the pH of the pre-treated sample of step a) to reach a pH from about 8 to about 9.

In a preferred embodiment, the denaturing detergent is a non-ionic surfactant, preferably polysorbate such as polysorbate 20.

In a preferred embodiment, the chaotropic agent is guanidinium thiocyanate.

In a preferred embodiment, the amplification enhancer is bovine serum albumin.

In a preferred embodiment, the pre-heating of the raw sample is performed at about 60°C to about 100°C.

In a preferred embodiment, the pre-heating in step a) is carried out simultaneously, prior to or after contacting the raw sample with the basic buffer, the denaturing detergent, the chaotropic agent or the amplification enhancer, or any combination thereof. In a preferred embodiment, the micro-organism to be detected is a virus or a bacterium. For example, a preferred virus to be detected according to the invention is a coronavirus, such as SARS-CoV-1, SARS-CoV-2, MERS, preferably is SARS-CoV-2.

In a preferred embodiment, the sample is an environmental sample or a biological sample. For example, the biological sample can be a sample from a subject, preferably a saliva sample, a nasal sample, a nasopharyngeal sample, a sputum sample, or an oropharyngeal sample, especially when the micro-organism is SARS-CoV-2. Alternatively, the environmental sample can be a sample from a surface or from air, especially when the micro-organism is SARS-CoV- 2.

In a preferred embodiment, the method of the invention further comprises, prior to step a), pre-treating the raw sample with a proteinase K, especially when the sample is a biological sample. According to this embodiment, step a) preferably comprises optionally contacting the sample with a basic buffer, and pre-heating the sample. More preferably, said method further comprises, prior to step b), the filtration of the pre-treated sample of step a).

In a preferred embodiment, step b) of the method of the invention comprises contacting the pre-treated sample of step a) with a set of loop-mediated isothermal amplification primers specific for the micro-organism under conditions sufficient for amplification of the DNA or RNA of the micro-organism, wherein the set of primers comprises at least a forward inner primer (FIP), a forward outer primer, a backward inner primer (BIP) and a backward outer primer, thereby producing an amplification product.

In a preferred embodiment, when the micro-organism is SARS-CoV-2, the set of primers is capable of detecting the RdRp gene encoding the RNA-dependent RNA polymerase and the N gene encoding the nucleoprotein of SARS-CoV-2.

LEGENDS TO THE FIGURES

Figure 1. LAMP method according to the invention, using a basic pH.

Figure 2. LAMP method according to the invention, using a denaturing detergent or a chaotropic agent.

Figure 3. LAMP method according to the invention, using an amplification enhancer.

Figure 4. LAMP method according to the invention, using a pre-heating step. Figure 5. LAMP method according to the invention, using a proteinase K pre-treatment and a pre-heating step.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, nomenclatures used herein, and techniques of molecular biology are those well-known and commonly used in the art.

The present invention may be more readily understood by reference to the following detailed description, including preferred embodiments of the invention.

Samples and micro-organisms to be detected

The term "sample" means any sample containing at least a nucleic acid, such as DNA and/or RNA. In the context of the present invention, the sample can more particularly be a biological sample or an environmental sample. For example, the biological sample can be a sample from a subject, preferably a saliva sample, a nasal sample, a nasopharyngeal sample, a sputum sample, an oropharyngeal sample, a blood sample, a bronchial aspirate sample, a bronchoalveolar lavage sample, a urine sample, or a feces sample, more preferably a saliva sample, a nasal sample, a nasopharyngeal sample, a sputum sample, or an oropharyngeal sample. A saliva sample is particularly preferred as it allows for a self and painless collection by the tested subject. The term “subject” encompasses herein any animal, preferably a mammal, more preferably a human. Said subject may be asymptomatic. Alternatively, the environmental sample can be a sample from a surface or from air, especially when the micro-organism to be detected can be transmitted by touching a surface or is an airborne micro-organism.

The sample according to the invention is a raw sample. By “raw sample", it means herein that the sample can contain not only the micro-organism to be detected, but also other biological material such as cells and/or environment impurities, depending on the nature of the sample collected. The raw sample can be collected according to conventional methods in the art, such as by applying a swab on a surface or on a nasopharyngeal mucosa, nasal mucosa (etc.), or by collecting aerosols (Verreault et al., Microbiol Mol Biol Rev . 2008 Sep; 72(3): 413-444). The micro-organism to be detected may be a virus or a bacterium. A particularly preferred virus to be detected according to the invention is a coronavirus, such as SARS-CoV-1, SARS- CoV-2, MERS, preferably SARS-CoV-2.

Pre-treatment step a)

The method according to the invention requires a minimal pre-treatment of the raw sample by: a) contacting the raw sample with a basic buffer, a denaturing detergent, a chaotropic agent or an amplification enhancer, or any combination thereof; and/or pre-heating the raw sample. This step a) ensures that the DNA or RNA of the micro-organism becomes sufficiently accessible to the amplification reagents so as to allow detection of the micro-organism, while also lowering the activity of RNAses, DNAses and polymerase inhibiting elements that may be present in the sample.

In a preferred embodiment, no purification step that is directed at purifying the DNA or RNA is carried out, especially prior to step b). Indeed, standard nucleic acid detection methods typically require to isolate pure DNA or RNA from a raw sample prior to the amplification step. The omission of such step in the present method allows the fast-track of the nucleic acid detection, while providing a sufficient sensitivity to detect micro-organism loads in the sample. For example, according to this embodiment, there is no need to carry out (i) an DNA or RNA extraction via e.g. chemical lysis and precipitation by chaotropic agents, followed by (ii) a neutralization of amplification inhibitors that may be present in the lysate, and (iii) a subsequent filtration of the lysate to e.g. remove aggregates of cell debris and reagents precipitated during neutralization.

In a preferred embodiment, the basic buffer is used under conditions sufficient for the final pH of the pre-treated sample of step a) to reach a pH from about 8 to about 9. For example, the basic buffer is a buffer having a pH from about 10 to about 12.

In a preferred embodiment, the denaturing detergent is a non-ionic surfactant such as polysorbate, preferably polysorbate 20. For example, the non-ionic surfactant is polysorbate 20, at a final concentration of about 0.05 to about 0.5%, preferably of about 0.4%, in the pre-treated sample of step a).

In a preferred embodiment, the chaotropic agent is guanidinium thiocyanate. In a preferred embodiment, the amplification enhancer is bovine serum albumin. For example, the amplification enhancer is bovine serum albumin, at a final concentration of about 0.2 mg/ml to about 5 mg/ml, preferably of about 2 mg/ml, in the pre-treated sample of step a).

In a preferred embodiment, the pre-heating of the raw sample is performed at about 60°C to about 100°C. For example, the pre-heating of a raw sample, especially of an environmental sample, can be performed at about 60°C to 90°C, preferably at 65°C, preferably for about 2 to about 20 minutes, more preferably for about 10 to about 15 minutes. In another example, the pre-heating of a raw sample, especially of a biological sample that has been pre-treated with a proteinase K as further detailed below, can be performed at about 60°C to 95°C, preferably at 95°C, preferably for about 2 to about 10 minutes, more preferably for about 5 minutes.

The above preferred embodiments may be performed independently from each other, or combined together.

For example, in a preferred embodiment, the method comprises: a) contacting the raw sample with a basic buffer.

For example, in another preferred embodiment, the method comprises: a) contacting the raw sample with a denaturing detergent or a chaotropic agent.

For example, in another preferred embodiment, the method comprises: a) contacting the raw sample with an amplification enhancer.

For example, in another preferred embodiment, the method comprises: a) contacting the raw sample with any combination of a basic buffer, a denaturing detergent, a chaotropic agent or an amplification enhancer.

For example, in another preferred embodiment, the method comprises: a) pre-heating the raw sample.

For example, in another preferred embodiment, the method comprises: a) contacting the raw sample with a basic buffer, a denaturing detergent, a chaotropic agent or an amplification enhancer, or any combination thereof; and pre-heating the raw sample. According to this latter embodiment, the pre-heating in step a) can be carried out simultaneously, prior to or after contacting the raw sample with the basic buffer, the denaturing detergent, the chaotropic agent or the amplification enhancer, or any combination thereof.

When the sample is a biological sample, additional steps may be required so as to enhance the detection of the micro-organism. More particularly, it may be desirable to inactivate nucleases (RNAses, DNAses) that may be present in the sample, using for example an enzyme such as a proteinase K.

Thus, in a preferred embodiment, the method further comprises, prior to step a): pretreating the raw sample with a proteinase K, especially when the sample is a biological sample. Such pre-treatment may be performed by contacting the raw sample with a proteinase K at room temperature, preferably for at least 1 to 5 minutes, more preferably for 1 to 2 minutes.

In a preferred embodiment, when the sample is pre-treated with proteinase K, the method comprises: a) pre-heating the sample. To do so, the pre-treated sample may be heated as described above, for example at 95°C for about 5 minutes.

In a preferred embodiment, when the sample is pre-treated with proteinase K, the method comprises: a) contacting the sample with a basic buffer, and pre-heating the sample. For example, the basic buffer may comprise borate buffered saline, Bovine Serum Albumin (BSA) and Ethylenediaminetetraacetic acid (EDTA), and preferably be at pH9. The basic buffer may be used to dilute the sample pre-treated with proteinase K, for example at a 1/2 ratio.

Still, in a preferred embodiment, when the sample is pre-treated with proteinase K, the method further comprises, between steps a) and b), the filtration of the pre-treated sample of step a). For example, the filtration may be performed by passing the pre-treated sample of step a) through a 0.1 to 0.5 pm filter, such as a 0.22 pm filter.

The amplification step b)

Following the sample pre-treatment in step a), the method according to the invention produces amplicons specific to the micro-organism by: b) selectively amplifying the DNA or RNA of the micro-organism from the pre-treated sample of step a) by a loop-mediated isothermal amplification (LAMP) or a reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

The term “amplification” or “amplifying” refers herein to a process that increases the representation of a population of specific nucleic acid sequences in a sample by producing multiple (i.e., at least 2) copies of the desired sequences. A “copy” or “amplicon” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.

A loop-mediated isothermal amplification (LAMP) assay amplifies DNA by relying on an autocyclic strand displacement reaction, which is performed at a constant temperature, usually between 60°C and 65°C (Notomi T. et al., Nucleic acids Res. 2000, Vol. 28(12)-e63). To do so, this well-known technology employs a DNA polymerase with strand displacement activity, and typically a set of four oligonucleotides, termed inner and outer primers, specifically designed to recognize six different recognition sites on the target nucleic acid. When RNA is to be amplified, the assay is referred as a reverse-transcription loop-mediated isothermal amplification (RT-LAMP), as it requires the additional presence of a reverse transcriptase to generate cDNA.

The DNA polymerase used in a LAMP or RT-LAMP assay can preferably be selected from the group consisting of Bst large fragment polymerase, Bst 2.0, Bst 3.0, Bea (exo-), Vent, Vent (exo-), Deep Vent, Deep Vent (exo-), 29 phage, MS-2 phage, Z-Taq, KOD, Klenow fragment, GspSSD, GspF, OmniAmp Polymerase, SD Polymerase and any combination thereof.

The outer primers play a role in strand displacement during the non- cyclic step only while the inner primers include both sense and antisense sequences and contribute to formation of typical LAMP or RT-LAMP amplification products having stem-loop structures.

Thus, in a preferred embodiment, step b) comprises contacting the pre-treated sample of step a) with a set of loop-mediated isothermal amplification primers specific for the microorganism under conditions sufficient for amplification of the DNA or RNA (i.e. cDNA) of the micro-organism, wherein the set of primers comprises at least a forward inner primer (FIP), a forward outer primer, a backward inner primer (BIP) and a backward outer primer, thereby producing an amplification product.

For example, when the micro-organism is SARS-CoV-2, the set of outer and inner primers is capable of detecting the RdRp gene encoding the RNA-dependent RNA polymerase (T target) and the N gene encoding the nucleoprotein (E target) of SARS-CoV-2.

In addition to the four oligonucleotide primers, the LAMP or RT-LAMP assay may involve the use of two additional primers, so-called loop primers, to improve amplification efficiency, thereby resulting in a total of six primers per target sequence. Such a combination of different primers, which span eight distinct sequences on the target nucleic acid, provides for a remarkable degree of assay specificity.

The detection step c)

Following the amplification of DNA or RNA in step b), the method according to the invention detects the presence of the micro-organism in the tested sample by: c) detecting the amplification product of step b).

To this end, several methods can be employed for the detection of LAMP or RT-LAMP amplification products including visual examination or turbidity monitoring of precipitated magnesium pyrophosphate, fluorescence detection of double-stranded DNA (dsDNA) with an intercalating fluorophore, bioluminescence reporting through pyrophosphate conversion.

Typical strategies for the indirect detection of LAMP or RT-LAMP amplification that can be used in the present invention rely on the formation of pyrophosphate as a reaction byproduct. As LAMP or RT-LAMP reactions proceed, pyrophosphate ions are released by incorporation of deoxynucleotide triphosphates (dNTPs) into the DNA strand during nucleic acid polymerization and these ions react with divalent metal ions, particularly magnesium ions, present in the reaction mix to produce a white, insoluble magnesium pyrophosphate precipitate. This product results in a progressive increase in the turbidity of the reaction solution and pyrophosphate precipitates can be measured quantitatively in terms of turbidity or observed by the naked eye as a pellet after centrifugation. In an alternative embodiment, the detection of LAMP or RT-LAMP amplification is achieved through the incorporation of manganese ions and calcein in the reaction. Calcein's fluorescence is naturally quenched by binding of manganese ions. Pyrophosphate production as a by-product of LAMP or RT-LAMP reaction removes manganese ions form the buffer through precipitation, and the increased turbidity coupled with restored calcein fluorescence enables an easy visual read-out upon excitation with either visible or UV light. In still another detection embodiment, the enzymatic conversion of pyrophosphate into ATP, which is produced during DNA synthesis, is monitored through the bioluminescence generated by thermostable firefly luciferase.

LAMP or RT-LAMP amplification products may also be detected through direct approaches which act via fluorescence reporting. The majority of such approaches are based on the use of intercalating dyes, such as ethidium bromide, SYBR Green, EvaGreen and YO-PRO- I. Typically, intercalating dyes are non-sequence-specific fluorescent dyes that exhibit a large increase in fluorescence emission upon binding into dsDNA. Such property may be used to monitor the nucleic acid amplification in real time by continuously measuring the fluorescence during the LAMP or RT-LAMP reaction. Fluorescence-based detection of LAMP or RT-LAMP amplification may also rely on the mechanism of Forster resonance energy transfer (FRET).

Among detection methods based on fluorescence energy transfer, hybridization-induced fluorescence quenching has also been exploited in LAMP or RT-LAMP applications, particularly through the principle of guanine quenching. In such approach, the fluorescence emitted by a 5' labeled loop-mediated isothermal amplification loop primer is progressively quenched upon hybridization to a complementary target sequence containing a guanine. The extent of the quenching effect depends on the number and positions of the adjacent G bases on the complementary target sequence. As target sequences accumulate in a real-time LAMP or RT-LAMP assay, measurements of nucleic acid amplification may be achieved by monitoring the amount of quenched fluorescence as a consequence of the incorporation of the dye-labeled loop primer in the amplification products.

The present invention will be better understood in light of the following detailed experiments. Nevertheless, the skilled artisan will appreciate that the present examples are not limitative and that various modifications, substitutions, omissions, and changes may be made without departing from the scope of the invention.

EXAMPLES

RAPID DETECTION OF SARS-CoV-2

The following molecular diagnostic method allows the rapid detection of SARS-CoV-2 by a RT-LAMP technology targeting 2 genes of SARS-CoV-2. This method can be directly performed from a raw biological sample taken e.g. on a swab, and thus does not require the isolation of pure RNA prior to amplification. It can be completed in at most 30 minutes, from sampling to result, and its clinical performance shows high sensitivity and specificity compared to conventional RT-q-PCR. It can be applied to any “dirty” biological sample containing DNA or RNA, and is thus not limited to the detection of SARS-CoV-2. MATERIALS AND METHODS

Sample collection

The swab was soaked into water and applied on an environmental surface (e.g. 10x10 cm surface), or on human tissue (e.g. saliva, nasal or nasopharyngeal sample), so as to collect a sample to be analyzed. Human tissue samples tested herein were collected from over a thousand symptomatic or contact-cases subject.

LAMP reaction

5 distinct protocols were performed, as detailed below. Protocols 1) to 4) were applied to an environmental sample, while protocol 5) was applied to a biological sample (herein, a saliva or nasal sample).

Protocol 1) based on a basic pH (Figure 1) lOOpl of basic buffer pHlO was directly added into a tube containing 150pL of a LAMP master Mix (ISO-004 master mix from Optigene). The swab was then discharged into the tube, followed by vortexing. 25 pL of the mixture was subsequently transferred into each reaction micro-tube which contains all the reagents to perform the RT-LAMP reaction (external, internal and loop primer mixture at final concentrations of 200, 1600 and 800 nM, respectively; and 5 pL of RNA template): 3 micro-tubes corresponding respectively to (1) a positive control (C+),

(2) T target (gene common to all SARS viruses), and (3) E target (gene specific to SARS-CoV- 2). The RT-LAMP amplification was performed at 65°C, for about 5 to 20 minutes depending on the viral load of the sample being analyzed.

Protocol 2) based on a denaturing detergent or chaotropic agent (Figure 2) lOOpl of a solution of polysorbate 20 (Tween20) 0.4% was directly added into a tube containing 150pL of a LAMP master Mix (ISO-004 master mix from Optigene). The swab was then discharged into the tube, followed by vortexing. 25 pL of the mixture was subsequently transferred into each reaction micro-tube which contains all the reagents to perform the RT- LAMP reaction (external, internal and loop primer mixture at final concentrations of 200, 1600 and 800 nM, respectively; and 5 pL of RNA template): 3 micro-tubes corresponding respectively to (1) a positive control (C+), (2) T target (gene common to all SARS viruses), and

(3) E target (gene specific to SARS-CoV-2). The RT-LAMP amplification was performed at 65°C, for about 5 to 20 minutes depending on the viral load of the sample being analyzed. Protocol 3) based on an amplification enhancer (Figure 3)

1 OO .1 of a Bovine Serum Albumin (400 ng/L) was directly added into a tube containing 150pL of a LAMP master Mix (ISO-004 master mix from Optigene). The swab was then discharged into the tube, followed by vortexing. 25 pL of the mixture was subsequently transferred into each reaction micro-tubes which contains all the reagents to perform the RT-LAMP reaction (external, internal and loop primer mixture at final concentrations of 200, 1600 and 800 nM, respectively; and 5 pL of RNA template): 3 micro-tubes corresponding respectively to (1) a positive control (C+), (2) T target (gene common to all SARS viruses), and (3) E target (gene specific to SARS-CoV-2). The RT-LAMP amplification was performed at 65°C, for about 5 to 20 minutes depending on the viral load of the sample being analyzed.

Protocol 4) based on a pre-heating step (Figure 4)

The swab was discharged in a tube containing 500 pl to ImL of water, and said tube was then pre-heated at 65°C. 100 pL of the heated sample solution was then transferred into a tube containing 150pL of a LAMP master Mix (ISO-004 master mix from Optigene), followed by vortexing. 25 pL of the mixture was subsequently transferred into each reaction micro-tubes which contains all the reagents to perform the RT-LAMP reaction (external, internal and loop primer mixture at final concentrations of 200, 1600 and 800 nM, respectively; and 5 pL of RNA template): 3 micro-tubes corresponding respectively to (1) a positive control (C+), (2) T target (gene common to all SARS viruses), and (3) E target (gene specific to SARS-CoV-2). The RT- LAMP amplification was performed at 65°C, for about 5 to 20 minutes depending on the viral load of the sample being analyzed.

Protocol 5) based on using a proteinase K pre-treatment and pre-heating step (Figure 5) 300pL of the saliva or nasal sample was transferred into an Eppendorf tube, to which 15 pL of proteinase K (50 mg/ml) was added. The tube was then vortexed and incubated at room temperature for 1 to 2 minutes, so that the proteinase K inactivates nucleases in the saliva sample. 300pL of a basic buffer pH9 comprising 0.2 M BBS (borate buffered saline), 8 mg/mL BSA and 2mM EDTA was subsequently added to the tube, and the resulting mix was heated at 95°C for about 5 minutes so as to inactivate the proteinase K. The pre-treated mix was then filtered via a 0.22 pm filter. 100 pL of the filtrated mix was directly transferred into a tube containing 150pL of a LAMP master Mix (ISO-004 master mix from Optigene), followed by vortexing. 25 pL of the mixture was subsequently transferred into each reaction micro-tubes which contains all the reagents to perform the RT-LAMP reaction (external, internal and loop primer mixture at final concentrations of 200, 1600 and 800 nM, respectively; and 5 pL of RNA template): 3 micro-tubes corresponding respectively to (1) a positive control (C+), (2) T target (gene common to all SARS viruses), and (3) E target (gene specific to SARS-CoV-2). The RT- LAMP amplification was performed at 65°C, for about 5 to 20 minutes depending on the viral load of the sample being analyzed.

Protocol 5) has been identified by the inventors as the optimized protocol for analyzing biological samples. Said protocol may nevertheless be simplified by including at least the treatment with proteinase K and the pre-heating step that inactivates said enzyme. Statistical methods

Analyses were conducted using GraphPad Prism 9.1.2 software (LA Jolla, CA, USA). The nonparametric Mann-Whitney test was used to compare the distribution of positive sample from NP RT-qPCR and saliva RT-LAMP. P-values less than 0.05 were considered significant.

RESULTS The analysis of the results was performed according to the criteria set forth in Table 1.

Table 1.

Table 2. SARS-CoV-2 LAMP detection according to Protocol 1. The virus was discharged either in buffer pHlO or water from a clean or dirty swab (*: based on an extraction yield of 100%).

Table 3. SARS-CoV-2 LAMP detection according to Protocol 2.

No interference was detected between the Tween and the LAMP reaction.

Table 4. SARS-CoV-2 LAMP detection according to Protocol 3 (*: based on an extraction yield of 100%).

No interference was detected between the BSA and the LAMP reaction. Table 5. SARS-CoV-2 LAMP detection according to Protocol 4. The virus was discharged in ImL of water from a clean or dirty swab.

Table 6. SARS-CoV-2 LAMP detection according to Protocol 5 (Sp: specificity; Se: sensibility).

Similar results were obtained whether the biological sample was a saliva or nasal sample. Preclinical results based on protocol 5) showed that i) sensitivity of the test reaches 100% at low Ct values (19 to 21), and that ii) specificity of that test reaches 100% regardless of the nature of the biological sample (e.g. nasopharyngeal or saliva sample). By contrast, conventional assays with RT-qPCR do not reach such specificity level: false positives were reported (Little et al., Radiology 2021, E160-E1612021), as well as positivity in saliva sample concomitant to a negative test from a nasopharyngeal sample (Azzi et al., Oral Diseases 2020. doi: 10.1111/odi.13368; Medeiros da Silva et al., Travel Med Infect Dis 2020, 38:101920).

Each of the above protocols 1 to 5 can be performed in under less than 20 to 30 minutes.

Claims

CLAIMS . An in vitro method of detecting the presence of a micro-organism in a sample, comprising: a) contacting a raw sample with a basic buffer, a denaturing detergent, a chaotropic agent or an amplification enhancer, or any combination thereof; and/or pre-heating the raw sample; b) selectively amplifying the DNA or RNA of the micro-organism from the pretreated sample of step a) by a loop-mediated isothermal amplification (LAMP) or a reverse-transcription loop-mediated isothermal amplification (RT- LAMP); and c) detecting the amplification product of step b), thereby detecting the presence of the micro-organism in the sample. The method according to claim 1, wherein no purification step that is directed at purifying DNA or RNA is carried out, especially prior to step b). The method according to claim 1 or 2, wherein the basic buffer is used under conditions sufficient for the pH of the pre-treated sample of step a) to reach a pH from about 8 to about 9. The method according to any one of the preceding claims, wherein the denaturing detergent is a non-ionic surfactant, preferably polysorbate, more preferably polysorbate 20. The method according to any one of the preceding claims, wherein the chaotropic agent is guanidinium thiocyanate. The method according to any one of the preceding claims, wherein the amplification enhancer is bovine serum albumin. The method according to any one of the preceding claims, wherein the pre-heating of the raw sample is performed at about 60°C to about 100°C. 8. The method according to any one of the preceding claims, wherein the pre-heating in step a) is carried out simultaneously, prior to or after contacting the raw sample with the basic buffer, the denaturing detergent, the chaotropic agent or the amplification enhancer, or any combination thereof.

9. The method according to any one of the preceding claims, wherein the microorganism is a virus or a bacterium.

10. The method according to claim 9, wherein the virus is a coronavirus, such as SARS- CoV-1, SARS-CoV-2, MERS, preferably is SARS-CoV-2.

11. The method according to any one of the preceding claims, wherein the sample is an environmental sample or a biological sample, preferably an environmental sample.

12. The method according to any one of the preceding claims, wherein the biological sample is a sample from a subject, preferably a saliva sample, a nasal sample, a nasopharyngeal sample, a sputum sample, or an oropharyngeal sample, especially when the micro-organism is SARS-CoV-2.

13. The method according to any one of the preceding claims, wherein the environmental sample is a sample from a surface or from air, especially when the micro-organism is SARS-CoV-2.

14. The method according to any one of the preceding claims, further comprising, prior to step a), pre-treating the raw sample with a proteinase K, especially when the sample is a biological sample.

15. The method according to claim 14, wherein step a) comprises: optionally contacting the sample with a basic buffer, and pre-heating the sample.

16. The method according to claim 14 or 15, further comprising, prior to step b), the filtration of the pre-treated sample of step a). 17. The method according to any one of the preceding claims, wherein step b) comprises contacting the pre-treated sample of step a) with a set of loop-mediated isothermal amplification primers specific for the micro-organism under conditions sufficient for amplification of the DNA or RNA of the micro-organism, wherein the set of primers comprises at least a forward inner primer (FIP), a forward outer primer, a backward inner primer (BIP) and a backward outer primer, thereby producing an amplification product. 18. The method according to any one of the preceding claims, wherein, when the microorganism is SARS-CoV-2, the set of primers is capable of detecting the RdRp gene encoding the RNA-dependent RNA polymerase and the N gene encoding the nucleoprotein of SARS-CoV-2.