WO2022189784A1

WO2022189784A1 - Nucleic acid amplification, kits, methods, and uses

Info

Publication number: WO2022189784A1
Application number: PCT/GB2022/050608
Authority: WO
Inventors: Jing Zhang
Original assignee: Phoenix Dx Ltd
Priority date: 2021-03-10
Filing date: 2022-03-09
Publication date: 2022-09-15
Also published as: GB202203292D0; GB2607391A

Abstract

The present invention relates to kits for use in the isothermal amplification of nucleic acids in a sample comprising a liquid transport medium for pathogens, the kit comprising: a chelating agent and a primer mix. The invention also relates to the use of agents able to improve nucleic acid amplification by isothermal techniques. The methods of the invention may be used in the detection of pathogens, including viruses such as a coronavirus. In addition, the invention relates to methods of isothermal amplification of nucleic acids, and to methods of detecting SARS-CoV-2 in vitro.

Description

NUCLEIC ACID AMPLIFICATION, KITS, METHODS, AND USES

FIELD OF THE INVENTION

The present invention relates to kits for use in the isothermal amplification of nucleic acids. The invention also relates to the use of agents able to improve nucleic acid amplification by isothermal techniques. In addition, the invention relates to methods of isothermal amplification of nucleic acids, and to methods of detecting SARS-CoV-2 in vitro.

INTRODUCTION

Control of diseases, such as the COVID-19 pandemic caused by the coronavirus SARS-CoV- 2, relies upon the ability to rapidly and accurately detect the presence of pathogens in clinical samples. The amplification and detection of specific nucleic acid sequences representative of pathogens of interest has recently become a particularly important approach to such detection programmes.

There are several types of clinical samples that can be used in traditional tests for upper respiratory viruses. Swabs (throat, nasal, and combined throat-nasal swabs), washes (nasal and throat washes), aspirates (mainly nasopharyngeal aspirates), serum (obtained from a blood sample), and tissues (tissues of the up and lower respiratory tract mainly of lungs, bronchus and trachea). Most of the samples can be preserved in the viral transport medium (VTM) after the collection. Different commercial VTM have many acceptable formulations that are suitable for different conditions of individual laboratories. In most of the cases, VTM can contain one or more of the following components: cell culture medium, salt solution, tryptose- phosphate broth, veal infusion broth, protein (bovine albumin or gelatin), antibiotics, and antimycotics.

VTM is very important for the successful diagnosis of viruses, where the key factors influencing success are the quality of the specimen and the conditions for the specimen transportation and storage before being processed in the laboratory. VTM allows the safe transfer of viruses and other pathogens (such as chlamydia and mycoplasma) for further research, including conventional cell culture methods, diagnostic tests, and molecular biology techniques.

During the COVID-19 outbreak, VTM has been widely used for the clinical sample collections and written in the standard procedure of clinical sample collection in WHO and CDC recommended instructions. However, samples comprising VTM have proven to inhibit the effectiveness of certain nucleic acid amplification techniques, particularly isothermal nucleic acid amplification techniques, such as loop-mediated isothermal amplification (LAMP). Since isothermal nucleic acid amplification techniques would otherwise constitute very desirable approaches to be used in the detection of pathogens, there is a need to provide conditions and reagents that render these techniques suitable for use with samples such as VTM.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 sets out effect of VTM on LAMP. Increasing concentration of VTM has an inhibitory effect on detection of SARS-CoV-2 virus by LAMP as seen in the delayed time to detection.

Figure 2 sets out effect of different formulation of VTM, with and without calcium, on LAMP performance. SARS-CoV-2 LAMP reactions with VTM formulations containing Ca²⁺ (VTM 2, VTM 4, VTM 5, VTM 7, VTM 12, and VTM 13) had a delayed Td compared to reactions without Ca²⁺.

Figure 3 sets out reduction of inhibitory effect of VTM on LAMP by EGTA. Different concentrations of EGTA were tested in LAMP reactions, with or without VTM. Adding EGTA to the reaction mixture containing VTM reduces detection time of SARS-CoV-2 RNA down to almost the same level as that of LAMP without VTM.

Figure 4 sets out determining optimal concentration of EGTA with different amounts of VTM. SARS-CoV-2 LAMP reaction with 50% and 25% VTM were tested with varying concentrations of EGTA.

Figure 5 sets out validation of effect of 2 mM EGTA on LAMP with different concentrations of VTM.

Figure 6 sets out EGTA reduces inhibitory effect of different types of VTM. Inactivated SARS- CoV-2 virus was resuspended in different commercial VTM formulations (25% v/v) and tested with LAMP reactions containing 2 mM EGTA. All reactions containing VTM showed similar performance to those without VTM. Figure 7 sets out testing VTM spiked with different amounts of SARS-CoV-2 virus. VTM or nuclease-free water were spiked with different amounts of heat-inactivated SARS-CoV-2 virus and used as template for LAMP reactions containing 2 mM EGTA.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a kit for use in the isothermal amplification of nucleic acids in a sample comprising a liquid transport medium for pathogens, the kit comprising:

• a chelating agent; and

• a primer mix.

According to a second aspect of the invention, there is provided the use of a chelating agent as an additive to an isothermal nucleic acid amplification reaction mixture comprising a sample of a liquid transport medium for pathogens, to improve amplification of nucleic acids.

According to a third aspect of the invention, there is provided a method of isothermal amplification of nucleic acids, the method comprising:

• a step of providing a sample of a liquid transport medium for pathogens;

• a step of providing a primer set for amplifying nucleic acids present in the transfer medium sample;

• a step of performing an isothermal nucleic acid amplification reaction using the sample and primer set; wherein the isothermal nucleic acid amplification reaction is performed in the presence of a chelating agent.

The methods of the third aspect of the invention may be used in the detection of pathogens. Suitably such a pathogen may be a virus, such as a coronavirus. As demonstrated in the Examples, the methods of the third aspect of the invention are particularly suitable for use in the detection of coronavirus SARS-CoV-2.

The use of the methods of the third aspect of the invention in the detection of SARS-CoV-2 is so beneficial that it gives rise to a fourth aspect of the invention, which provides a method of detecting SARS-CoV-2 in vitro, the method comprising:

• a step of providing a sample of a liquid transport medium for pathogens from a subject requiring SARS-CoV-2 detection; • a step of providing a primer set for amplifying SARS-CoV-2 nucleic acids present in the sample;

• a step of performing an isothermal nucleic acid amplification reaction using the sample and primer set;

• a step of comparing the results of the nucleic acid amplification reaction with data indicative of the presence or absence of SARS-CoV-2; and

• determining whether or not the subject has a SARS-CoV-2 infection on the basis of this comparison; wherein the nucleic acid amplification reaction is performed in the presence of a chelating agent.

Kits according to the first aspect of the invention are suitable to be employed in the uses of the second aspect of the invention, or in the methods of the third or fourth aspects of the invention.

The kits, uses or methods of the invention may make use of at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof, as considered in more detail below.

Detailed description

The present invention is based upon the inventors’ finding that chelating agents, such as EGTA, can be used to improve methods of isothermal amplification of nucleic acids carried out on samples comprising liquid transport medium for pathogens. This observation gives rise to a number of advantages that offer significant benefits in clinical practice.

As discussed elsewhere in the specification, transport media (such as viral transport medium, or universal transport medium) are regularly used for the transfer to testing sites of samples, such as swabs, suspected of containing pathogens. During the COVID-19 outbreak, VTM has been widely used in methods for the collection of clinical samples, and its use has been specified in procedures carried out in accordance with World Health Organisation (WHO) and Center for Disease Control (CDC) guidelines.

Unfortunately, transport media appear to disrupt the ability of techniques for isothermal amplification of nucleic acids. These techniques (exemplified by loop-mediated isothermal amplification - LAMP) allow efficient and rapid amplification of nucleic acids under simple conditions. As such, the methods are well-suited to use in point of care contexts, such as diagnostic or disease surveillance applications. However, the inhibitory effects of transport media, such as VTM, have, to date, had an adverse impact upon the capacity to use these techniques in practice.

When seeking to overcome this problem, the inventors have found that chelating agents, and particularly chelating agents that preferentially bond to calcium ions, are able to improve amplification of nucleic acids by such isothermal techniques. In particular, and as discussed further below, the inventors have found that the kits, uses and methods of the invention, all of which employ chelating agents such as EGTA, are able to increase both the rate of amplification by such reactions and the sensitivity of the reactions. It will be appreciated that these advantages are broadly applicable, and are particularly beneficial in the context of diagnostic uses of isothermal nucleic acid amplification techniques.

While some of the effects of the kits, uses and methods of the invention may arise from the chelating agents reversing the inhibitory activity of constituents of the transport media, the results that they have achieved indicate that this is not the sole mode of action. Surprisingly, the inventors have found that the kits, uses and methods of the invention are able to improve isothermal nucleic acid amplification techniques carried out in the presence of a sample of a liquid transport medium for pathogens to the extent that such methods are even more effective than when the same methods are carried out in the absence of such a sample.

DEFINITIONS

The following provides some definitions that may be usefully considered when practicing the invention. References in the following paragraphs to “the present invention” should be interpreted as relating to any appropriate aspect of the invention (kits, uses, or methods).

By the same token, except for where the context requires otherwise, features described with respect to one of the aspects of the invention (whether kits of the first aspect of the invention, uses of the second aspect of the invention, or methods of the third or fourth aspects of the invention) should be taken as also being disclosed and applicable to the other aspects of the invention set out herein. For example, features described in connection with the kits of the invention should be taken as applicable also to the uses of the invention, or methods of the third or fourth aspects of the invention. Similarly, features described in connection with the uses of the invention should be taken as applicable also to the kits or methods of the invention, and features described in connection with a method of the invention should be taken as applicable to the “other” method of the invention, and to the kits or uses of the invention

Isothermal amplification of nucleic acids

Techniques for the isothermal amplification of nucleic acids allow nucleic acid populations to be expanded without the need for temperature cycling during the performance of the method. This offers a number of significant advantages as compared to techniques that require thermal cycling of samples in order to achieve amplification. Among the advantages of isothermal techniques are their improved simplicity. These techniques may be performed in a single tube (which reduces the potential for contamination of samples - which is important in diagnostic contexts), and can generally be performed for relatively low cost. The simplicity and ruggedness of isothermal amplification techniques means that they are also well suited to use outside laboratory settings. This makes them particularly suitable for use in point of care applications. Furthermore, isothermal amplification techniques are generally rapid, avoiding undue delays, and making these techniques suitable for use in contexts in which it is necessary to undertake high numbers of tests.

The skilled person will be aware of a number of different techniques for isothermal amplification of nucleic acids that may potentially be utilised in the context of the present invention. Without limitation, a suitable technique for isothermal amplification of nucleic acids may be selected from the group consisting of: loop-mediated isothermal amplification (LAMP); helicase-dependent amplification (HDA); rolling circle amplification (RCA); multiple displacement amplification (MDA); recombinase polymerase amplification (RPA); and nucleic acid sequence-based amplification (NASBA).

There are ample resources available to allow the skilled person to select or design appropriate reagents for the application of these techniques to contexts such as diagnostic uses, for example in the diagnosis of coronavirus diseases such as COVID-19, caused by SARS-CoV- 2.

The inventors have shown that the present invention is particularly effective in the context of LAMP. Information provided below and in the Examples, as well as the common general knowledge, will allow the skilled person to select appropriate reagents and reaction conditions to allow the present invention to be used in LAMP techniques. Isothermal nucleic amplification reactions take place using a reaction mixture. In the context of the present invention, the reaction mixture will comprise at least in part, a sample of a liquid transport medium for pathogens.

Sample

A sample in the context of the present invention may be any sample capable of providing information regarding the presence or absence of nucleic acids of interest. Typically, the sample may be a clinical sample, such as a clinical sample collected by swab (for example, throat, nasal, or combined throat-nasal swabs), lavage (for example nasal and throat washes), aspirate (mainly nasopharyngeal aspirates), blood samples (for the collection of serum), or tissue samples (for example tissue biopsies of the up or lower respiratory tract mainly of lungs, bronchus and trachea).

The nucleic acids of interest may be nucleic acids associated with a pathogen, such as a viral, chlamydia, mycoplasma, or ureaplasma pathogen. In a suitable embodiment, such a pathogen is a virus, for example a coronavirus, such as SARS-CoV-2.

Liquid transport media for pathogens

Clinical samples, such as swabs, aspirates or the like, suspected of containing pathogens will typically be placed in liquid transport media for collection and transfer to testing sites, as well as maintenance and storage of the samples. Suitable liquid transfer media may be selected with reference to the nature of the suspected pathogen.

Suitable examples of liquid transport media that may be utilised in embodiments of the various methods, kits or uses of the invention include, but are not limited to, those selected from the group consisting of: viral transport medium (VTM); and universal transport medium (UTM).

In a suitable embodiment, the transfer medium is selected from the group consisting of: Centers for Disease Control (CDC) VTM; Gibco Viral Transport Medium; MWE Virocult VTM; Copan Universal Transport Medium; Remel MicroTest™ M4RT™; BD universal viral transport; Biocomma VTM; and EO Laboratories VTM.

VTM may be utilised in instances in which a sample is suspected of containing viral, chlamydia, or mycoplasma pathogens. UTM may be utilised in instances in which a sample is suspected of containing viral, chlamydia, mycoplasma, or ureaplasma pathogens.

The liquid transport medium for pathogens may comprise a source of calcium ions. That said, the inventors have found that the invention is also effective in the context of media that lack calcium ions.

As set out elsewhere, the inventors have found that the invention allows isothermal amplification of nucleic acids to be achieved in samples containing relatively high concentrations of transfer medium samples, such as VTM samples or UTM samples. This is advantageous in that it reduces the degree of dilution of the original sample that is necessary prior to practice of the chosen nucleic acid amplification technique. Since the original sample is the source of the nucleic acids of interest, the ability to reduce dilution provides advantages in that the starting concentration of nucleic acids is increased, even prior to amplification.

The invention may be practiced effectively in embodiments in which the transfer medium sample constitutes up to 25% of the reaction mixture by volume. The invention may be practiced effectively in embodiments in which the transfer medium sample constitutes up to 26%, up to 27%, up to 28%, up to 29%, up to 30%, up to 31%, up to 32%, up to 33%, up to 34%, up to 35%, up to 36%, up to 37%, up to 38%, up to 39%, up to 40%, up to 41%, up to 42%, up to 43%, up to 44%, or up to 45% of the reaction mixture by volume. Indeed, the invention may be practiced effectively in embodiments in which the transfer medium sample constitutes up to 46%, up to 47%, up to 48%, up to 49%, or even up to 50% of the reaction mixture by volume.

In a suitable embodiment, the transfer medium sample constitutes between approximately 5% and approximately 50% of the total reaction mixture by volume. For example, the transfer medium sample may constitute between approximately 10% and approximately 40% of the total reaction mixture by volume, between approximately 12% and approximately 35% of the total reaction mixture by volume, between approximately 15% and approximately 30% of the total reaction mixture by volume, or between approximately 20% and approximately 27% of the total reaction mixture by volume.

Suitably, the transfer medium sample may constitute at least 5% of the total reaction mixture by volume. For example, the transfer medium sample may constitute at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% of the total reaction mixture by volume. In a suitable embodiment, the sample of liquid transfer medium for pathogens constitutes approximately 25% of the total reaction mixture by volume.

For the avoidance of doubt, the embodiments considered in the preceding paragraphs are applicable to the first, second, third and fourth aspects of the invention.

Chelating agents

The invention makes use of a chelating agent in the kits, uses and methods described herein. The skilled person will be aware of a large number of chelating agents.

Suitably he chelating agent preferentially binds to calcium ions. In the context of the present invention, this should be taken as indicating that the binding affinity of the chelating agent is higher for calcium ions than for other metal ions. The skilled person will be well aware of methods by which binding affinity may be investigated in practice. In a suitable embodiment, the chelating agent may bind exclusively to calcium ions.

In a suitable embodiment, the chelating agent is selected from the group consisting of: EGTA (ethylene g!ycol-bis^-aminoethyl ether)-A/,/V,,V,A/'-tetraacetic add, also known as egtazic add); BAPTA (1,2-bis(o-aminophenoxy)ethane-/\/,/\/,/\/',/\/'-tetraacetic acid); 5,5’-Dimethyl- BAPTA; Tetraacetoxymethyl Bis(2-aminoethyl) Ether N,N,N',N'-Tetraacetic Acid; FURA-2 pentapotassium salt; FLUO 3, Pentaammonium Salt; and INDO 1 pentapotassium salt.

The inventors have found that EGTA is a particularly useful chelating agent that may be employed in embodiments of the various aspects of the invention.

The chelating agent may be provided in an amount sufficient to effectively sequester calcium ions present in the sample of transport medium present within an isothermal nucleic acid amplification reaction mixture.

Suitably, the chelating agent (such as EGTA) is provided in an amount sufficient to achieve a concentration of between 0.5 mM and 8 mM of the chelating agent in an isothermal nucleic acid amplification reaction mixture. For example, the chelating agent may be provided in an amount sufficient to achieve a concentration of between 1 mM and 6 mM, or between 1.5 mM and 4 mM, of the chelating agent in the isothermal nucleic acid amplification reaction mixture. Suitably, the chelating agent is provided in an amount sufficient to achieve a concentration of approximately 2 mM of the chelating agent (such as EGTA) in the isothermal nucleic acid amplification reaction mixture.

These embodiments set out in the preceding paragraphs are, except for where the context requires otherwise, applicable to the first, second, third and fourth aspects of the invention.

Primer mixes and primer sets

The kits of invention comprise a primer mix. A primer mix may also be employed in producing reaction mixtures made use of in the uses or methods of the invention.

A suitable primer mix may be prepared based upon the isothermal nucleic acid amplification technique to be used, and based upon the target nucleic acids to be amplified.

For example, in the case that LAMP is to be used for isothermal nucleic acid amplification, a suitable primer mix will comprise LAMP primers. In the case that HDA is to be used for isothermal nucleic acid amplification, a suitable primer mix will comprise HDA primers. If RCA is to be used for isothermal nucleic acid amplification, a suitable primer mix will comprise RCA primers. If MDA is to be used for isothermal nucleic acid amplification, a suitable primer mix will comprise MDA primers. In the case of RPA being used for isothermal nucleic acid amplification, a suitable primer mix will comprise RPA primers, and in the case of that NASBA is to be used for isothermal nucleic acid amplification, a suitable primer mix will comprise NASBA primers.

Similarly, in the case that isothermal nucleic acid amplification is to be used to amplify viral nucleic acids, the primer mix should comprise primers for viral nucleic acids. In the case that the nucleic acids to be amplified are chlamydia nucleic acids, the primer mix should comprise primers for chlamydia nucleic acids. When the nucleic acids to be amplified are mycoplasma nucleic acids, the primer mix should comprise primers for mycoplasma nucleic acids, and when the nucleic acids to be amplified are ureaplasma nucleic acids, the primer mix should comprise primers for ureaplasma nucleic acids.

Suitably, the primer mix comprises primers capable of amplifying coronavirus nucleic acids. In particular, a suitable primer mix may comprise primers, such as LAMP primers, for SARS- CoV-2. It will be appreciated that, in the context of the present disclosure, references to nucleic acids of pathogens (such as SARS-CoV-2) where the primary nucleic acids are RNA should also be taken as encompassing nucleic acids complementary to the SARS-CoV-2 nucleic acids. Similarly, primer mix suitable for amplification of such nucleic acids should also be taken as encompassing mixes that comprise primers for the amplification of nucleic acids of the pathogen (such as SARS-CoV-2), or for the amplification of nucleic acids complementary to those of the pathogen (such as SARS-CoV-2).

Many isothermal nucleic acid amplification techniques, such as LAMP, require the use of sets of primers in which different primers perform different roles in the amplification. LAMP primer sets require the presence of: a forward inner prime (FIP); a backward inner primer (BIP); a forward outer primer (F3); and a backward outer primer (B3). Typically, LAMP primer sets also comprise loop primers (a forward loop primer, FL, and a backward loop primer, BL) which increase the rated of the reaction.

In the context of the present invention, a primer mix may comprise one or more primer sets for amplifying nucleic acids. In such an embodiment, the provision of a primer set in a method of the invention may be achieved by providing a suitable primer mix of the sort described herein. In particular, a primer mix in accordance with the present invention may comprise a primer set of the sort described below.

The inventors have generated a set of LAMP primers for SARS-CoV-2 that are particularly effective. These are set out in SEQ ID NOs: 1 to 6, and this primer set was used to generate the results set out in the Examples. In a suitable embodiment of the present invention, a primer mix comprises at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

It will be appreciated that one or more of the primers of SEQ I D NOs: 1 to 6 may comprise one or more modifications while still providing effective for the amplification of nucleic acids associated with SARS-CoV-2. Accordingly, the invention may also make use of primer sets that comprise at least one primer having a base sequence selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

The base sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6 are set out in Table 1 below:

Table 1

In the context of the present invention, a primer “having a base sequence” according to a recited sequence (e.g. any one of SEQ ID NOs: 1 to 6, or their variants) may comprise the sequence in question. Thus, a primer set of the invention may comprise at least one primer comprising a base sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6. For example, a primer set of the invention may comprise at least one primer comprising the base sequence of SEQ I D NO: 1 , and/or at least one primer comprising the base sequence of SEQ I D NO: 2, and/or at least one primer comprising the base sequence of SEQ ID NO: 3, and/or at least one primer comprising the base sequence of SEQ ID NO: 4, and/or at least one primer comprising the base sequence of SEQ ID NO: 5, and/or at least one primer comprising the base sequence of SEQ ID NO: 6.

In a suitable embodiment, a primer “having a base sequence” according to a recited sequence may consist of the sequence in question. In keeping with this, it will be recognised that a primer set of the invention may comprise at least one primer consisting of a base sequence selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6. For example, a primer set of the invention may comprise at least one primer consisting of the base sequence of SEQ I D NO: 1 , and/or at least one primer consisting of the base sequence of SEQ ID NO: 2, and/or at least one primer consisting of the base sequence of SEQ ID NO: 3, and/or at least one primer consisting of the base sequence of SEQ ID NO: 4, and/or at least one primer consisting of the base sequence of SEQ ID NO: 5, and/or at least one primer consisting of the base sequence of SEQ ID NO: 6.

For the purposes of the present disclosure, a variant of a recited sequence may incorporate one, two, or three alterations in the base sequence as compared to the recited sequence. Thus a primer set of the invention may comprise at least one primer that is a variant of SEQ ID NO: 1 that varies from the base sequence of SEQ ID NO: 1 by one, two, or three alterations; and/or may comprise at least one primer that is a variant of SEQ ID NO: 2 that varies from the base sequence of SEQ ID NO: 2 by one, two, or three alterations; and/or may comprise at least one primer that is a variant of SEQ ID NO: 3 that varies from the base sequence of SEQ ID NO: 3 by one, two, or three alterations; and/or may comprise at least one primer that is a variant of SEQ ID NO: 4 that varies from the base sequence of SEQ ID NO: 4 by one, two, or three alterations; and/or may comprise at least one primer that is a variant of SEQ ID NO: 5 that varies from the base sequence of SEQ ID NO: 5 by one, two, or three alterations; and/or may comprise at least one primer that is a variant of SEQ ID NO: 6 that varies from the base sequence of SEQ ID NO: 6 by one, two, or three alterations.

Constituents of primer sets suitable to be employed in the kits, uses and methods of the invention

A suitable primer set may comprise at least one primer having a base sequence selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof. A primer set of the invention may comprise multiple primers meeting these criteria.

A suitable primer set may comprise at least two primers having base sequences selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

Suitably, a primer set may comprise at least three primers having base sequences selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

For example, a primer set may comprise at least four primers having base sequences selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof. In a suitable embodiment a primer set may comprise at least five primers having base sequences selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

Indeed, a primer set may comprise at least six primers having base sequences selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

A suitable primer set may comprise a mixture of primers having base sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and of primers having base sequences selected from the group consisting of: a variant of SEQ ID NO: 1 , a variant of SEQ ID NO: 2, a variant of SEQ ID NO: 3, a variant of SEQ ID NO: 4, a variant of SEQ ID NO: 5, and a variant of SEQ ID NO: 6.

Suitably a primer set may comprise each of SEQ ID NOs: 1 to 6.

In a suitable embodiment, a primer set comprises SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof, at a ratio of approximately 1:1:4:4:2:2.

A primer set may comprise SEQ ID NO: 1 or a variant thereof in an amount sufficient to produce a concentration of approximately 0.2 mM in a LAMP reaction mixture; SEQ ID NO: 2 or a variant thereof in an amount sufficient to produce a concentration of approximately 0.2 pM in a LAMP reaction mixture; SEQ ID NO: 3 or a variant thereof in an amount sufficient to produce a concentration of approximately 0.8 pM in a LAMP reaction mixture; SEQ ID NO: 4 or a variant thereof in an amount sufficient to produce a concentration of approximately 0.8 pM in a LAMP reaction mixture; SEQ ID NO: 5 or a variant thereof in an amount sufficient to produce a concentration of approximately 0.4 pM in a LAMP reaction mixture; SEQ ID NO: 6 or a variant thereof in an amount sufficient to produce a concentration of approximately 0.4 pM in a LAMP reaction mixture.

Except for where any context requires otherwise, the consideration set out in the preceding paragraphs may be employed in all kits, uses or methods of the present invention that require the use of a primer mix or a primer set for the amplification of nucleic acids associated with SARS-CoV-2.

Optional constituents of kits of the invention or reaction mixtures

The uses and methods of the invention employ reaction mixtures to perform isothermal amplification of nucleic acids. The kits of the invention provide suitable means by which some or all of the constituents of such reaction mixtures may be provided. Accordingly, in the following paragraphs, considerations set out in respect of the kits of the invention should also be taken as applicable to the reaction mixtures employed in the uses or methods of the invention, and considerations set out in relation to these reaction mixtures should also be taken as applicable to the kits of the invention and their constituents.

In addition to the constituents required by the statements of invention set out above, the kits of the invention, or reaction mixtures employed in the uses or methods of the invention, may comprise one or more further components selected from the group consisting of:

• a dNTP mixture;

• a buffer solution;

• a DNA polymerase;

• a reverse transcriptase;

• a nucleic acid dye;

• a preservative;

• positive control nucleic acid; and

• distilled water.

The skilled person will be able to select appropriate examples of many of these components on the basis of the isothermal nucleic acid amplification technique that is to be used.

By way of non-limiting example, the buffer solution may comprise a Tris buffer. Such a buffer may comprise one or more component from the group consisting of: Tris-HCI, (NH4)2S04, KCI, MgS04, and Tween 20. Indeed, a suitable buffer solution may comprise Tris-HCI, (NH4)2S04, KCI, MgS04, and Tween 20, as discussed further below. An appropriate DNA polymerase may be selected so that it is effective in the chosen isothermal nucleic acid amplification technique. In a suitable example the DNA polymerase may be a Bst polymerase, for example Bst 2.0 DNA polymerase, as considered below.

In a suitable embodiment, the nucleic acids to be amplified are RNA nucleic acids. In this case, a reverse transcriptase may be used to allow generation of corresponding DNA, which can then be amplified. Commercially available reverse transcriptases, such as WarmStart® RTx, may be used in such embodiments.

Amplified nucleic acids generated may be visualised by any suitable nucleic acid dye. Merely by way of example, a suitable dye may be an intercalating dye. In a suitable example of such an embodiment, the dye is SYTO 62.

The selection of components set out above are all suitable for use in combination in embodiments in which LAMP is the selected isothermal nucleic acid amplification technique.

A suitable positive control nucleic acid may be selected with a view to the target nucleic acids to be amplified. For example, in the case that the nucleic acids to be amplified are pathogen nucleic acids, the positive control nucleic acids may be provided in the form of nucleic acids extracted from the pathogen, or in the form of inactivated pathogens comprising the nucleic acids. In the case of a virus, such as SARS-CoV-2, the positive control nucleic acid may be provided in the form of a sample of inactivated virus.

Any suitable preservative may be used increase the shelf-life or stability of a kit of the invention. Suitable preservatives may be selected with reference to the conditions in which it is desired that the kit be preserved. Suitably, the preservative is selected from the group consisting of: a cryopreservative; and a lyopreservative.

A kit of the invention, such as one described in any of the embodiments set out above, may be provided in lyophilised form. For example, a suitable kit of the invention may be provided in the form of lyophilised beads. Kits of the invention in accordance with these embodiments are stable at room temperature for prolonged periods without the need for cold chain transport or storage. Furthermore, they provide significant advantages in ease of use, in that they need only be reconstituted in a predetermined volume of a suitable liquid, such as a sample of a liquid transport medium for pathogens, in order to provide a reaction mixture that comprises requisite constituents at concentration well suited to perform the required amplification reaction. In such embodiments, a lyophilised bead may comprise all required constituents of a kit of the invention. Alternatively, the constituents of a kit of the invention may be provided in separate beads (each bead comprising one or more of the necessary constituents).

Suitably the constituents of a kit of the invention may be integrated into a test device, or a component thereof (such as a microfluidic cartridge). Kits of the invention in lyophilised form are particularly suitable for use in such embodiments. For example, a kit of the invention in the form of lyophilised beads may be provided in a microfluidic cartridge that can be used in a method of the invention.

A suitable embodiment of a kit of the invention for use in LAMP is provided in the form of lyophilised beads. These may each be of approximately 3 mm diameter. The beads contain all the components of a reaction mixture (save for the target nucleic acids) to allow amplification of nucleic acids by means of a LAMP reaction. The beads, and hence the reaction mixture, are reconstituted with a suitable volume (for example, 25 pi) of the liquid transfer medium sample containing the target nucleic acid.

Kits of the invention (such for example in the form of lyophilised beads of the sort considered in the embodiment above), or reaction mixtures to be employed in the uses or methods of the invention, may comprise some or all of the constituents in the list set out below.

• EGTA;

• a primer mix comprising a set of primers for SARS-CoV-2;

• a dNTP mixture;

• a buffer solution comprising Tris-HCI, (NH4)2S04, KCI, MgS04, and Tween 20;

• a Bst DNA polymerase;

• a WarmStart ® RTx reverse transcriptase;

• SYTO 62 nucleic acid dye; and

• an excipient such as a cryopreservative or lyopreservative.

In use, the beads are reconstituted in the liquid sample, and incubated at a constant temperature for a desired time (suitably at least 30 minutes). Presence of the target nucleic acids leads to their amplification, and the accumulation of double stranded DNA. This generates a fluorescent signal produced by the intercalating dye present in the bead. In a suitable embodiment, a reaction mixture to be employed in the uses or methods of the invention, may comprise some or all of the constituents set out in the following list at the concentrations referred to. In a suitable embodiment a kit of the invention (for example in the form of lyophilised beads) may comprise some or all of the constituents set out in the following list in proportions such that when the kit is used (for example by reconstitution of lyophilised beads) to produce a reaction mixture, the constituents are present within the reaction mixture at the concentrations referred to.

• dNTPs at a concentration of 1 mM or approximately 1 mM,

• Tris-HCI at a concentration of 20 mM or approximately 20 mM,

• (NhU^SCUat a concentration of 10 mM or approximately 10 mM,

• KCI at a concentration of 50 mM or approximately 50 mM,

• MgSCUat a concentration of 6 mM or approximately 6 mM,

• Tween^® 20at a concentration of 0.1 % or approximately 0.1 %,

• 0.2 mM F3 primer, 0.2 pM B3 primer, 0.8 pM FIP primer, 0.8 pM BIP primer, 0.4 pM LF primer, 0.4 pM LB primer,

• Bst 2.0 DNA polymerase at a concentration of 320 U/pl or approximately 320 U/pl,

• WarmStart® RTx Reverse Transcriptase at a concentration of 300 U/pl or approximately 300 U/pl,

• SYTO 62 at a concentration of 2.5 pM or approximately 2.5 pM,

• EGTA at a concentration of 2 mM or approximately 2 mM, and

• an excipient such as a cryopreservative or lyopreservative at a concentration of 3% or approximately 3%.

Each of these embodiments may suitably comprise a primer set comprising at least one primer having a base sequence selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof. Each of these embodiments may suitably comprise a primer set comprising at least one primer selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6. References to the F3, B3, FIP, BIP, LF and LB primers may be construed with reference to these primers within the set of SEQ ID NOs: 1 to 6. Further considerations regarding the primer mixes or sets that may be used can be found elsewhere in the specification. The lists provided above are not to be considered exhaustive, and the skilled person will appreciate replacement constituents or further components that may be used in the kits of the invention or reaction mixtures employed in the uses or methods of the invention.

“Improving” amplification of nucleic acids

As set out above, the invention is based upon the inventors’ finding that the addition of a chelating agent, such as EGTA, to an isothermal nucleic acid amplification reaction mixture, comprising a sample of a liquid transport medium for pathogens, is able to improve amplification of nucleic acids within the reaction mixture. This improvement may be identified or assessed with respect to any appropriate parameter, including any one or more selected from the following.

An improvement in nucleic acid amplification may be demonstrated by a reduction in the time taken for an amplification reaction to generate a predetermined quantity of amplified nucleic acid (“rate of amplification reaction”).

Alternatively, or additionally, an improvement in nucleic acid amplification may be demonstrated by a reduction in the lowest concentration of a nucleic acid of that may be used to generate a detectable quantity of amplified nucleic acid (“limit of detection”).

Without being bound by any hypothesis, the inventors believe that the kits, uses or methods of the invention employing a chelating agent (such as EGTA) achieve some of their effect by removing the inhibition of isothermal nucleic acid amplification techniques (such as LAMP) that may otherwise arise as the result of the presence in the reaction mixture of a sample of a liquid transport medium for pathogens. The capacity of an aspect or embodiment of the invention to bring about such a removal of inhibition may be investigated and assessed (and quantified, if desired) by a skilled person using any suitable methods known to them.

However, the inventors’ results (set out in Figure 6, and discussed in the Examples) have surprisingly demonstrated that isothermal nucleic amplification reactions (such as LAMP) carried out in the presence of both a sample of a liquid transport medium for pathogens and a chelating agent (such as EGTA) are able to generate a predetermined amount of amplified nucleic acids in less time than comparable control reaction carried out in the absence of a sample of such a medium. This is a truly surprising finding, in that it clearly indicates that the kits, uses and methods of the invention are not simply removing inhibition caused by constituents of the sample, but that they are themselves having a direct positive effect upon the rate of the isothermal nucleic acid amplification reaction. Thus, in a suitable embodiment, kits, uses or methods of the invention may be employed to increase the rate of an isothermal nucleic acid reaction as compared to suitable controls in which a chelating agent is not present.

It will be appreciated that, as set out in the examples, the improvements made available by the kits, uses or methods of the invention are, in some conditions, sufficient to allow an amplification reaction to be performed that would not be possible in the absence of a chelating agent. In particular, the kits, uses and methods of the invention allow effective isothermal nucleic acid amplification reactions to occur in the presence of concentrations of liquid transport medium for pathogens that would otherwise

An improvement in the rate of a nucleic acid amplification reaction achieved by an embodiment of the invention may be demonstrated by a reduction in the time taken for an amplification reaction to generate a predetermined quantity of amplified nucleic acid. Suitably, an improvement in the rate of nucleic acid amplification may be demonstrated by a reduction of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% as compared to the time taken to generate the same quantity of amplified nucleic acid in comparable control conditions lacking the presence of a chelating agent.

An improvement in the rate of nucleic acid amplification may be demonstrated by a reduction of at least 0.5 minutes, at least 1.0 minutes, at least 1.5 minutes, at least 2.0 minutes, at least

2.5 minutes, at least 3.0 minutes, at least 3.5 minutes, at least 4.0 minutes, at least 4.5 minutes, at least 5.0 minutes, at least 5.5 minutes, at least 6.0 minutes, at least 6.5 minutes, at least 7.0 minutes, at least 7.5 minutes, at least 8.0 minutes, at least 9.5 minutes, at least 10.0 minutes 10.5 minutes, at least 11.0 minutes, at least 11.5 minutes, at least 12.0 minutes, at least 12.5 minutes, at least 13.0 minutes, at least 13.5 minutes, at least 14.0 minutes, at least 14.5 minutes, at least 15.0 minutes, at least 15.5 minutes, at least 16.0 minutes, at least

16.5 minutes, at least 17.0 minutes, at least 17.5 minutes, at least 18.0 minutes, at least 19.5 minutes, or at least 20.0 minutes as compared to the time taken to generate the same quantity of amplified nucleic acid in comparable control conditions lacking the presence of a chelating agent.

An increase in the rate of an isothermal nucleic acid reaction (as compared to suitable controls in which a chelating agent is not present) may be demonstrated in respect of the kits, uses or methods of the invention in the event that these are able to increase the rate of reaction as compared to the control by at least at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% as compared to a control isothermal nucleic acid reaction in which a chelating agent is not present.

Similarly, an increase in the rate of an isothermal nucleic acid reaction (as compared to suitable controls in which a chelating agent is not present) may be demonstrated in respect of the kits, uses or methods of the invention in the event that these are able to achieve a reduction of at least 0.5 minutes, at least 1.0 minutes, at least 1.5 minutes, at least 2.0 minutes, at least

16.5 minutes, at least 17.0 minutes, at least 17.5 minutes, at least 18.0 minutes, at least 19.5 minutes, or at least 20.0 minutes as compared to the time taken to generate the same quantity of amplified nucleic acid as compared to a control isothermal nucleic acid reaction in which a chelating agent is not present.

The preceding paragraphs refer to the ability of an isothermal nucleic acid amplification technique to generate a predetermined quantity of nucleic acid. Such a quantity may, for example be the quantity that constitutes the threshold amount sufficient for detection of a pathogen within a sample.

Thus, alternatively or additionally to the consideration set out above, an improvement in nucleic acid amplification may be demonstrated by a reduction in the lowest concentration of a nucleic acid of interest (such as a nucleic acid from a pathogen) within a sample that may be used to generate a detectable quantity of amplified nucleic acid. As mentioned above, this quantity may be referred to as the limit of detection of the amplification technique. The limit of detection of a technique is important, as it correlates with the sensitivity of assays (such as diagnostic assays) making use of the technique in question. Thus, when the limit of detection of an amplification technique is lowered by employing the kits, uses or methods of the invention, so sensitivity of the technique is increased. An improvement may be confirmed to have been achieved in respect of the kits, uses or methods of the invention in the case that they reduce the limit of detection as compared to controls using techniques in which a chelating agent is not provided. Suitably, such a reduction of the limit of detection may be a reduction of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more as compared to the appropriate control lacking a chelating agent.

Similarly, an improvement may be confirmed to have been achieved in respect of the kits, uses or methods of the invention in the case that they increase the sensitivity of an assay employing an isothermal nucleic acid amplification technique as compared to a control assay in which a chelating agent is not provided. Suitably, such an increase in the sensitivity of the assay may be an increase of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more as compared to the appropriate control.

Methods of amplifying nucleic acids, and methods of detecting SARS-CoV-2 in vitro

The third and fourth aspects of the invention respectively relate to a method of isothermal amplification of nucleic acids and a method of detecting SARS-CoV-2 in vitro using isothermal nucleic acid amplification. The methods of both the third and fourth aspects of the invention involve performing an isothermal nucleic acid amplification reaction using sample of a liquid transport medium for pathogens, this reaction being carried out in the presence of a chelating agent.

The methods of the third aspect of the invention may have utility in the typing and monitoring of pathogens with reference to their expression of particular characteristic mutations. The monitoring of particular forms of pathogens, such as particular mutant forms of SARS-CoV-2 or potentially pandemic influenza viruses, represents an important step in disease control.

While the fourth aspect of the invention is directed to methods of detecting SARS-CoV-2 that clearly have diagnostic applications, the methods of the third aspect of the invention also have utility in diagnosis. Accordingly, in a suitable embodiment a method in accordance with the third aspect of the invention may be a method of isothermal amplification of nucleic acids for diagnostic purposes. The diagnosis may be based upon a determination of the presence or absence of nucleic acids indicative of a pathogen within the sample. Purely for purposes of illustration, the pathogen may be selected from the groups considered elsewhere in the present disclosure. Suitable primer sets for the amplification of the nucleic acids may be selected with reference to the isothermal amplification technique chosen (for example LAMP) and the pathogen to be detected (such as a viral, chlamydia, mycoplasma, or ureaplasma pathogen).

The skilled person will appreciate that the improvements in isothermal nucleic acid techniques considered above are able to offer significant advantages in the context of the methods of the invention, whether of the third or fourth aspect.

The improvements in rate and sensitivity of reaction are particular applicable to use of the methods of the third or fourth aspects of the invention for diagnostic purposes. It will be appreciated that the ability to accelerate the rate of detection of pathogens (such as SARS- CoV-2) and to increase the sensitivity of such rapid diagnostic methods offers considerable benefits to clinicians in the fight against infection.

The capacity of the kits, uses and methods of the invention to allow amplification techniques to be practiced on reaction mixtures comprising higher proportions of samples than could otherwise be used also confers an advantage in that sensitivity is improved since the extent to which the sample must be diluted is reduced.

The methods of the fourth aspect of the invention comprise steps of comparing the results of the nucleic acid amplification reaction with data indicative of the presence or absence of SARS-CoV-2, and determining whether or not the subject has a SARS-CoV-2 infection on the basis of this comparison.

In a similar fashion, methods of the third aspect of the invention may optionally include steps of:

• comparing the results of the nucleic acid amplification reaction with data indicative of the presence or absence of a pathogen of interest; and

• determining whether or not the subject has an infection with the pathogen on the basis of this comparison.

In both cases, the comparison and determination may involve the production of an amplification curve that reflects the generation of amplified target nucleic acids. The curve may be determined by assaying for labelling of amplified nucleic acids with a suitable nucleic acid dye or label (as considered elsewhere in the specification). For example, the requisite curve may be produced by monitoring fluorescence in the case of methods in which the generation of amplified target nucleic acids is determined by incorporation of a fluorescent nucleic acid dye, such as SYTO 62. In such embodiments, once the amplification curve has been produced, it may be analysed to enable the differentiation of true positive from false positive results, using standard techniques such as those based upon known curve properties and signal intensity.

The invention will now be further described with reference to the following Examples.

EXAMPLES

1 Introduction

1.1 Clinical sample collection and preparation and viral transport medium

There are several types of clinical samples can be used in the traditional test for the upper respiratory viruses. Swabs (throat, nasal, and combined throat-nasal swabs), washes (nasal and throat washes), aspirates(mainly nasopharyngeal aspirates), serum (obtained from a blood sample), and tissues (tissues of the up and lower respiratory tract mainly of lungs, bronchus and trachea). Most of the samples can be preserved in the viral transport medium (VTM) after the collection. Different commercial VTM have many acceptable formulations that are suitable for different conditions of individual laboratories. In most of the cases, VTM can contain the following components: cell culture medium, salt solution, tryptose-phosphate broth, veal infusion broth, protein (bovine albumin or gelatin), antibiotics, and antimycotics.

VTM is very important for the successful diagnosis of viruses, which is the key factor influences the quality of the specimen and the conditions for the specimen transportation and storage before being processed in the laboratory. VTM allows the safe transfer of viruses, chlamydia and mycoplasma for further research, including conventional cell culture methods, diagnostic tests, and molecular biology techniques.

During the COVID-19 outbreak, VTM is widely used for the clinical sample collections and written in the standard procedure of clinical sample collection in WHO and CDC recommended instructions.

1.2 Hanks' Balanced Salt solution based VTM

Hanks' Balanced Salt solution (HBSS) is one of the components often used in the VTM. The formular of HBSS contains several different salts, including Sodium Chloride (NaCI), Potassium Chloride (KCI), Potassium Phosphate monobasic (KH2P04), Sodium Phosphate dibasic (Na2HP04), Magnesium Sulfate (MgS04-7H20), Magnesium Chloride (MgCI2-6H20), Calcium Chloride (CaCI2), Sodium Bicarbonate (NaHC03), and Dextrose (Table 2). The CDC recommended VTM recipe is one of the HBSS based VTM, which contains 1X HBSS, 2% FBS, 100pg/mL Gentamicin, and 0.5pg/ml_ Amphotericin B.

Table 2. The components and concentrations of commercial HBSS. 1.3 Removal of the nucleic acid amplification inhibitors

The inventors hypothesised that calcium ions may be inhibiting the LAMP reaction by competing with magnesium ions and binding to the polymerase. Were this the case, the inhibitory effects of calcium ions could be partially reversed by increasing the magnesium concentration in the reaction to well above the standard levels normally required for LAMP. Another way of removing calcium ions would be to use appropriate chelating agents. The inventors investigated both these approaches in the studies set out herein.

1.4 Ca2+ precipitation methods

There are several calcium chelating agents, including edetic acid (EDTA), citric acid, and edetate disodium anhydrous (EDTA-Na2) and EDTA salts are commonly used precipitation agents in laboratory, which can bind to several metal ions including calcium and magnesium.

However, the inventors found that EDTA’s ability to bind to both calcium and magnesium makes it unsuitable for use in isothermal nucleic acid amplification such as LAMP reactions. Hence, they considered using the egtazic acid (EGTA) instead of EDTA to precipitate the calcium ions. EGTA, ethylene glycol-bis (b-aminoethyl ether)-N, N, N', N'-tetraacetic acid, is a chelating agent related to the better known EDTA. EGTA’s preferential binding to calcium ions and lower affinity for magnesium compared to EDTA, provided desirable properties for these experiments.

Ca²⁺ + EGTA⁴ < [Ca-EGTA]^2-

2. Methods and materials

2.1 Salt solution stock preparation

• CaCl₂-2H₂0 (Sigma-Aldrich, St. Louis, Missouri, US)

The molecular weight of CaCl₂-2H₂0 is 147.01 g/mol. The solubility of CaCL at 20°C is 0.745 g/mL. The stock is kept on the chemical shelf in the Microbiology Lab. To make 1.26126126 M stock (1000X), add 1.854 g CaCl₂-2H₂0 into 10 mL water. Mix thoroughly by inverting the 15ml_ tube several times. Filter sterilise it. Dilute 10 times to make 100X CaCL solution. Keep the stock in 4°C fridge.

• NaHCC>3 (Sigma-Aldrich, St. Louis, Missouri, US)

The molecular weight of NaHC03 is 84.007 g/mol. The solubility of NaHC03 at 25°C is 0.1 g/mL. The stock is kept on the chemical shelf in the Microbiology Lab. To make 4.166666667 M stock (100X), add 0.7 g NaHC03 into 20 mL water. Mix thoroughly by inverting the 15 mL tube several times. Filter sterilise it. Keep the stock in 4°C fridge.

• MgCI2 (Sigma-Aldrich, St. Louis, Missouri, US)

The original stock concentration of MgCL solution is 1 M. The stock is kept on the liquid chemical shelf in the Microbiology Lab. To make 0.492610837 M stock (1000X), add 4.926 mL MgCL stock solution into 5.074 mL water. Mix thoroughly by inverting the 15 mL tube several times. Filter sterilise it. Dilute 10 times to make 100X MaCL solution. Keep the tubes in 4°C fridge.

• MgSCL (New England Biolabs, Ipswich, Massachusetts, US)

The MgSCL solution is the same MgSCL as what we used in the LAMP and qPCR experiments. The original stock concentration of MgSCL solution is 100 mM. The stocks are kept in the - 20°C freezer in 1.7 mL tubes. To make 40.6504065 mM stock (100X), thaw the MgSCL solution at room temperature and add 406.5 pL stock into 593.5 pL water. Keep the stock in 4°C fridge.

• Fungizone (Amphotericin B) (Sigma-Aldrich, St. Louis, Missouri, US)

The Amphotericin B stock is kept in -20°C freezer. The original concentration is 250 pg/mL. To make the 50 pg/mL (100X) fungizone, add 1 mL stock into 4 mL water. Aliquot into 1 mL Eppendorf tubes and keep the tubes in -20°C freezer.

• Gentamicin (Sigma-Aldrich, St. Louis, Missouri, US)

The Gentamicin stock is kept at room temperature. The original concentration is 10 mg/mL (100X). 2.2 VTM preparation

All VTM samples were prepared using biosafety cabinet, and proper PPE worn during the procedure to guarantee the sterilisation conditions.

4 different commercial VTM were purchased from different companies, including LSP (Life Science Group Ltd., Barnet, Herts, UK) Viral Transport Medium CDC Protocol (VTA-001), E&O (E&O Laboratories Ltd., Burnhouse, Bonnybridge, UK) Virus Transport Medium (BM1672), Biocomma (Biocomma Ltd., Shenzhen, Guangdong Province, China) Virus Transport and Preservation Medium (YVJ-E), and MWE (Medical Wire & Equipment Co. Ltd., Corsham, Wltshire, UK) Sigma Virocult® Media (MW951T). If not mentioned otherwise, the LSP Viral Transport Medium CDC Protocol was used as the standard VTM in all the VTM experiments.

11 different HBSS based VTM were prepared according to the different formulations in Table 3 and Table 4. All the VTM were stored at 4 °C for the later usage.

Table 3. The formulae of different VTM.

Table 4. Continuation of Table 3.

2.3 SARS-CoV-2 genomic RNA and heat inactivated virus

The genomic RNA and the heat inactivated virus of Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) were ordered from American Type Culture Collection (ATCC, Manassas, Virginia, US).

The genomic RNA (ATCC® VR-1986D™) is isolated from a preparation of Severe acute respiratory syndrome-related coronavirus 2 strain 2019-nCoV/USA-WA1/2020. The quantified concentration of the two vials of ATCC® VR-1986D™ are 8.07 x 10³ genome copies/pL and 4.73 x 10³ genome copies/pL.

The heat inactivated SARS-CoV-2 virus (ATCC® VR-1986HK™) is a preparation of Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) strain 2019-nCoV/USA- WA1/2020 that has been inactivated by heating to 65°C for 30 minutes and is therefore unable to replicate. The product contains heat-inactivated, clarified cell lysate and supernatant from Vero E6 cells infected with SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020. The quantified concentration of the two vials of ATCC® VR-1986HK™ are 1.6 x 10⁵ TCIDso/mL - 3.75 x 10⁵ genome copies/pL and 1.6 x 10⁵ TCIDso/mL - 1.77 x 10⁵ genome copies/pL.

2.4 LAMP Assay The primers SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 (designed by the inventors) were used to target and amplify the N gene of SAR- CoV-2.

For the LAMP reactions without EGTA and performed in 10 pL total per reaction, 1 pL of 10X primer mix (consisting of 2 pM F3/B3, 8 pM FIP/BIP and 4 pM LF/LB) was added to 1 pL Isothermal Amplification Buffer containing 2 mM MgSCL (New England Biolabs, Ipswich, Massachusetts, US), 1.0 pL of dNTP mix (New England Biolabs), 0.4 pL of 100pM MgSCL (New England Biolabs), 0.4 pL Bst WarmStart Polymerase (New England Biolabs), 0.2 pL of reverse transcriptase, 0.2 pL of 50 pM SYTO 62 fluorescent dye to make the Master Mix. Template was diluted by 100% VTM. 1.0 pL, 1.5 pL, 2.0 pL, or 2.5 pL of template was added correspondingly to the 10%, 15%, 20%, and 25% of VTM reactions, and nuclease-free water was added to make a total reaction volume of 10 pL.

For the different EGTA concentration experiment, 1 pL of 10X primer mix (consisting of 2 pM F3/B3, 8 pM FIP/BIP and 4 pM LF/LB) was added to 1 pL Isothermal Amplification Buffer containing 2 mM MgS0₄ (New England Biolabs, Ipswich, MA, US), 1.0 pL of dNTP mix (New England Biolabs), 0.4 pL of 100pM MgS0₄ (New England Biolabs), 0.4 pL Bst WarmStart Polymerase (New England Biolabs), 0.2 pL of reverse transcriptase, 0.2 pL of 50 pM SYTO 62 fluorescent dye to make the Master Mix. Template was diluted by 100% VTM. 0.05 pL, 0.1 pL, 0.15 pL, or 0.2 pL of 100mM EGTA was added to the reaction correspondingly to the 0.5 mM, 1 mM, 1.5 mM, or 2 mM EGTA final concentration reaction. 2.5 pL template and nuclease- free water were added to make a total reaction volume of 10 pL.

For the LAMP reactions with EGTA and performed in 10 pL total per reaction, 1 pL of 10X primer mix (consisting of 2 pM F3/B3, 8 pM FIP/BIP and 4 pM LF/LB) was added to 1 pL Isothermal Amplification Buffer containing 2 mM MgS0₄ (New England Biolabs, Ipswich, MA, US), 1.0 pL of dNTP mix (New England Biolabs), 0.4 pL of 100pM MgS0₄ (New England Biolabs), 0.4 pL Bst WarmStart Polymerase (New England Biolabs), 0.2 pL of reverse transcriptase, 0.2 pL of 50 pM SYTO 62 fluorescent dye, and 0.2 pL of 100 mM EGTA to make the Master Mix. Template was diluted by 100% VTM. 2.5 pL, 3.0 pL, 4.0 pL, or 5.0 pL of template was added correspondingly to the 25%, 30%, 40%, and 50% of VTM reactions. Nuclease-free water was added to make a total reaction volume of 10 pL.

For the LoD determination experiment, LAMP was performed in 25 pL total per reaction. 2.5 pL of 10X primer mix (consisting of 2 pM F3/B3, 8 pM FIP/BIP and 4 pM LF/LB) was added to 2.5 pL Isothermal Amplification Buffer containing 2 mM MgS0₄ (New England Biolabs, Ipswich, MA, US), 2.5 pl_ of dNTP mix (New England Biolabs), 1 mI_ of 100mM MgSCU (New England Biolabs), 1 mI_ of Bst WarmStart Polymerase (New England Biolabs), 0.5 mI_ of reverse transcriptase, 0.5 mI_ of 500 mM SYTO 62 fluorescent dye, 0.5 mI_ of 100mM EGTA, 6.25 mI_ of template, and nuclease-free water to make a total reaction volume of 25 mI_. ddhhO was used for no-template controls (NTCs). The LAMP assay was performed at 59.3 °C for 30 minutes followed by melt curve analysis over a 60-95 °C temperature gradient (2 seconds per 0.5 °C) using a Bio-Rad CFX96 Real-Time PCR System (Rio-Rad Laboratories, Berkeley, CA, USA). The fluorescence detection channel was Cy5. The time-to-detection (T_d) was defined as the time in minutes where the reporting dye fluorescence first exceeds the calculated back-ground level. All experiments were set up on ice. All LAMP assays were carried out in triplicate (three technical replicates) with three repeats including NTCs if not mentioned otherwise. All the LAMP reaction setting up were performed in biosafety cabinet and proper PPE were worn during the procedure to guarantee the sterilisation conditions.

2.5 Assay Validation

Analytical sensitivity was tested with 20 replicates. Specific optimised conditions for each assay were used to test performance across six serial dilutions of template. The limit of detection (LoD) was determined by testing 20 replicates at the lowest detectable concentration and confirming positive results in ³95% of all replicates (n³19). Assay precision was evaluated by collating mean T_d and standard deviation (SD) for each repeat at each dilution of template. The coefficients of variation (CoV) determine both average intra-assay variability and inter assay variability. Linear regression analysis was performed on average T_d results for template concentrations within the LoD range (linear range). One-way annova test

2.6 Statistical Analysis

All statistical analyses and graphs were conducted using GraphPad Prism v8 (GraphPad Software Inc., USA).

3. Results

3.1 VTM inhibit LAMP reaction The SARS-CoV-2 genomic RNA (ATCC® VR-1986D™, 8.07 x 103 genome copies/pL) was used as the template and tested with the different concentrations of LSP™ commercial VTM (Figure 1). The initial template concentration was 300 genomic RNA copies per 10 pL reaction. 10%, 15%, 20%, and 25% VTM were tested. As the VTM concentration increased, the LAMP detection time (Td) became larger, which means the amplification was slower. Without the VTM, the signal could be detected as soon as around 10 minutes. A 2.4 minutes of Td delay could be observed even with only 10% VTM. When the VTM concentration increased to 15%, 20%, and 25%, the Td was delayed by 3.6, 5.7, and 7.8 minutes correspondingly (Figure 1). Any concentration above or equal to 50% VTM could inhibit LAMP completely in the absence of chelating agents (data not shown in this report).

3.2 Effect of HBSS on SARS-CoV-2 LAMP

To determine the major inhibitor of LAMP in the VTM, an experiment was designed using 25% VTM with different components in the LAMP (Table 5). The heat inactivated SARS-CoV-2 virus (ATCC® VR-1986HK™, 1.6 x 10⁵ TCI Dso/mL - 3.75 x 10⁵ genome copies/pL) was used as the template. 2.5 pL of the template was added in the final volume 10 pL per reaction. The initial template concentration was 300 genome copies per 10 pL reaction and was calculated as 51.2 TCI Dso/mL.

According to the data in figure 2, 25% of VTM 2, VTM 4, VTM 5, VTM 7, VTM 12, and VTM 13, which all contain Ca²⁺, had the largely delayed T_d compared with other VTM or no VTM control. The VTM 1 , VTM 3, VTM 6, VTM 8, VTM 9, VTM 10, and VTM 11 contain no Ca²⁺, and had a slightly delayed T_d compared with the no VTM control. Therefore, the presence of Ca²⁺ was determined as a major inhibitor, though not the only inhibitor, of the LAMP amplification technique.

Table 5. The different formulations of VTM used in this experiment.

3.3 Removal of the LAMP inhibition caused by VTM samples

The inventors investigated two potential ways to revive the DNA amplification when Ca²⁺ exist in LAMP reaction conditions, adding of the chelating agent to precipitate the Ca²⁺, or increasing the Mg²⁺ concentration to compensate for the presence of Ca²⁺.

3.3.1 Increasing of Mg²+ cannot remove the Ca2+ inhibition on LAMP

To test if the increased Mg²⁺ concentration can compensate the Ca²⁺ existence, different concentrations of extra Mg²⁺ were introduced to the LAMP reaction. The final concentration of 6 mM, 8 mM, 10 mM, and 12 mM of Mg²⁺ have been tested and the performances of LAMP were not promoted by increased Mg²⁺ concentration. On the contrast, the high concentration of Mg²⁺ inhibited the LAMP reaction, showing the delayed T_d compared with the original 6 mM final Mg²⁺ concentration (Data not shown in this report). To test if the Mg²⁺ concentrations were too high, another LAMP experiment was designed, with the final concentration of 6 mM, 6.5 mM, 7 mM, 7.5 mM, and 8 mM of Mg²⁺ final concentration. The lower final concentration of Mg2+ with 2mM, 3 mM, 4 mM, 5 mM, and 6 mM were tested at the same time. Either the higher or lower Mg²⁺ concentration promoted the LAMP performance (Data not shown in this report).

3.3.2 Application of EGTA to LAMP

To test if addition of the chelating agent EGTA was able to overcome the inhibitory effects of VTM on LAMP, different EGTA concentrations were tested on LAMP reaction mixtures comprising 25% VTM. The heat inactivated SARS-CoV-2 virus (ATCC® VR-1986HK™, 1.6 x 10⁵ TCID5o/mL - 3.75 x 10⁵ genome copies/pL) was used as the template. The final EGTA concentration of 0.5 mM, 1.0 mM, 1.5 mM, and 2.0 mM were tested and the 2.0 mM EGTA gave the best result (Figure 3).

To test the effect of EGTA on LAMP with concentrations higher than 2 mM, an experiment using 0, 2mM , 4 mM, 6 mM, 8 mM, and 10 mM EGTA testing with 50%, 25%, and 0 VTM in LAMP was designed and done (Figure 4). With 50% VTM, no signal can be detected with 10 mM EGTA or without EGTA. 9 out of 9 positive samples were detected with 2 mM, 4 mM, and 6 mM EGTA. Only 3 out of 9 positive samples were detected with 8 mM EGTA. With 25% VTM, 9 out of 9 positive samples were detected with all different EGTA concentrations. With 2 mM EGTA, T_d had the lowest value of 11.559 minutes among all the EGTA concentrations, indicating the 2 mM EGTA had the best performance among all the different concentrations. When there was no VTM in the LAMP reactions, with all different EGTA concentrations, 9 out of 9 positive samples were detected. The T_{d O}f all the samples with no VTM were below 15 minutes, indicating the existence of EGTA did not downshift the LAMP performance when there was no VTM in the reaction. (Figure 4)

Then the effects of different VTM concentrations on the LAMP performances with 2 mM EGTA were tested (Figure 5). The heat inactivated SARS-CoV-2 virus (ATCC® VR-1986HK™, 1.6 x 10⁵ TCID5o/mL - 3.75 x 10⁵ genome copies/pL) was used as the template. According to the figure 5, 50% VTM could largely delay the LAMP T_d even with the EGTA existence, while the T_d of 25% VTM LAMP had no significant difference compared to the control group (no VTM control) when added with 2 mM EGTA. The T_d of LAMP was delayed by different extents with the different percentages of VTM between 50% and 25% (30% and 40%).

Dunnett's multiple Below Adjusted

Mean Diff. 95.00% Cl of diff. Summary comparisons test threshold? P Value no VTM vs. 50% VTM -8.558 -10.96 to -6.153 Yes

<0.0001 no VTM vs. 40% VTM -4.032 -6.437 to -1.627 Yes 0.0013 no VTM vs. 30% VTM -1.813 -4.218 to 0.5923 No ns 0.1691 no VTM vs. 25% VTM -0.5062 -2.911 to 1.899 No ns 0.9406

Table 6. Multiple comparison analysis between the different VTM concentrations and the no VTM control.

Four different commercial VTM were uses to test their effects on LAMP performances (Figure 6). The heat inactivated SARS-CoV-2 virus (ATCC® VR-1986HK™, 1.6 x 10⁵ TCIDso/mL - 1.77 x 10⁵ genome copies/pL) was used as the template. According to the data shown in Figure 6, with the existence of 2 mM EGTA in the LAMP reaction, there was no significant difference of T_d between LAMP with or without 25% VTM, showing the EGTA can efficiently remove the LAMP inhibition caused by VTM.

Dunnett's multiple

Mean Diff. 95.00% Cl of diff. Summary

comparisons test

no VTM vs. LSP VTM CDC protocol -1 .372 -5.112 to 2.369 No ns 0.6706 no VTM vs. E&O VTM 1 .347 -2.303 to 4.997 No ns 0.6666 no VTM vs. Biocomma VTM 0.4380 -3.212 to 4.088 No ns 0.9873 no VTM vs. Virocult VTM 0.9505 -2.700 to 4.601 No ns 0.8488

Table 7. Multiple comparison analysis between the different VTM and the no VTM control.

3.4 Limit of detection of SARS-CoV-2 LAMP with VTM

To determine the limit of detection (LoD) of SARS-CoV-2 LAMP, VTM or nuclease-free water were spiked with a serial dilution of heat-inactivated SARS-CoV-2 virus (ATCC® VR- 1986HK™, 1.6 x 10⁵ TCIDso/mL - 1.77 x 10⁵ genome copies/pL), and. added to the reaction

at 25% (v/v) (Figure 7). The LoD of LAMP with VTM was determined as 7.2 TCIDso/mL (Table 8). The LoD of LAMP without VTM was determined as 3.6 TCIDso/mL (Table 9).

Table 8. A summary table of results for LAMP assay SARS-CoV-2 analytical sensitivity testing with 25% VTM. The 95% LoD is indicated by shading. CoV = coefficient of variation.

Table 9. A summary table of results for LAMP assay SARS-CoV-2 analytical sensitivity testing without VTM. The 95% LoD is indicated by shading. CoV = coefficient of variation.

ASPECTS AND EMBODIMENTS OF THE INVENTION

The following paragraphs do not constitute claims in respect of the present invention, but are useful in understand the subject matter of the invention, and particularly for understand subject matter in respect of which protection may be sought.

1. A kit for use in the isothermal amplification of nucleic acids in a sample comprising a liquid transport medium for pathogens, the kit comprising:

• a chelating agent; and

• a primer mix.

2. A kit according to paragraph 1, wherein the chelating agent preferentially binds to calcium ions.

3. A kit according to paragraph 2, wherein the chelating agent is selected from the group consisting of: EGTA; BAPTA; 5,5’-Dimethyl-BAPTA; Tetraacetoxymethyl Bis(2-aminoethyl) Ether N,N,N',N'-Tetraacetic Acid; FURA-2 pentapotassium salt; FLUO 3, Pentaammonium Salt; and IN DO 1 pentapotassium salt.

4. A kit according to paragraph 3, wherein the chelating agent is EGTA.

5. A kit according to any of paragraphs 1 to 4, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of between approximately 0.5 mM and approximately 8 mM when the kit is used to produce a nucleic acid amplification reaction mixture.

6. A kit according to paragraph 5, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of between approximately 1 mM and approximately 6 mM when the kit is used to produce a nucleic acid amplification reaction mixture.

7. A kit according to paragraph 6, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of approximately 2 mM when the kit is used to produce a nucleic acid amplification reaction mixture.

8. A kit according to any preceding paragraph, wherein the liquid transport medium for pathogens is selected from the group consisting of: a viral transfer medium (VTM); and a universal transport medium (UTM). 9. A kit according to paragraph 8, wherein the transport medium sample is a VTM sample.

10. A kit according to any of paragraphs 8 to 9, wherein the transfer medium comprises a source of calcium ions.

11. A kit according to paragraph 10, wherein the transfer medium is selected from the group consisting of: Centers for Disease Control (CDC) VTM; Gibco Viral Transport Medium; MWE Virocult VTM; Copan Universal Transport Medium; Remel MicroTest™ M4RT™; BD universal viral transport; Biocomma VTM; and EO Laboratories VTM.

12. A kit according to any preceding paragraph, wherein the nucleic acids are those of a pathogen selected from the group consisting of: a viral pathogen; chlamydia; and mycoplasma.

13. A kit according to paragraph 12, wherein the nucleic acids are viral nucleic acids.

14. A kit according to paragraph 13, wherein the nucleic acids are nucleic acids of SARS- CoV-2.

15. A kit according to any preceding paragraph, wherein the kit is for loop-mediated isothermal amplification (LAMP) of nucleic acids.

16. A kit according to paragraph 15, wherein the primers are LAMP primers for SARS- CoV-2.

17. A kit according to paragraph 16, wherein the primer mix comprises primers for the amplification of SARS-CoV-2 nucleic acids, or nucleic acids complementary to SARS-CoV-2 nucleic acids.

18. A kit according to paragraph 16 or paragraph 17, wherein the primer mix comprises at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

19. A kit according to any preceding paragraph, further comprising at least one additional component selected from the group consisting of: • a dNTP mixture;

• a buffer solution;

• a DNA polymerase;

• a reverse transcriptase;

• a nucleic acid dye;

• a preservative;

• positive control nucleic acid; and

• distilled water.

20. A kit according to paragraph 19, wherein the DNA polymerase is Bst DNA polymerase.

21. A kit according to paragraph 19 or paragraph 20, wherein the nucleic acid dye is an intercalating dye.

22. A kit according to paragraph 21 , wherein the nucleic acid dye is SYTO 62.

23. A kit according to any preceding paragraph, wherein the constituents of the kit are provided in lyophilised form.

24. A kit according to paragraph 23, wherein the kit is provided in the form of lyophilised beads.

25. A kit according to paragraph 24, wherein the lyophilised beads comprise:

• EGTA;

• a primer mix comprising primers for the amplification of SARS-CoV-2 nucleic acids, or nucleic acids complementary to SARS-CoV-2 nucleic acids;

• a dNTP mixture;

• a buffer solution comprising Tris-HCI, (NhU^SCU, KCI, MgSCU, and Tween 20;

• a Bst DNA polymerase;

• a WarmStart ® RTx reverse transcriptase;

• SYTO 62 nucleic acid dye; and

• a cryopreservative.

26. A kit according to paragraph 25, wherein the primer mix comprises at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

27. A kit according to paragraph 25, wherein the lyophilised beads are for reconstitution with a sample comprising a liquid transport medium for pathogens to provide conditions for isothermal nucleic acid amplification as follows:

• EGTA at a concentration of approximately 2 mM;

• a dNTP mixture at a concentration of approximately 1 mM;

• a buffer solution comprising Tris-HCI at a concentration of approximately 20 mM, (NH₄)₂S0₄ at a concentration of approximately 10 mM, KOI at a concentration of approximately 50 mM, MgS0₄ at a concentration of approximately 6 mM, and Tween 20 at a concentration of approximately 0.1%;

• approximately 320 U/mI Bst 2.0 DNA polymerase;

• approximately 300U/pl WarmStart ® RTx reverse transcriptase;

• SYTO 62 nucleic acid dye at a concentration of approximately 2.5 mM; and

• a cryopreservative at a concentration of approximately 3%.

28. A kit according to paragraph 27, wherein the primer mix is provided in an amount such that on reconstitution the condition for isothermal nucleic acid amplification comprise: SEQ ID NO: 1 or a variant thereof at a concentration of approximately 0.2 mM; SEQ ID NO: 2 or a variant thereof at a concentration of approximately 0.2 pM; SEQ ID NO: 3 or a variant thereof at a concentration of approximately 0.8 pM; SEQ ID NO: 4 or a variant thereof at a concentration of approximately 0.8 pM; SEQ ID NO: 5 or a variant thereof at a concentration of approximately 0.4 pM; and SEQ ID NO: 6 or a variant thereof at a concentration of approximately 0.4 pM.

29. A kit according to any preceding paragraph, wherein the transfer medium sample constitutes up to 50% of the reaction mixture by volume.

30. A kit according to use according to paragraph 29, wherein the transfer medium sample constitutes up to 40% of the reaction mixture by volume.

31. A kit according to paragraph 30, wherein the transfer medium sample constitutes up to 30% of the reaction mixture by volume. 32. A kit according to paragraph 31, wherein the transfer medium sample constitutes up to 25% of the reaction mixture by volume.

33. A kit according to any preceding paragraph, wherein the transfer medium sample constitutes between approximately 5% and approximately 50% of the reaction mixture by volume.

34. A kit according to paragraph 33, wherein the transfer medium sample constitutes between approximately 10% and approximately 40% of the reaction mixture by volume.

35. A kit according to paragraph 34, wherein the transfer medium sample constitutes between approximately 15% and approximately 30% of the reaction mixture by volume.

36. A kit according to any preceding paragraph, wherein the transfer medium sample constitutes at least 5% of the reaction mixture by volume.

37. A kit according to paragraph 36, wherein the transfer medium sample constitutes at least 20% of the reaction mixture by volume.

38 A kit according to any of paragraphs 29 to 37, wherein the transfer medium constitutes approximately 25% of the reaction mixture by volume

39. The use of a chelating agent as an additive to an isothermal nucleic acid amplification reaction mixture comprising a sample of a liquid transport medium for pathogens, to improve amplification of nucleic acids.

40. The use according to paragraph 39, wherein the chelating agent preferentially binds to calcium ions.

41. The use according to paragraph 40, wherein the chelating agent is selected from the group consisting of: EGTA; BAPTA; 5,5’-Dimethyl-BAPTA; Tetraacetoxymethyl Bis(2- aminoethyl) Ether N,N,N',N'-Tetraacetic Acid; FURA-2 pentapotassium salt; FLUO 3, Pentaammonium Salt; and INDO 1 pentapotassium salt.

42. The use according to paragraph 41 , wherein the chelating agent is EGTA. 43. The use according to any of paragraphs 39 to 42, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of between 0.5 mM and 8 mM of the chelating agent in the isothermal nucleic acid amplification reaction mixture.

44. The use according to paragraph 43, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of between 1 mM and 6 mM of the chelating agent in the isothermal nucleic acid amplification reaction mixture.

45. The use according to paragraph 44, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of approximately 2 mM of the chelating agent in the isothermal nucleic acid amplification reaction mixture.

46. The use according to any of paragraphs 39 to 45, wherein the liquid transport medium for pathogens is selected from the group consisting of: a VTM; and a UTM.

47. The use according to paragraph 46, wherein the transport medium sample is a VTM sample.

48. The use according to any of paragraphs 39 to 47, wherein the transfer medium comprises a source of calcium ions.

49. The use according to paragraph 48, wherein the transfer medium is selected from the group consisting of: Centers for Disease Control (CDC) VTM; Gibco Viral Transport Medium; MWE Virocult VTM; Copan Universal Transport Medium; Remel MicroTest™ M4RT™; BD universal viral transport; Biocomma VTM; and EO Laboratories VTM.

50. The use according to any of paragraphs 39 to 49, wherein the nucleic acids are those of a pathogen selected from the group consisting of: a viral pathogen; chlamydia; and mycoplasma.

51. The use according to any of paragraph 50, wherein the nucleic acids are viral nucleic acids.

52. The use according to paragraph 51, wherein the nucleic acids are nucleic acids of SARS-CoV-2. 53. The use according to any of paragraphs 39 to 52, wherein the isothermal nucleic acid amplification mixture is a loop-mediated isothermal amplification mixture.

54. The use according to paragraph 53, wherein the reaction mixture comprises LAMP primers for SARS-CoV-2.

55. The use according to paragraph 54, wherein the LAMP reaction mixture comprises at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

56. The use according to paragraph 55, wherein the LAMP reaction mixture comprises a primer mix comprising primers for the amplification of SARS-CoV-2 nucleic acids, or nucleic acids complementary to SARS-CoV-2 nucleic acids.

57. The use according to any of paragraphs 39 to 56, wherein the transfer medium sample constitutes up to 50% of the reaction mixture by volume.

58. The use according to paragraph 57, wherein the transfer medium sample constitutes up to 40% of the reaction mixture by volume.

59. The use according to paragraph 58, wherein the transfer medium sample constitutes up to 30% of the reaction mixture by volume.

60. The use according to paragraph 59, wherein the transfer medium sample constitutes up to 25% of the reaction mixture by volume.

61. The use according to any of paragraphs 39 to 60, wherein the transfer medium sample constitutes between approximately 5% and approximately 50% of the reaction mixture by volume.

62. The use according to paragraph 61, wherein the transfer medium sample constitutes between approximately 10% and approximately 40% of the reaction mixture by volume.

63. The use according to paragraph 62, wherein the transfer medium sample constitutes between approximately 15% and approximately 30% of the reaction mixture by volume. 64. The use according to any of paragraphs 39 to 63, wherein the transfer medium sample constitutes at least 5% of the reaction mixture by volume.

65. The use according to paragraph 64, wherein the transfer medium sample constitutes at least 20% of the reaction mixture by volume.

66. The use according to any of paragraphs 39 to 65, wherein the transfer medium constitutes approximately 25% of the reaction mixture by volume

67. A method of isothermal amplification of nucleic acids, the method comprising:

• a step of providing a sample of a liquid transport medium for pathogens;

68. A method according to paragraph 67, wherein the chelating agent preferentially bonds to calcium ions.

69. A method according to paragraph 68, wherein the chelating agent is selected from the group consisting of: EGTA; BAPTA; 5,5’-Dimethyl-BAPTA; Tetraacetoxymethyl Bis(2- aminoethyl) Ether N,N,N',N'-Tetraacetic Acid; FURA-2 pentapotassium salt; FLUO 3, Pentaammonium Salt; and INDO 1 pentapotassium salt.

70. A method according to paragraph 69, wherein the chelating agent is EGTA.

71. A method according to any of paragraphs 67 to 70, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of between 0.5 mM and 8 mM of the chelating agent during the step of performing the nucleic acid amplification reaction.

72. A method according to paragraph 71, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of between 1mM and 6mM of the chelating agent during the step of performing the nucleic acid amplification reaction. 73. A method according to paragraph 72, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of approximately 2mM of the chelating agent during the step of performing the nucleic acid amplification reaction.

74. A method according to any of paragraphs 67 to 73, wherein the liquid transport medium for pathogens is selected from the group consisting of: a VTM; and a UTM.

75. A method according to paragraph 74, wherein the transport medium sample is a VTM sample.

76. A method according to any of paragraphs 67 to 75, wherein the transfer medium comprises a source of calcium ions.

77. A method according to paragraph 76, wherein the transfer medium is selected from the group consisting of: Centers for Disease Control (CDC) VTM; Gibco Viral Transport Medium; MWE Virocult VTM; Copan Universal Transport Medium; Remel MicroTest™ M4RT™; BD universal viral transport; Biocomma VTM; and EO Laboratories VTM.

78. A method according to any of paragraphs 67 to 77, wherein the nucleic acids are those of a pathogen selected from the group consisting of: a viral pathogen; chlamydia; and mycoplasma.

79. A method according to paragraph 78, wherein the nucleic acids are viral nucleic acids.

80. A method according to paragraph 79, wherein the nucleic acids are nucleic acids of SARS-CoV-2.

81. A method according to any of paragraphs 67 to 80, wherein the isothermal amplification is LAMP.

82. A method according to paragraph 81 , wherein the primer set comprises LAMP primers for SARS-CoV-2.

83. A method according to paragraph 82, wherein the primer set comprises a primer mix comprising primers for the amplification of SARS-CoV-2 nucleic acids, or nucleic acids complementary to SARS-CoV-2 nucleic acids. 84. A method according to paragraph 82, wherein the primer set comprises at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; SEQ ID NO: 6 or a variant thereof.

85. A method according to any of paragraphs 69 to 84, wherein the transfer medium sample provided constitutes up to 50% of the reaction mixture by volume.

86. A method according to paragraph 85, wherein the transfer medium sample provided constitutes up to 40% of the reaction mixture by volume.

87. A method according to paragraph 86, wherein the transfer medium sample provided constitutes up to 30% of the reaction mixture by volume.

88. A method according to paragraph 87, wherein the transfer medium sample provided constitutes up to 25% of the reaction mixture by volume.

89. A method according to any of paragraphs 67 to 88, wherein the transfer medium sample provided constitutes between approximately 5% and approximately 50% of the reaction mixture by volume.

90. A method according to paragraph 89, wherein the transfer medium sample provided constitutes between approximately 10% and approximately 40% of the reaction mixture by volume.

91. A method according to paragraph 90, wherein the transfer medium sample provided constitutes between approximately 15% and approximately 30% of the reaction mixture by volume.

92. A method according to any of paragraphs 67 to 91, wherein the transfer medium sample provided constitutes at least 5% of the reaction mixture by volume.

93. A method according to paragraph 92, wherein the transfer medium sample provided constitutes at least 20% of the reaction mixture by volume.

94. A method according to any of paragraphs 63 to 88, wherein the transfer medium provided constitutes approximately 25% of the reaction mixture by volume 95. A method of detecting SARS-CoV-2 in vitro, the method comprising:

• a step of providing a sample of a liquid transport medium for pathogens from a subject requiring SARS-CoV-2 detection;

• a step of providing a primer set for amplifying SARS-CoV-2 nucleic acids present in the sample;

96. A method according to paragraph 95, wherein the chelating agent preferentially bonds to calcium ions.

97. A method according to paragraph 96, wherein the chelating agent is selected from the group consisting of: EGTA; BAPTA; 5,5’-Dimethyl-BAPTA; Tetraacetoxymethyl Bis(2- aminoethyl) Ether N,N,N',N'-Tetraacetic Acid; FURA-2 pentapotassium salt; FLUO 3, Pentaammonium Salt; and INDO 1 pentapotassium salt.

98. A method according to paragraph 97, wherein the chelating agent is EGTA.

99. A method according to any of paragraphs 95 to 98, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of between 0.5 mM and 8 mM of the chelating agent during the step of performing the isothermal nucleic acid amplification reaction.

100. A method according to paragraphs 99, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of between 1mM and 6mM of the chelating agent during the step of performing the isothermal nucleic acid amplification reaction. 101. A method according to paragraph 100, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of approximately 2mM of the chelating agent during the step of performing the isothermal nucleic acid amplification reaction.

102. A method according to any of paragraphs 95 to 101, wherein the isothermal nucleic acid amplification reaction is a LAMP reaction.

103. A method according to paragraph 97, wherein the primer set comprises a primer mix comprising primers for the amplification of SARS-CoV-2 nucleic acids, or nucleic acids complementary to SARS-CoV-2 nucleic acids.

104. A method according to paragraph 103, wherein the primer set comprises at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof

105. A method according to any of paragraphs 95 to 104, wherein the liquid transport medium for pathogens is selected from the group consisting of: a VTM; and a UTM.

106. A method according to paragraph 105, wherein the transport medium sample is a VTM sample.

107. A method according to any of paragraphs 95 to 106, wherein the transfer medium comprises a source of calcium ions.

108. A method according to paragraph 107, wherein the transfer medium is selected from the group consisting of: Centers for Disease Control (CDC) VTM; Gibco Viral Transport Medium; MWE Virocult VTM; Copan Universal Transport Medium; Remel MicroTest™ M4RT™; BD universal viral transport; Biocomma VTM; and EO Laboratories VTM.

109. A method according to any of paragraphs 95 to 108, wherein the transfer medium sample provided constitutes up to 50% of the reaction mixture by volume.

110. A method according to paragraph 106, wherein the transfer medium sample provided constitutes up to 40% of the reaction mixture by volume. 111. A method according to paragraph 110, wherein the transfer medium sample provided constitutes up to 30% of the reaction mixture by volume.

112. A method according to paragraph 111, wherein the transfer medium sample provided constitutes up to 25% of the reaction mixture by volume.

113. A method according to any of paragraphs 95 to 112, wherein the transfer medium sample provided constitutes between approximately 5% and approximately 50% of the reaction mixture by volume.

114. A method according to paragraph 113, wherein the transfer medium sample provided constitutes between approximately 10% and approximately 40% of the reaction mixture by volume.

115. A method according to paragraph 114, wherein the transfer medium sample provided constitutes between approximately 15% and approximately 30% of the reaction mixture by volume.

116. A method according to any of paragraphs 95 to 115, wherein the transfer medium sample provided constitutes at least 5% of the reaction mixture by volume.

117. A method according to paragraph 116, wherein the transfer medium sample provided constitutes at least 20% of the reaction mixture by volume.

118. A method according to any of paragraphs 95 to 117, wherein the transfer medium provided constitutes approximately 25% of the reaction mixture by volume.

Claims

• a chelating agent; and

• a primer mix.

2. A kit according to claim 1 , wherein the chelating agent preferentially binds to calcium ions.

3. A kit according to claim 2, wherein the chelating agent is selected from the group consisting of: EGTA; BAPTA; 5,5’-Dimethyl-BAPTA; Tetraacetoxymethyl Bis(2-aminoethyl) Ether N,N,N',N'-Tetraacetic Acid; FURA-2 pentapotassium salt; FLUO 3, Pentaammonium Salt; and IN DO 1 pentapotassium salt.

4. A kit according to claim 3, wherein the chelating agent is EGTA.

5. A kit according to any of claims 1 to 4, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of approximately 2 mM when the kit is used to produce a nucleic acid amplification reaction mixture.

6. A kit according to any preceding claim, wherein the liquid transport medium for pathogens is selected from the group consisting of: a viral transfer medium (VTM); and a universal transport medium (UTM).

7. A kit according to claim 6, wherein the transfer medium is selected from the group consisting of: Centers for Disease Control (CDC) VTM; Gibco Viral Transport Medium; MWE Virocult VTM; Copan Universal Transport Medium; Remel MicroTest™ M4RT™; BD universal viral transport; Biocomma VTM; and EO Laboratories VTM.

8. A kit according to any preceding claim, wherein the nucleic acids are those of a pathogen selected from the group consisting of: a viral pathogen; chlamydia; and mycoplasma.

9. A kit according to claim 8, wherein the nucleic acids are nucleic acids of SARS-CoV- 2.

10. A kit according to any preceding claim, wherein the kit is for loop-mediated isothermal amplification (LAMP) of nucleic acids.

11. A kit according to claim 10, wherein the primer mix comprises at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

12. A kit according to claim 11 , wherein the kit is provided in the form of lyophilised beads comprising:

• EGTA;

• a primer mix comprising at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof;

• a dNTP mixture;

• a buffer solution comprising Tris-HCI, (NhU^SO^ KOI, MgS0₄, and Tween 20;

• a Bst DNA polymerase;

• a WarmStart ® RTx reverse transcriptase;

• SYTO 62 nucleic acid dye; and

• a cryopreservative.

13. A kit according to claim 12, wherein the lyophilised beads are for reconstitution with a sample comprising a liquid transport medium for pathogens to provide conditions for isothermal nucleic acid amplification as follows:

• EGTA at a concentration of approximately 2 mM;

• a dNTP mixture at a concentration of approximately 1 mM;

• approximately 320 U/mI Bst 2.0 DNA polymerase; • approximately 300U/pl WarmStart ® RTx reverse transcriptase;

• SYTO 62 nucleic acid dye at a concentration of approximately 2.5 mM; and

• a cryopreservative at a concentration of approximately 3%.

14. The use of a chelating agent as an additive to an isothermal nucleic acid amplification reaction mixture comprising a sample of a liquid transport medium for pathogens, to improve amplification of nucleic acids.

15. The use according to claim 14, wherein the chelating agent is EGTA.

16. The use according to claim 14 or claim 15, wherein the chelating agent is provided in an amount sufficient to achieve a concentration of approximately 2 mM of the chelating agent in the isothermal nucleic acid amplification reaction mixture.

17. The use according to claim 16, wherein the transfer medium is selected from the group consisting of: Centers for Disease Control (CDC) VTM; Gibco Viral Transport Medium; MWE Virocult VTM; Copan Universal Transport Medium; Remel MicroTest™ M4RT™; BD universal viral transport; Biocomma VTM; and EO Laboratories VTM.

18. The use according to any of claims 14 to 17, wherein the nucleic acids are nucleic acids of SARS-CoV-2.

19. The use according to any of claims 14 to 18, wherein the isothermal nucleic acid amplification mixture is a loop-mediated isothermal amplification mixture.

20. The use according to claim 19, wherein the reaction mixture comprises LAMP primers for SARS-CoV-2 comprising at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof.

21. The use according to any of claims 14 to 20, wherein the transfer medium sample constitutes between approximately 5% and approximately 50% of the reaction mixture by volume.

22. A method of isothermal amplification of nucleic acids, the method comprising: • a step of providing a sample of a liquid transport medium for pathogens;

• a step of providing a primer set comprising at least one primer selected from the group consisting of: SEQ ID NO: 1 or a variant thereof; SEQ ID NO: 2 or a variant thereof; SEQ ID NO: 3 or a variant thereof; SEQ ID NO: 4 or a variant thereof; SEQ ID NO: 5 or a variant thereof; and SEQ ID NO: 6 or a variant thereof;

23. A method of detecting SARS-CoV-2 in vitro, the method comprising:

24. A method according to claim 22 or claim 23, wherein the chelating agent is EGTA.

25. A method according to any of claims 22 to 24, wherein:

• the chelating agent is provided in an amount sufficient to achieve a concentration of approximately 2mM of the chelating agent during the step of performing the nucleic acid amplification reaction; and/or

• the transfer medium is selected from the group consisting of: Centers for Disease Control (CDC) VTM; Gibco Viral Transport Medium; MWE Virocult VTM; Copan Universal Transport Medium; Remel MicroTest™ M4RT™; BD universal viral transport; Biocomma VTM; and EO Laboratories VTM; and/or • the isothermal amplification is LAMP; and/or

• the transfer medium sample provided constitutes between approximately 5% and approximately 50% of the reaction mixture by volume.