WO2021225430A1

WO2021225430A1 - Method for detection of sars-cov-2 infection

Info

Publication number: WO2021225430A1
Application number: PCT/MY2021/050037
Authority: WO
Inventors: Yee Ling LAU; Meng Yee LAI; Nur Izati BINTI MUSTAPA; Ilyiana BT ISMAIL; Afifah HAJI HASSAN; Kalaiarasu A/L M.PEARIASAMY
Original assignee: Universiti Malaya
Current assignee: Universiti Malaya
Priority date: 2020-05-05
Filing date: 2021-04-30
Publication date: 2021-11-11
Anticipated expiration: 2022-11-05
Also published as: MY204210A; CN115989323A

Abstract

The invention relates to an oligonucleotide primer set comprising: an inner primer set comprising a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 1 and a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 2; and an outer primer set comprising a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 3 and a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 4; wherein the oligonucleotide primer set is suitable for amplification of a nucleic acid with a loop-mediated isothermal amplification method.

Description

METHOD FOR DETECTION OF SARS-CoV-2 INFECTION

FIELD OF INVENTION

The present invention relates to a method for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. More particularly, the present invention relates to oligonucleotide primers and assay kit for detection of SARS-CoV- 2 infection.

BACKGROUND OF THE INVENTION

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by a novel coronavirus, SARS-CoV-2, also known as severe acute respiratory syndrome coronavirus 2. The disease is reported to cause respiratory illness ranging from mild symptoms such as cough, fever, fever, to severe illness such as breathing difficulty, or death. Other symptoms may include fatigue, muscle pain, diarrhoea, sore throat, loss of smell, and abdominal pain. According to United States Centers for Disease Control and Prevention (CDC), the symptoms may appear around 2 to 14 days after exposure. There are also cases where those infected may appear asymptomatic.

Several real-time reverse transcription polymerase chain reaction (RT-PCR) methods have been developed and recommended by CDC and World Health Organisation (WHO) for detecting SARS-like coronaviruses. Particularly, methods using RT-PCT are highly sensitive and specific, but are too expensive and tedious in terms of maintenance of reagents in cold storage facility and use of a high precision thermal cycler. Furthermore, RT-PCT also requires to operate by an experienced personnel for better result accuracy. A journal publication, Corman el. al (2020) “Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR”, discloses an assay for detecting 2019-nCoV in the absence of physical sources of viral genomic nucleic acid using real time RT-PCR.

Loop-mediated isothermal amplification (LAMP) is an alternative isothermal amplification method for nucleic acid detection, and it generally takes less than an hour to complete under a constant temperature. As compared to RT-PCR, LAMP does not require the use of major equipment and its ease of handling makes an ideal diagnostic tool for use under scarce diagnostic resources. There exists a need to provide a method for detection of SARS-CoV-2 infection through providing oligonucleotide primer set and amplifying the nucleotide sequence specific to SARS-CoV-2 by a LAMP method.

SUMMARY OF INVENTION

The main objective of the invention is to provide a rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, using an oligonucleotide primer set comprising primers that bind complementary to N1 nucleoprotein region of SARS-CoV-2, and performing an amplification reaction using a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technique, such that time taken to diagnose the presence or absence of virus infection is speed up by 1.5 fold faster than a real-time reverse transcription polymerase chain reaction (RT-PCR). Furthermore, the invention provides a colorimetric detection of the amplification reaction, in which the detection prevents contamination of the surroundings and provides indication of a positive amplification reaction detectable by naked eyes. In addition, the invention eliminates the use of sophisticated equipment and facilitates the detection method to be conveniently performed under a condition of insufficient resources.

The first aspect of the invention describes an oligonucleotide primer set comprising: an inner primer set comprising a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 1 and a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 2; and an outer primer set comprising a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 3 and a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 4; wherein the oligonucleotide primer set is suitable for amplification of a nucleic acid with a loop-mediated isothermal amplification method.

Preferably, the oligonucleotide primer set further comprises a loop primer set comprising a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 5 and a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 6.

Preferably, the oligonucleotide primer set further comprises a swarm primer set comprising a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 7 and a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 8.

Preferably, the nucleic acid has a nucleotide sequence as set forth in SEQ ID NO. 9.

The second aspect of the invention describes a method for detection of a severe acute respiratory syndrome coronavirus 2 infection comprising performing a loop-mediated isothermal amplification of a target nucleic acid region of the vims, wherein the amplification reaction comprises the oligonucleotide primer set according to the first aspect of the invention.

Preferably, the target nucleic acid region has a nucleotide sequence as set forth in SEQ ID NO. 9.

The third aspect of the invention describes a method for vims detection comprising detecting amplification of a target nucleic acid region of SARS-CoV-2 by loop- mediated isothermal amplification, wherein the amplification comprises the oligonucleotide primer set according to the first aspect of the invention, and thereby diagnosing the presence or absence of infection with SARS-CoV-2. Preferably, the target nucleic acid region has a nucleotide sequence as set forth in SEQ ID NO. 9.

The fourth aspect of the invention describes an assay kit for vims detection comprising the oligonucleotide primer set according to the first aspect of the invention.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiment described herein is not intended as limitations on the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

FIGURE 1 shows the nucleotide sequence of oligonucleotide primers as set forth in SEQ ID NO. 1-8.

FIGURE 2 shows the nucleotide sequence as set forth in SEQ ID NO. 9 representing N1 nucleoprotein region complementary to the oligonucleotide primers.

FIGURE 3 shows the nucleotide sequence as set forth in SEQ ID NO. 10 representing SARS-CoV-2 (Genbank accession no. MN988713.1).

FIGURE 4 shows the nucleotide sequence as set forth in SEQ ID NO. 11 representing SARS-CoV-2 (Genbank accession no. LC528233.1).

FIGURE 5 shows the nucleotide sequence as set forth in SEQ ID NO. 12 representing SARS-CoV-2 (Genbank accession no. MT123293.1).

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

The invention describes a method for detection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using an oligonucleotide primer set comprising an oligonucleotide primer that binds complementary to a nucleotide sequence as set forth in SEQ ID NO. 9 representing N1 nucleoprotein region of SARS-CoV-2. The nucleotide sequence of N1 nucleoprotein region is located at the 28195th to 28394th, 28203rd to 28402nd, and 28267th to 28466th positions, in a nucleotide sequence of SARS-CoV-2 as depicted in SEQ ID NO. 10 (Genbank accession no. MN988713.1), SEQ ID NO. 11 (Genbank accession no. LC528233.1) and SEQ ID NO. 12 (Genbank accession no. MT123293.1), respectively. Preferably, the oligonucleotide primer set is suitable for amplification of a nucleic acid with a loop-mediated isothermal amplification (LAMP) method, in which the nucleic acid is a target nucleic acid consisting of the nucleotide sequence as set forth in SEQ ID NO. 9.

As described herein, the oligonucleotide primers comprises an oligonucleotide selected from any one or combination of (a) an oligonucleotide comprising at least consecutive 17 bases selected from the nucleotide sequences represented by SEQ ID NO. 1-8 or complementary nucleotide sequences thereof; (b) an oligonucleotide capable of hybridizing under stringent conditions to the oligonucleotide (a); and (c) an oligonucleotide comprising a nucleotide sequence of the oligonucleotide (a) or (b) with the substitution, deletion, insertion, or addition of one or several bases and having a primer function. By way of example, but not by way of limitation, the oligonucleotide can be synthesized chemically, or alternatively cleaving a natural nucleic acid with a restriction enzyme and then modifying or linking resulting fragments to compose a desired nucleotide sequence.

As described herein, the oligonucleotide primer consisting of a nucleotide sequence selected from any one or combination of (a) a nucleotide sequence having F2 region at the 3’ end side and Flc region at the 5’ end side of the target nucleic acid; (b) a nucleotide sequence having F3 region of the target nucleic acid; (c) a nucleotide sequence having B2 region at the 3’ end side and Blc region at the 5’ end side of the target nucleic acid; and (d) a nucleotide sequence having B3 region of the target nucleic acid, when F3c, F2c, and Flc nucleotide sequence regions are selected from the 3’ end, and B3, B2, and B1 nucleotide sequence regions are selected from the 5’ end, of the target nucleic acid. Their respective complementary nucleotide sequences are defined as F3, F2, and FI, and B3c, B2c, and Blc, respectively.

In one preferred embodiment, the oligonucleotide primer set comprises at least four primers that recognize 6 distinct regions in the nucleotide sequence of the target nucleic acid, which the oligonucleotide primer set includes an inner primer set comprising a “forward inner primer (FIP)” consisting of a nucleotide sequence as set forth in SEQ ID NO. 1 and a “backward inner primer (BIP)” consisting of a nucleotide sequence as set forth in SEQ ID NO. 2; and an outer primer set comprises a “forward outer primer (FOP)” consisting of a nucleotide sequence as set forth in SEQ ID NO. 3 and a “backward outer primer (BOP)” consisting of a nucleotide sequence as set forth in SEQ ID NO. 4. Particularly, the FIP and BIP able to recognize F3c, F2c, Flc regions from the 3’ end side, and B3, B2 and B1 regions from the 5’ end side, respectively, of the target nucleic acid. More particularly, FIP consists of a nucleotide sequence selected from F2 and a nucleotide sequence selected from Flc, and BIP consists of a nucleotide sequence selected from B2 and a nucleotide sequence selected from Blc; and FOP consists of a nucleotide sequence selected from F3, and BOP consists of a nucleotide sequence selected from B3. In this context, F in each primer indicates that the primer complementarity binds to the sense strand of the target nucleic acid and provides a synthesis origin. On the other hand, B in each primer indicates that the primer complementarily binds to the antisense strand of the target nucleic acid and provides a synthesis origin.

In another preferred embodiment, a loop primer set comprising loop primers is used herein in addition to the inner primer set and outer primer set. The loop primers refer to two primers comprising, at the 3’ end, a nucleotide sequence complementary to a sequence in a loop formed by the annealing of complementary sequences present at the same strand of an amplification product obtained by the LAMP method. The use of the loop primer set increases nucleic acid synthesis origins in number and achieves reduction in reaction time and enhancement in detection sensitivity. Preferably, the loop primer set comprises a forward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 5, and a backward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 6.

In another preferred embodiment, the oligonucleotide primer set further includes swarm primer set comprising a forward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 7, and a backward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 8. The primers as described herein is used as an enhancement to the LAMP reaction for a variety of reasons, such as to increase amplicons production resulting in greater rates of early-stage amplicons, to increase ability to take advantage of nicks in double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and to provide a strand displacement. Particularly, the primers function as providing strand displacement downstream from the LAMP reaction site, annealing to the opposite DNA strand from the LAMP reaction site, and opening up the DNA or RNA in the opposite direction of the LAMP reaction site. Furthermore, the primers provide strand displacement of double-stranded DNA without the use of heat or chemical denaturation.

The invention also provides a method for detection of SARS-CoV-2 infection comprising performing a LAMP technique of a target nucleic acid region of the virus, wherein the amplification reaction comprises the oligonucleotide primer set of the invention. Preferably, the target nucleic acid region is the N1 nucleoprotein region having the nucleotide sequence as set forth in SEQ ID NO. 9. SARS-CoV-2 is a RNA vims, therefore RNA is used as a template and allowed a nucleic acid amplification reaction to proceed in the same way as template DNA by adding reverse transcriptase to a reaction solution for template DNA to provide a reverse-transcriptase loop- mediated isothermal amplification (RT-LAMP) method. Reverse transcriptase used herein is not particularly limited as long as it is an enzyme having activities of synthesizing complementary DNA (cDNA) with RNA as a template. In one exemplary embodiment, such enzyme may include AMY, Cloned AMV, and MMLV reverse transcriptase. Superscript II, ReverTra Ace, and Thermoscript and preferably includes AMV or Cloned AMV reverse transcriptase. Alternatively, the use of an enzyme having both reverse transcriptase and DNA polymerase activities, such as Bca DNA polymerase, can achieve an RT-LAMP reaction using a single enzyme. The enzyme or reverse transcriptase used in nucleic acid synthesis may be purified from viruses, bacteria, or the like or may be prepared by a gene recombination technique. These enzymes may be modified by fragmentation, amino acid substitution, and so on.

The LAMP method applied herein allows a complementary strand synthesis reaction to take place under isothermal conditions using the oligonucleotide primers. The method includes obtaining a sample, in which the sample can be derived from human or other living bodies suspected from being infected with SARS-CoV-2, for example, sputum, bronchi alveolar lavage fluids, nasal secretions, nasal aspirates, nasal lavage fluids, nasal swabs, pharyngeal swabs, throat washings, saliva, blood, serums, plasmas, spinal fluids, urine, faeces, and tissues. Alternatively, cells or culture solutions thereof used in experiment, or virus -containing samples separated from living body-derived samples or cultural cells may also be used as the sample. These samples may be subjected to pre-treatment such as separation, extraction, condensation, and purification.

In addition, the invention provides a method for virus detection comprising detecting amplification of a target nucleic acid region of SARS-CoV-2 by LAMP, wherein the amplification comprises the oligonucleotide primer set of the invention and thereby diagnosing the presence or absence of infection with SARS-CoV-2. Preferably, the target nucleic acid region is the N 1 nucleoprotein region consisting of the nucleotide sequence as set forth in SEQ ID NO. 9.

The invention also provides an assay kit for virus detection comprising an oligonucleotide primer set comprising an inner primer set comprising a forward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 1 and a backward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 2; and an outer primer set comprising a forward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 3 and a reverse primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 4, wherein the oligonucleotide primer set is suitable for amplification of a nucleic acid with a LAMP method. Preferably, the nucleic acid has a nucleotide sequence as set forth in SEQ ID NO. 9. The assay kit further comprises a loop primer set comprising a forward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 5 and a backward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 6; and a swarm primer set comprising a forward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 7, and a backward primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 8.

A dye can be further provided in the assay kit, such that to provide an indication for the vims detection. By way of example, but not by way of limitation, the dye is hydroxynaphthol blue (HNB) dye, preferably at an amount of 1 pL. Use of HNB dye eliminates the need to open tube lid and enables colorimetric detection of the amplification reaction, in which a positive amplification reaction can be indicated by a colour change from violet to sky blue.

A variety of reagents necessary for the detection of nucleic acid amplification by use of the primers of the invention can be packaged in advance into the assay kit, which include deoxynucleoside triphosphates (dNTPs) serving as substrates for nucleic acid synthesis. DN A polymerase for performing nucleic acid synthesis, an enzyme having reverse transcription activities, buffer solutions or salts that provide conditions suitable to an enzyme reaction, protective agents for stabilizing the enzymes or the templates and optionally comprises reagents necessary for the detection of reaction products.

Advantageously, the invention is sensitive to detect 1 copy of RNA per reaction specific to SARS-CoV-2 infection, which a result of 5-fold better than a real-time PCR is exhibited in the invention. Furthermore, the method provided herein shows rapid turnaround time and can be completed in less than one hour. As such, the invention provide early and accurate diagnosis for SARS-CoV-2 infection, such that a prompt institutionalization can be initiated for effective infection control and public health measurement.

The present disclosure includes as contained in the appended claims, as well as that of the foregoing description. Although this invention has been described in its preferred form with a degree of particularly, it is understood that the present disclosure of the preferred form has been made only by way of example and that numerous changes in the details of construction and the combination and arrangements of parts may be resorted to without departing from the scope of the invention.

EXAMPLE

The following non-limiting example has been carried out to illustrate the preferred embodiments of the invention.

EXAMPLE 1

Primers were designed using Primer Explorer V4 software (Eiken Chemical Co. Ltd., Tokyo, Japan) based on SARS-CoV-2 N1 marker (GenBank accession no. MN988713.1, LC528233.1 and MT123293.1). Reverse transcription LAMP (RT- LAMP) was carried out using Loopamp RNA amplification kit (Eiken Chemical Co. Ltd., Japan). The amplification was carried out in a Loopamp real-time turbidimeter (LA-320; Teramecs Co. Ltd., Tochigi, Japan) with 2X reaction mixture, 5m RNA template and species-specific primers. The primers are LIP consisting of a nucleotide sequence as set forth in SEQ ID NO. 1, BIP consisting of a nucleotide sequence as set forth in SEQ ID NO. 2, L3 consisting of a nucleotide sequence as set forth in SEQ ID NO. 3, B3 consisting of a nucleotide sequence as set forth in SEQ ID NO. 4, LLP consisting of a nucleotide sequence as set forth in SEQ ID NO. 5, and BLP consisting of a nucleotide sequence as set forth in SEQ ID NO. 6. Endpoint assessment was conducted using visual inspection followed by addition of 1 pL hydroxynaphthol blue (HNB) dye (Sigma, USA). A positive amplification was indicated by a colour change from violet to sky blue. EXAMPLE 2

Analytical sensitivity of the SARS-CoV-2 RT-LAMP assay was determined through limit of detection (LODs) using 10-fold serially diluted in vitro transcript ribonucleic acid (RNA) with known numbers of nucleic acid copies (10 cp/pL, 5 cp/pL, 2 cp/pL, 1 cp/pL and 0.1 cp/pL) and comparing the assay with RT-PCR. The reactions were carried out in duplicates.

Specificity of the LAMP-LF assay was determined by using genomic RNA of coronaviruses (HCoV-OC43 and SARS-CoV), adenovirus, human metapneumovirus, influenza A (A/Hlpdm2009 and A/H3) viruses, influenza B virus, parainfluenza virus 3, rhinovirus A, respiratory syncytial virus B and enterovirus D68.

Forty-seven RT-PCR positive and forty-two RT-PCR negative nasopharyngeal swabs samples from COVID-19 outbreak in Malaysia as of 2020 were collected by Hospital Sungai Buloh, Malaysia. Total RNA was extracted using QIAamp viral RNA minikit (Qiagen, Germany) according to the manufacturer's instructions and 50 pL elution was obtained for each sample. The RNA samples were analysed by RT-PCR. SARS-CoV-2 RT-LAMP reactions were run at 65°C for 30 min. Clinical sensitivity was calculated using a formula of (number of true positives)/(number of true positives + number of false negatives), and clinical specificity was calculated using a formula of (number of true negatives)/( number of true negatives + number of false positives) comparing to RT-PCR. Ethical approval for this study was obtained from Medical Research Ethics Committee (MREC) Ministry of Health Malaysia (NMRR-20-535-53855).

EXAMPLE 3

The SARS-CoV-2 RT-LAMP assay was able to detect 1 copy per reaction of SARS- CoV-2 RNA in 30 minutes while the LOD for RT-PCR was 5 copies per reaction. No cross-reactivity with other viruses was found in either assay. Addition of HNB to the LAMP reaction solution did not affect the time taken for amplification (data not shown). The RT-LAMP assay demonstrated a 100% sensitivity as all the RNA samples that were positive by RT-PCR were tested positive with RT-LAMP. None of the 42 RT-PCR negative samples were tested positive for SARS-CoV2 using this assay. No false positive reactions were noted.

EXAMPLE 4 RT-LAMP is prone to aerosol contamination due to its high sensitivity. Generally, a LAMP assay can be analysed through running an agarose gel or by adding S YBR Green I thereto. The addition of SYBR Green has to be added post-completion of LAMP reaction, thereby tubes used for performing RT-LAMP reaction needs to be opened for gel electrophoresis which the open tube can lead to contaminating the surrounding and subsequent reactions. To avoid contamination, HNB dye is used herein to enable result detection using naked eyes and does not affect amplification efficiency. This approach has been shown to be sensitive and simple for visual detection of turkey coronavirus RNA in tissues and faeces.

Claims

1. An oligonucleotide primer set comprising: an inner primer set comprising a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 1 and a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 2; and an outer primer set comprising a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 3 and a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 4; wherein the oligonucleotide primer set is suitable for amplification of a nucleic acid with a loop-mediated isothermal amplification method.

2. The oligonucleotide primer set according to claim 1, further comprising a loop primer set comprising a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 5 and a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 6.

3. The oligonucleotide primer set according to claim 1 or 2, further comprising a swarm primer set comprising a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 7 and a primer consisting of a nucleotide sequence as set forth in SEQ ID NO. 8.

4. The oligonucleotide primer set according to claim 1, wherein the nucleic acid has a nucleotide sequence as set forth in SEQ ID NO. 9.

5. A method for detection of a severe acute respiratory syndrome coronavirus 2 infection comprising performing a loop-mediated isothermal amplification of a target nucleic acid region of the vims, wherein the amplification reaction comprises the oligonucleotide primer set according to any one of claims 1 to 4.

6. The method according to claim 5, wherein the target nucleic acid region has a nucleotide sequence as set forth in SEQ ID NO. 9.

7. A method for vims detection comprising detecting amplification of a target nucleic acid region of severe acute respiratory syndrome coronavims 2 by loop- mediated isothermal amplification, wherein the amplification comprises the oligonucleotide primer set according to any one of claims 1 to 4 and thereby diagnosing the presence or absence of infection with severe acute respiratory syndrome coronavims 2.

8. The method according to claim 7, wherein the target nucleic acid region has a nucleotide sequence as set forth in SEQ ID NO. 9.

9. An assay kit for vims detection comprising the oligonucleotide primer set according to any one of claims 1 to 4.