CN115989323A - Method for detecting SARS-CoV-2 infection - Google Patents
Method for detecting SARS-CoV-2 infection Download PDFInfo
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Abstract
The invention discloses an oligonucleotide primer set, which comprises: an inner primer group comprising a primer of a nucleotide sequence shown as SEQ ID NO.1 and a primer of a nucleotide sequence shown as SEQ ID NO. 2; and an external primer set comprising a primer of a nucleotide sequence shown as SEQ ID NO.3 and a primer of a nucleotide sequence shown as SEQ ID NO. 4; wherein the oligonucleotide primer set is suitable for amplifying nucleic acid by using a loop-mediated isothermal amplification method.
Description
Technical Field
The invention relates to a method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. More specifically, the invention relates to oligonucleotide primers and assay kits for detecting SARS-CoV-2 infection.
Background
2019 coronavirus disease (COVID-19) is an infectious disease caused by a novel coronavirus SARS-CoV-2, which is also called severe acute respiratory syndrome coronavirus 2. It is reported that the disease causes respiratory diseases ranging from mild symptoms such as cough and fever to severe diseases such as dyspnea or death. Other symptoms may include fatigue, muscle aches, diarrhea, sore throat, loss of smell, and abdominal pain. According to the U.S. center for disease control and prevention (CDC), symptoms may appear around 2 to 14 days after exposure. In some cases, the infected person may be asymptomatic.
The CDC and the World Health Organization (WHO) developed and recommended several real-time reverse transcription polymerase chain reaction (RT-PCR) methods to detect SARS-like coronaviruses. In particular, the method using RT-PCT has high sensitivity and specificity, but is too expensive and cumbersome in terms of reagent maintenance in a refrigeration facility and use of a high-precision thermal cycler. In addition, RT-PCT also requires operation by experienced personnel to obtain better accuracy of results. Journal publication, corman et al (2020) "Detection of 2019novel coronavirus (2019-nCoV) by real-time RT-PCR", discloses a method for detecting 2019-nCoV using real-time RT-PCR in the absence of physically derived viral genomic nucleic acid.
Loop-mediated isothermal amplification (LAMP) is another isothermal amplification method for nucleic acid detection, and is usually completed in less than one hour at constant temperature. In contrast to RT-PCR, LAMP does not require the use of major equipment, and its ease of operation makes it an ideal diagnostic tool for use under scarce diagnostic resources. It is desirable to provide a method for detecting SARS-CoV-2 infection by providing an oligonucleotide primer set and amplifying a SARS-CoV-2 specific nucleotide sequence by the LAMP method.
Disclosure of Invention
The main object of the present invention is to provide rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using an oligonucleotide primer set comprising a primer complementarily binding to the N1 nucleoprotein region of SARS-CoV-2, and an amplification reaction using reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology, such that the time required for diagnosing the presence or absence of viral infection is 1.5 times faster than real-time reverse transcription polymerase chain reaction (RT-PCR). In addition, the present invention provides for colorimetric detection of amplification reactions, wherein the detection prevents contamination of the surrounding environment and provides an indication of a positive amplification reaction that can be detected by the naked eye. In addition, the invention omits the use of complex equipment, so that the detection method can be conveniently carried out under the condition of insufficient resources.
The first aspect of the present invention discloses an oligonucleotide primer set comprising: an inner primer set comprising a primer of a nucleotide sequence shown as SEQ ID NO.1 and a primer of a nucleotide sequence shown as SEQ ID NO. 2; and an external primer set comprising a primer of a nucleotide sequence shown as SEQ ID NO.3 and a primer of a nucleotide sequence shown as SEQ ID NO. 4; wherein the oligonucleotide primer set is suitable for amplifying nucleic acid by using a loop-mediated isothermal amplification method.
Preferably, the oligonucleotide primer set further comprises a circular primer set comprising a primer of the nucleotide sequence shown in SEQ ID NO.5 and a primer of the nucleotide sequence shown in SEQ ID NO. 6.
Preferably, the oligonucleotide primer set further comprises a group of primer sets comprising a primer of the nucleotide sequence shown as SEQ ID NO.7 and a primer of the nucleotide sequence shown as SEQ ID NO. 8.
Preferably, the nucleic acid has the nucleotide sequence shown as SEQ ID NO. 9.
In a second aspect of the invention, there is disclosed a method of detecting severe acute respiratory syndrome coronavirus 2 infection comprising loop-mediated isothermal amplification of a target nucleic acid region of the virus, wherein the amplification reaction comprises a set of oligonucleotide primers according to the first aspect of the invention.
Preferably, the target nucleic acid region has the nucleotide sequence shown in SEQ ID NO. 9.
In a third aspect the invention discloses a method for the detection of a virus comprising detecting amplification of a region of a SARS-CoV-2 target nucleic acid by loop-mediated isothermal amplification, wherein the amplification comprises a set of oligonucleotide primers according to the first aspect of the invention, and thereby diagnosing the presence or absence of SARS-CoV-2 infection.
Preferably, the target nucleic acid region has the nucleotide sequence shown in SEQ ID NO. 9.
In a fourth aspect of the invention, there is disclosed an assay kit for the detection of a virus comprising an oligonucleotide primer set according to the first aspect of the invention.
Those 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 embodiments described herein are not intended to limit the scope of the present invention.
Drawings
For the purposes of promoting an understanding of the invention, there is illustrated in the drawings embodiments which are presently preferred, the invention, both as to its construction and its operation, together with many of its advantages, will be readily understood and appreciated when considered in connection with the following description.
FIG. 1 schematically shows the nucleotide sequences of the oligonucleotide primers shown in SEQ ID NO. 1-8.
FIG. 2 schematically shows the nucleotide sequence shown in SEQ ID NO.9 representing the N1 nucleoprotein region complementary to the oligonucleotide primer.
FIG. 3 shows the nucleotide sequence representing SARS-CoV-2 (GenBank accession number MN 988713.1) as shown in SEQ ID NO. 10.
FIG. 4 shows the nucleotide sequence representing SARS-CoV-2 (GenBank accession number LC 528233.1) as shown in SEQ ID NO. 11.
FIG. 5 shows the nucleotide sequence representing SARS-CoV-2 (GenBank accession number MT 123293.1) as shown in SEQ ID NO. 12.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings according to preferred embodiments thereof. It should be understood, however, that the description is limited to the preferred embodiments of the present invention only for the purpose of facilitating the 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 claims.
The present invention discloses a method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using an oligonucleotide primer set comprising an oligonucleotide primer complementarily binding to a nucleotide sequence representing an N1 nucleoprotein region of SARS-CoV-2 as set forth in SEQ ID NO. 9. The nucleotide sequences of the N1 nucleoprotein region are located at positions 28195 to 28394, 28203 to 28402 and 28267 to 28466 in the SARS-CoV-2 nucleotide sequence shown in SEQ ID NO.10 (GenBank accession No. MN 988713.1), SEQ ID NO.11 (GenBank accession No. LC 528233.1) and SEQ ID NO.12 (GenBank accession No. MT 123293.1), respectively. Preferably, the oligonucleotide primer set is suitable for amplifying a nucleic acid by a loop-mediated isothermal amplification (LAMP) method, wherein the nucleic acid is a target nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO. 9.
As described herein, the oligonucleotide primer comprises an oligonucleotide selected from any one or a combination of the following: (a) An oligonucleotide comprising at least 17 contiguous bases selected from the nucleotide sequence set forth in SEQ ID nos. 1 to 8 or a nucleotide sequence complementary thereto; (b) An oligonucleotide capable of hybridizing to oligonucleotide (a) under stringent conditions; (c) An oligonucleotide consisting of the nucleotide sequence of the oligonucleotide (a) or (b), having substitution, deletion, insertion or addition of one or several bases, and having a primer function. For example, but not limited to, oligonucleotides can be chemically synthesized or by cleaving natural nucleic acids with restriction endonucleases and then modifying or ligating the resulting fragments to make up the desired nucleotide sequence.
As described herein, an oligonucleotide primer comprises a nucleotide sequence selected from any one or a combination of: (a) A nucleotide sequence having an F2 region on the 3 '-terminal side and an F1c region on the 5' -terminal side of the target nucleic acid; (b) a nucleotide sequence having a F3 region of the target nucleic acid; (c) A nucleotide sequence having a B2 region at the 3 '-terminal side and a B1c region at the 5' -terminal side of the target nucleic acid; (d) Having a nucleotide sequence of the B3 region of the target nucleic acid, when the F3c, F2c and F1c nucleotide sequence regions are selected from the 3 'end of the target nucleic acid and the 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 F1, and B3c, B2c and B1c, respectively.
In a preferred embodiment, the set of oligonucleotide primers comprises at least four primers that recognize 6 different regions in the target nucleic acid nucleotide sequence, wherein the set of oligonucleotide primers comprises: an inner primer set comprising a "Forward Inner Primer (FIP)" of the nucleotide sequence shown in SEQ ID NO.1 and a "reverse inner primer (BIP)" of the nucleotide sequence shown in SEQ ID NO. 2; and an outer primer set comprising a "Forward Outer Primer (FOP)" of the nucleotide sequence shown in SEQ ID NO.3 and a "reverse outer primer (BOP)" of the nucleotide sequence shown in SEQ ID NO. 4. Specifically, FIP and BIP can recognize F3c, F2c, and F1c regions on the 3 '-terminal side and B3, B2, and B1 regions on the 5' -terminal side of the target nucleic acid, respectively. More specifically, FIP comprises a nucleotide sequence selected from F2 and a nucleotide sequence selected from F1c, BIP comprises a nucleotide sequence selected from B2 and a nucleotide sequence selected from B1 c; and FOP comprises a nucleotide sequence selected from F3 and BOP comprises a nucleotide sequence selected from B3. In this case, F in each primer indicates that the primer binds complementary to the sense strand of the target nucleic acid and provides the synthesis origin. On the other hand, B in each primer means that the primer complementarily binds to the antisense strand of the target nucleic acid and provides the synthesis origin.
In another preferred embodiment, a circular primer set comprising circular primers is used herein in addition to the inner primer set and the outer primer set. The loop primers are two primers each having, at the 3' -end, a nucleotide sequence complementary to a sequence in a loop formed by annealing to a complementary sequence present on the same strand of an amplification product obtained by the LAMP method. The use of the circular primer set increases the number of nucleic acid synthesis origins and realizes a reduction in reaction time and an improvement in detection sensitivity. Preferably, the circular primer set comprises a forward primer of the nucleotide sequence shown as SEQ ID NO.5 and a reverse primer of the nucleotide sequence shown as SEQ ID NO. 6.
In another preferred embodiment, the oligonucleotide primer sets further comprise a group primer set comprising a forward primer of the nucleotide sequence shown as SEQ ID No.7 and a reverse primer of the nucleotide sequence shown as SEQ ID No. 8. The primers described herein are used as enhancers for the LAMP reaction for a variety of reasons, such as to increase amplicon production, thereby increasing the rate of early amplicons, to increase the ability to utilize double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) nicks, and to provide strand displacement. In particular, the function of the primers is to provide strand displacement downstream of the LAMP reaction site, annealing of the DNA strand opposite the LAMP reaction site, and opening of the DNA or RNA in the opposite direction of the LAMP reaction site. In addition, the primers provide strand displacement of double-stranded DNA without the use of heat or chemical denaturation.
The present invention also provides a method for detecting SARS-CoV-2 infection comprising subjecting a target nucleic acid region of a virus to LAMP technology, wherein the amplification reaction comprises the oligonucleotide primer set of the present invention. Preferably, the target nucleic acid region is an N1 nucleoprotein region having the nucleotide sequence shown as SEQ ID NO. 9. SARS-CoV-2 is an RNA virus, and therefore a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) method is provided by adding a reverse transcriptase to a reaction solution of a template DNA using RNA as a template to allow a nucleic acid amplification reaction to proceed in the same manner as the template DNA. The reverse transcriptase used herein is not particularly limited as long as it is an enzyme having an activity of synthesizing a complementary DNA (cDNA) using an RNA as a template. In one exemplary embodiment, such enzymes may include AMY, cloned AMV, and MMLV reverse transcriptase. Superscript II, reverTraAce and Thermoscript preferably comprise AMV or cloned AMV reverse transcriptase. Alternatively, using an enzyme having reverse transcriptase and DNA polymerase activities, such as Bca DNA polymerase, RT-LAMP reaction can be achieved using a single enzyme. The enzyme or reverse transcriptase used for nucleic acid synthesis may be purified from viruses, bacteria, etc., or may be prepared by gene recombination techniques. These enzymes can be modified by fragmentation, amino acid substitution, and the like.
The LAMP method applied herein allows complementary strand synthesis reaction to occur under isothermal conditions using oligonucleotide primers. The method includes obtaining a sample, which may be derived from a human or other living organism suspected of being infected with SARS-CoV-2, such as sputum, bronchoalveolar lavage, nasal secretions, nasal aspirates, nasal lavage, nasal swabs, pharyngeal swabs, throat washes, saliva, blood, serum, plasma, spinal fluid, urine, stool, and tissue. Alternatively, cells used in experiments, culture solutions thereof, or virus-containing samples isolated from living samples or cultured cells may be used. These samples may be subjected to pretreatment such as separation, extraction, concentration and purification.
Further, the present invention provides a virus detection method comprising detecting amplification of a SARS-CoV-2 target nucleic acid region by LAMP, wherein the amplification comprises the oligonucleotide primer set of the present invention, thereby diagnosing whether or not SARS-CoV-2 virus is infected. Preferably, the target nucleic acid region is an N1 nucleoprotein region comprising the nucleotide sequence shown in SEQ ID No. 9.
The invention also provides a determination kit for virus detection, which comprises an oligonucleotide primer group, wherein the primer group comprises an inner primer group, and the inner primer group comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.1 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 2; and an external primer set comprising a forward primer of the nucleotide sequence shown as SEQ ID NO.3 and a reverse primer of the nucleotide sequence shown as SEQ ID NO. 4. Wherein the oligonucleotide primer set is suitable for amplifying nucleic acid by using an LAMP method. Preferably, the nucleic acid has the nucleotide sequence shown as SEQ ID NO. 9. The determination kit further comprises a circular primer group, wherein the circular primer group comprises a forward primer of a nucleotide sequence shown as SEQ ID NO.5 and a reverse primer of a nucleotide sequence shown as SEQ ID NO. 6; and the determination kit comprises a group primer group of a forward primer of the nucleotide sequence shown as SEQ ID NO.7 and a reverse primer of the nucleotide sequence shown as SEQ ID NO. 8.
Dyes may also be provided in the assay kit to provide an indication for virus detection. For example, but not limited to, hydroxynaphthol blue (HNB) dye, preferably in an amount of 1. Mu.L. The use of HNB dyes eliminates the need to open the lid and enables colorimetric detection of amplification reactions, where a positive amplification reaction can be indicated by a change in color from purple to sky blue.
Various reagents necessary for detecting nucleic acid amplification using the primers of the present invention may be prepackaged into a detection kit, which includes deoxynucleoside triphosphates (dNTPs) as substrates for nucleic acid synthesis, DNA polymerase for performing nucleic acid synthesis, an enzyme having reverse transcription activity, a buffer solution or salt providing conditions suitable for the enzyme reaction, a protecting agent for stabilizing the enzyme or template, and optionally reagents necessary for detecting the reaction product.
Advantageously, the present invention is sensitive to detection of 1 RNA per reaction for SARS-CoV-2 infection, showing 5-fold better results in the present invention than real-time PCR. Furthermore, the methods provided herein exhibit fast turnaround times and can be completed in less than an hour. Thus, the present invention provides an early and accurate diagnosis of SARS-CoV-2 infection, enabling timely institutionalization for effective infection control and public health measures.
The disclosure of the present invention includes what is contained in the accompanying claims and the foregoing description. Although the present invention has been described in its preferred form with a certain degree of particularity, 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 arrangement of parts may be resorted to without departing from the scope of the invention.
Examples
The following non-limiting examples have been implemented to illustrate preferred embodiments of the present invention.
Example 1
Primers were designed based on the SARS-CoV-2N1 marker (GenBank accession numbers MN988713.1, LC528233.1 and MT 123293.1) using Primer Explorer V4 software (Eiken Chemical Co.Ltd., tokyo, japan). Reverse transcription LAMP (RT-LAMP) was performed using the Loopamp RNA amplification kit (Eiken Chemical Co. Ltd., japan). Amplification was performed in a Loopamp real-time turbidimeter (LA-320, teramecs co.ltd., tochigi, japan) using a 2X reaction mixture, a 5 μ RNA template, and species-specific primers. The primer comprises: FIP comprising the nucleotide sequence shown as SEQ ID NO.1, BIP comprising the nucleotide sequence shown as SEQ ID NO.2, F3 comprising the nucleotide sequence shown as SEQ ID NO.3, B3 comprising the nucleotide sequence shown as SEQ ID NO.4, FLP comprising the nucleotide sequence shown as SEQ ID NO.5 and BLP comprising the nucleotide sequence shown as SEQ ID NO. 6. Endpoint evaluation was performed using visual inspection, and then 1 μ Ι _ of hydroxynaphthol blue (HNB) dye (Sigma, USA) was added, the color changed from violet to sky blue indicating positive amplification.
Example 2
The analytical sensitivity of the SARS-CoV-2RT-LAMP detection was determined by serial dilution of 10-fold out-of-range transcribed ribonucleic acid (LODs) with known amounts of nucleic acid replicas (10 cp/. Mu.L, 5 cp/. Mu.L, 2 cp/. Mu.L, 1 cp/. Mu.L and 0.1 cp/. Mu.L) and comparison to the RT-PCR method. Reactions were performed in duplicate.
The specificity of LAMP-LF detection was determined by using the genomic RNA of coronavirus (HCoV-OC 43 and SARS-CoV), adenovirus, human metapneumovirus, influenza A virus (A/H1 pdm2009 and A/H3) virus, influenza B virus, parainfluenza virus 3, rhinovirus A, respiratory syncytial virus B and enterovirus D68.
The Malaysia double-drape Hospital collected 47 RT-PCR positive nasopharyngeal swab specimens and 42 RT-PCR negative nasopharyngeal swab specimens with up to 2020 CoVID-19 outbreaks in Malaysia. Total RNA was extracted using a QIAamp viral RNA mini kit (Qiagen, germany) according to the manufacturer's instructions, and 50. Mu.L of eluate was obtained for each sample. RNA samples were analyzed by RT-PCR. The SARS-CoV-2RT-LAMP reaction was run at 65 ℃ for 30 minutes. Clinical sensitivity was calculated using the formula (number of true positives)/(number of true positives + number of false negatives), and clinical specificity was calculated using the formula (number of true negatives)/(number of true negatives + number of false positives), compared to RT-PCR. Ethical approval for this study was obtained from the ethical committee on Medical Research (MREC) (NMRR-20-535-53855), ministry of health, malaysia.
Example 3
SARS-CoV-2RT-LAMP assay 1 part per SARS-CoV-2RNA reaction was detectable within 30 minutes, while the LOD of RT-PCR method was 5 parts per reaction. Neither assay found cross-reactivity with the other virus. The addition of HNB to the LAMP reaction solution did not affect the time required for amplification (data not shown). The RT-LAMP assay showed 100% sensitivity, since all RNA samples positive by RT-PCR were positive in RT-LAMP detection. Using this assay, none of the 42 RT-PCR negative samples detected positive for SARS-CoV 2. No false positive reaction was found.
Example 4
RT-LAMP is susceptible to aerosol contamination due to its high sensitivity. Typically, LAMP assays can be analyzed by running an agarose gel or by adding SYBR Green I thereto. The addition of SYBR Green must be added after the completion of the LAMP reaction, and therefore the tube for performing the RT-LAMP reaction needs to be opened for gel electrophoresis, which may cause contamination of the surroundings and subsequent reactions. To avoid contamination, herein, naked eye detection results can be achieved using HNB dyes without affecting amplification efficiency. This method has proven to be sensitive and simple for the visual detection of turkey coronavirus RNA in tissues and feces.
Claims (9)
1. An oligonucleotide primer set comprising:
an inner primer group comprising a primer of a nucleotide sequence shown as SEQ ID NO.1 and a primer of a nucleotide sequence shown as SEQ ID NO. 2; and
an external primer set comprising a primer of a nucleotide sequence shown as SEQ ID NO.3 and a primer of a nucleotide sequence shown as SEQ ID NO. 4;
wherein the oligonucleotide primer set is suitable for amplifying nucleic acid by using a loop-mediated isothermal amplification method.
2. The oligonucleotide primer set according to claim 1, further comprising a circular primer set comprising a primer of a nucleotide sequence shown as SEQ ID No.5 and a primer of a nucleotide sequence shown as SEQ ID No. 6.
3. The oligonucleotide primer set according to claim 1 or 2, further comprising a group primer set comprising a primer of a nucleotide sequence set forth as SEQ ID No.7 and a primer of a nucleotide sequence set forth as SEQ ID No. 8.
4. The oligonucleotide primer set according to claim 1, wherein the nucleotide sequence of the nucleic acid is represented by SEQ ID No. 9.
5. A method for detecting severe acute respiratory syndrome coronavirus 2 infection, comprising performing loop-mediated isothermal amplification of a target nucleic acid region of a virus, said amplification reaction comprising the oligonucleotide primer set of any one of claims 1-4.
6. The method of claim 5, wherein the target nucleic acid region has a nucleotide sequence set forth as SEQ ID No. 9.
7. A method for detecting a virus, comprising detecting amplification of a target nucleic acid region of severe acute respiratory syndrome coronavirus 2 by loop-mediated isothermal amplification, wherein said amplification comprises the oligonucleotide primer set of any one of claims 1-4, thereby diagnosing the presence or absence of infection with severe acute respiratory syndrome coronavirus 2.
8. The method of claim 7, wherein the target nucleic acid region has a nucleotide sequence set forth as SEQ ID No. 9.
9. A virus detection kit comprising the oligonucleotide primer set of any one of claims 1 to 4.
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| CN111057798A (en) * | 2020-01-20 | 2020-04-24 | 复旦大学附属华山医院 | LAMP primer combination and kit for detecting novel coronavirus |
| CN111088406A (en) * | 2020-02-17 | 2020-05-01 | 深圳麦科田生物医疗技术有限公司 | Probe, primer, kit and detection method for detecting novel coronavirus based on double loop-mediated isothermal amplification technology |
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| CN111088406A (en) * | 2020-02-17 | 2020-05-01 | 深圳麦科田生物医疗技术有限公司 | Probe, primer, kit and detection method for detecting novel coronavirus based on double loop-mediated isothermal amplification technology |
| CN110982944A (en) * | 2020-03-03 | 2020-04-10 | 中国农业科学院北京畜牧兽医研究所 | Novel visualized constant-temperature rapid detection kit for coronavirus |
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