KR102226356B1 - Primers for detecting Dengue virus by LAMP - Google Patents

Abstract

The present application discloses a set of primers capable of commonly detecting dengue virus types 1, 2, 3, and 4. The primer set according to the present application has high sensitivity and specificity, so that it is possible to quickly and conveniently detect the presence of dengue virus infection, thereby increasing the speed and convenience of response.

Description

Primers for detecting Dengue virus by LAMP {Primers for detecting Dengue virus by LAMP}

It is a technology related to detection of dengue virus based on nucleic acid detection technology.

Dengue virus is the most prevalent pathogen of mosquito-borne diseases in the world, causing dengue virus infection. Dengue virus infections are classified into dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), depending on the symptoms. Dengue fever is a disease that recovers 2 to 4 weeks after the onset. It occurs mainly in Asia, the South Pacific region, Africa and the tropical regions of the Americas. Dengue hemorrhagic fever has been reported in most of Asia, the Pacific region, and Latin America. It is a more fatal disease than dengue, and is accompanied by high fever and hemorrhagic fever symptoms. The mosquito that mediates the dengue virus is mainly Aedes aegypti ( A. agypti ), and depending on the region, Ae. albopictus, Ae. polynesiensis, Ae. pseudoscutellares, etc. are also known as vector vectors.

Dengue fever has been spreading gradually over the past 10 years, with epidemic outbreaks in more than 100 countries including Africa, the Americas, and Southeast Asia. Currently, about two-fifths of the world's population, or 2.5 billion people, are at risk of dengue infection. The WHO estimates that 50 million people are infected with the dengue virus every year.

Diagnosis of dengue virus infection is required for appropriate treatment management by quickly and accurately discriminating and identifying the causative pathogen. In particular, there is no case of indigenousization in Korea, so it is very important in terms of monitoring overseas inflows. In particular, there are four types of dengue virus serologically different (DENV 1, DENV 2, DENV 3 and DENV 4).

Methods to check for dengue virus infection include virus isolation from blood and cerebrospinal fluid, antibody detection from serum, and virus gene detection in serum and cerebrospinal fluid.

The method of detecting antibodies is based on serological detection of anti-dengue IgM and IgG in serum by ELISA. However, when similar viruses such as West Nile virus, Japanese encephalitis virus, and Chikungunya virus are present in the sample sample, the reliability of the diagnosis result is low due to duplicate detection. However, this serological method has a disadvantage in that it cannot detect infection with dengue virus in the early stages of the disease.

Recently, RT-PCR (reverse transcriptase polymerase chain reaction) has been used to test a number of RNA viruses. Although RT-PCR has the advantage of being able to quickly detect a small amount of virus compared to antibodies, it has a long reaction time and a false-positive result due to non-specific hybridization is a problem.

Because dengue virus causes similar symptoms, it is not possible to distinguish the serotype of infected dengue virus by symptoms alone, so a method to distinguish them is needed. In addition, for rapid screening, it is necessary to develop a method to detect infection very quickly regardless of serotype. In particular, since the drug prescription method according to the dengue serotype is not different, further development of a method for detecting the presence or absence of a dengue virus is needed.

The present application aims to provide a technique capable of very rapidly detecting the presence of infection at the nucleic acid level regardless of serotype.

In one aspect, the present application provides a primer set for LAMP analysis comprising a total of 8 primers capable of detecting all four serotypes of dengue virus of serotypes 1, 2, 3 and 4. In one embodiment, the primer set is the F3 primer of SEQ ID NO: 1, the B3 primer of SEQ ID NO: 2, the FIP primer of SEQ ID NO: 3 and SEQ ID NO: 4, the BIP primer of SEQ ID NO: 5, the LF primer of SEQ ID NO: 6 and SEQ ID NO: 7 And the LB primer of SEQ ID NO: 8.

The primer set according to the present application can quickly and accurately detect whether a sample is infected with dengue virus, regardless of the serotype, and does not particularly show cross-reactivity to the 18 kinds of microorganisms listed in Table 3, and thus dengue virus infection. You can more efficiently discriminate whether or not.

One or more primers of the primer set according to the present application may be labeled with a detectable labeling substance, and in particular, may be efficiently used for screening a large amount of specimens.

In another aspect, the present application also provides a method for detecting the presence or absence of four serotype dengue viruses in vitro using the primer set disclosed herein.

In one embodiment, the method comprises the steps of: providing a sample derived from a subject in need of detection of the virus; Providing a set of primers disclosed herein; Performing a LAMP reaction using the primer and sample: And if there is a difference by analyzing the LAMP reaction result and comparing it with a negative control sample, for example, if the LAMP reaction is positive, the sample is infected with dengue virus. And determining as.

When using the method according to the present application, with a ^{sensitivity of at least 10 3} copies under the conditions disclosed herein, it is possible to specifically detect dengue virus without cross-reactivity to infectious microorganisms as defined by the FDA.

In one embodiment, the method according to the present application is that the synthesis of cDNA from the RNA genome and the LAMP reaction using the same as a template are performed as a single reaction, which is a reverse transcription LAMP reaction.

In one embodiment, the analysis of the LAMP reaction result of the method according to the present application may be determined through measurement using at least one of color change measurement or turbidity of the reactant.

We are able to detect dengue virus types 1, 2, 3, and 4 with high sensitivity and specificity using one type of primer set, so it is possible to quickly and easily detect dengue virus infection, increasing the speed and convenience of response. do. In particular, it is recognized as a reliable diagnostic method and can be used in the field only if it does not exhibit cross-reactivity to 18 kinds of microorganisms that are similar to the nucleotide sequence of dengue virus or that cause symptoms similar to those in the case of infection.The primer set according to the present application can be used for these infectious microorganisms. Dengue virus infection can be specifically detected without cross-reactivity.

Figure 1 shows the sequence of the four serotype types of dengue virus and shows the location of the primer set designed herein to be able to detect all four types at the same time.
Figure 2a is a result of testing the specificity of the LAMP reaction using the primer set according to the present application, it shows that all four serotypes of the dengue virus can be detected.
2B is a result of an experiment on the cross-reactivity of the primer set according to the present application, indicating that it is specific for dengue virus without cross-reactivity against infectious microorganisms defined by the FDA.
3 is an experiment result of the sensitivity of the LAMP reaction using the primer set according to the present application.
4 is an analysis of the primer set according to the present application for each of the four types of viruses in terms of sensitivity to the existing primer set, and when the primer set according to the present application was used, it was found that the sensitivity was 100 times excellent in a total reaction of 40 minutes. . Due to the characteristic of infectious diseases, it is very important to show high sensitivity with a short reaction time because a large number of samples must be processed. In particular, since the number of copies of the virus is not high at the beginning of infection, accurate diagnosis cannot be made if the sensitivity is low. It is very important to prevent fatal complications such as high fever and hemorrhagic fever by treating antibiotics through accurate diagnosis in the early stages of infection.
5 shows the registration status of the samples analyzed using the primer set according to the present application.

The present application uses the LAMP (Loop Mediated Isothermal Amplification, Loop Mediated Isothermal Amplification Method) method and the detection site used in the conventional PCR method, using different detection sites that match all of the base sequences of the four serotypes. It is based on the discovery that dengue virus can be detected quickly and easily with high accuracy and sensitivity.

Since all four serotypes of the dengue virus have different nucleotide sequences, it is difficult to prepare a primer for detecting the four serotypes dengue viruses with one type of primer set. To solve this problem, 4 serotype sequences were sorted to find a 3'UTR region with a similar base sequence, and from this, 6 existing primers were used to create primers that satisfies all 4 serotype sequences. In the set of LAMP method (F3, B3, FIP, BIP, LF, LB), FIP and LF of types 1, 3 and 2, 4 were separately produced and added, so that a total of 8 primers (F3, B3, FIP-1) , FIP-2, BIP-1, LF-1, LF-2, LB) were able to detect all types 1,2,3,4 with one set. Since it is not possible to find a sequence that matches all of the base sequences using six primers, it is impossible to prepare a primer that can detect the four serotypes of the dengue virus. By making each, it was possible to prepare a primer that satisfies all four serotype nucleotide sequences.

Thus, in one aspect, the present application relates to a primer set for LAMP analysis capable of detecting dengue virus types 1, 2, 3, and 4 regardless of serotype.

The term "primer" as used herein refers to a nucleic acid molecule that is a single-stranded oligonucleotide in which a nucleotide is added to the 3'end by covalent bonds in a nucleic acid amplification or synthesis reaction using a polymerase. The primer set according to the present application is used for the amplification of nucleic acids, that is, LAMP analysis.

As used herein, the term "nucleic acid" refers to an RNA or DNA molecule comprising one or more nucleotides in any form, including single-stranded or double-stranded oligonucleotides or polynucleotides, or analogs thereof and derivatives thereof. The nucleic acid of dengue virus for which the primer set according to the present application is used includes RNA of dengue virus and cDNA synthesized therefrom. It also includes all or part of the viral genome.

As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably, and either one includes both. The term "primer" may also be used interchangeably with "oligonucleotide" and "polynucleotide", and includes nucleic acids (RNA or DNA), or aptamers.

As used herein, the term “detection” refers to whether or not to be infected with dengue virus, or to determine the prognosis of an infected subject, to recur after treatment after infection, or to therametrics (e.g., to provide information about the efficacy of the treatment). Monitoring).

As used herein, the term "target" or "target nucleic acid" refers to a nucleic acid sequence to which the primer set herein is bound and specifically amplified by the primer set according to the present application, and includes RNA or DNA, especially DNA.

The primers according to the present invention specifically bind to the target nucleic acid through complementarity and amplify the target nucleic acid in the LAMP manner. Here, in particular, only genes specific to each type of virus described in FIG. 1 can be specifically detected.

The target amplified using the primer set according to the present application is 3'UTR (Untranslated Region, untranslated region). In the present application, as a result of designing a primer set including a total of 8 primers at the above region, all four serotypes of viruses can be detected with high sensitivity and specificity. In particular, in order to increase sensitivity and specificity, in the present application, FIP and LF were separately grouped among the 4 serotypes to produce a total of 8 primers that match the base sequences of the 4 serotypes. By producing and performing the reaction, it was possible to detect dengue virus with high sensitivity and specificity.

This is superior to the previously disclosed primer set in terms of sensitivity, reaction time, cost, and convenience of reaction. The primers produced by Teoh et al., BMC Infectious Diseases 2013, 13:387, which disclose previously published LAMP reaction primers that detect all four types of dengue, have a total of 9 primers in one set, and the sensitivity is side by side. In compared to the primers disclosed herein, all 4 types were found to be 100 times lower. In another document (Dauner et al., Diagnostic Microbiology and Infectious Disease 83 (2015) 30-36), a total of 11 primers are one set, as well as in terms of time, the primer set disclosed herein is 10 ³ copies in a 40-minute reaction. On the other hand, the sensitivity is the same only when the reaction for 1 hour disclosed in the above document shows the same sensitivity.

If there are many types of primers included in a primer set in one reaction, problems such as an increase in cost, a decrease in amplification reaction due to a decrease in concentration that can be used for amplification of each primer, a non-specific reaction due to binding between primers, and a decrease in primer concentration, etc. This happens.

The primers according to the present application contain a small number of primers compared to those disclosed in the existing literature, but exhibit high specificity, sensitivity, and rapid reaction time.

LAMP is a nucleic acid amplification method capable of amplifying a target nucleic acid with high sensitivity and specificity under isothermal conditions (Notomi, T. et al. 2000. Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Res 28, E63). displacement) active DNA polymerase and six sites of the target nucleic acid, that is, in the direction of 5′→3′, the B3 region, the B2 region, the B1 region, the F1c region, the F2c region and the F3c region (the region complementary to the region is B3c , B2c, B1c, F1, F2 and F3) specifically designed primer sets of at least 4 to 6 are used (Korean Patent No. 612551; Nagamine, K. et al. 2002. Accelerated reaction by Loop mediated isothermal amplification using loop primers.See Mol Cell Probes 16, 223-9, etc.). The standard LAMP reaction includes at least four types of two inner primers FIP (forward inner primer), BIP (backward inner primer), and two outer primers F3 (forward outer primer) and B3 (backward outer primer). For rapid reaction (Accelerated LAMP), a total of 6 primers can be used, including additionally LF (Loop F) and LB (Loop B).

The LAMP reaction largely includes an initial structure production step, a cycling amplification step, and an extension and recycling step. In the initiation step, the F2 portion constituting the inner primer FIP binds to the F2c region of the target nucleic acid to initiate the reaction. Subsequently, the outer primer F3 primer binds to the F3c region of the target nucleic acid to initiate strand-replacement DNA synthesis, thereby generating a single-stranded nucleic acid. This single strand forms a ring at the 5'end through F1/F1c bonding, and the second inner primer BIP and the outer primer B3 bind to this, whereby DNA synthesis and strand replacement DNA synthesis occur. As a result, a single strand with a stem-loop formed at both ends of the dumbbell shape is formed, and DNA synthesis starts from the 3'end of the F1 region. Then, in the cycling amplification step, only two inner primers are used, and at first, one inner primer binds to the ring, resulting in the synthesis of strand-replacement DNA, forming the original ring, forming a single strand and a new ring that doubled the stem portion in the ring structure. Forming single strands are generated, and the amplification products are amplified up to ^{about 10 9} within 1 hour by continuing to synthesize strand-replacement DNA using this as a template. For more detailed principles and characteristics and functions of each F3 primer, B3 primer, FIP primer, BIP primer, Loop F primer (LF), and Loop B (LB) primer, the previously mentioned documents Notomi et al., 2000 and Nagamine et al. al., 2002 may be referred to. RT (reverse transcription) LAMP is a template that first synthesizes RNA into DNA and then performs LAMP, and a method of synthesizing RNA into DNA is known in the art.

As used herein, the term "complementary" or "complementary" refers to a state in which a primer or oligonucleotide can sequence-specifically bind to a target nucleic acid through pairing of a Watson-Crick base under a predetermined hybridization (hybridization), binding or annealing condition. As a term, it includes all of partial complementarity, substantially complementary, and perfectly complementary. Substantially complementarity means complementarity to the extent that the two strands of nucleic acid sequences are not completely complementary to each other, but the effect according to the present application by binding to the target nucleic acid, that is, the target sequence can be amplified through the LAMP method.

Due to the characteristic of the LAMP reaction, since the length of the amplification product increases in each step by the length of the BIP in FIP, it is important to select the gene for which the LAMP primer is to be designed and the region in which the primer is to be designed. The primer according to the present application was selected from the 3'UTR region as described herein, a region that is easy to analyze and does not form a secondary structure, and in one embodiment according to the present application, F2, a template that is continuously used in the LAMP reaction The size between B2 and B2 can be selected to be from 160 bp to 250 bp, especially about 200 bp. For example, in one embodiment according to the present invention, when the total reaction time is 20 to 40 minutes, the region is determined so that the length is from a minimum of 160 bp to a maximum of 250 bp, and a primer is designed therefrom. If the region in which the primer is designed is out of the above range, there is a disadvantage that the reaction time becomes longer. As the size of the final product increases, the amplification time increases, and the change in the buffer color due to the amplification product is delayed. This is because the larger the size of the region, the longer the amplification time and the buffer color change due to the amplification product becomes slower.

The length of the primer specifically binding to the target nucleic acid or its complementary sequence according to the present application can be appropriately determined in consideration of reaction conditions such as reaction time, temperature, and base distribution of the target nucleic acid, and in the case of F3 and B3, it is at least 15 nucleotides. , FIP and BIP are at least 30 nucleotides, and LF and LB are at least 15 nucleotides, but are not limited thereto.

In addition, when the GC content of the primer exceeds 45% <Primer <60%, the sensitivity decreases when the content is high, whereas the specificity decreases when the concentration is low because of the characteristic of mutually binding by triple bonds between G and C nucleic acid molecules.

In addition, in relation to the Tm of the primer, due to the characteristics of the LAMP method that reacts under isothermal conditions at 63°C, when the Tm value of the primer is lowered outside 60°C <Primer <65°C, the specificity is lowered due to the influence of dimers, etc. When the temperature is over 65℃, the efficiency of binding to the gene decreases and the sensitivity decreases, and a primer can be designed in consideration of this.

In particular, since hybridization of the four primers to the template is important in the initiation stage, it is important to design the Tm value to fall within a specific range, and the size and sequence can be determined in consideration of this. For example, the Tm of the F2 and B2 sequences included in BIP and FIP is determined in the range of about 60 to 65 degrees, which is the optimum temperature for the polymerase used, for example, when Bst polymerase is used, and F2 and B2 F1c and B1c located outside of the Tm value are determined to be slightly higher than this, so that a looped out structure can be formed immediately after the single strand is released from the template. In addition, the Tm of the outer primers F3 and B3 is set lower compared to the Tm of F2 and B2 to induce an initial reaction to occur in the inner primers rather than the outer primers.

In the method according to the present application, LF and LB primers may additionally be used. In this case, LF can be manufactured in the region between F2 and F1c, and the intervals between F2 and F1c are manufactured in 1bp to 10bp, respectively, and in the region between F2 and F1c, GC content (45% <Primer <60%) and Tm (60℃) <Primer <65°C) can be prepared to prepare a primer having a length of about 15bp-25bp. In the case of LB, it may be manufactured in a region between B1c and B2, and manufacturing conditions may refer to LF. When LF and LB are added, there is an effect of improving the sensitivity of LAMP and detecting the result in a faster time.

The term "hybridization" or "hybridization" as used herein refers to a reaction in which two strands of complementary nucleic acid molecules form a sequence-specific complex through hydrogen bonding. Hybridization may constitute the step of binding of a target nucleic acid or a template by the primer in the LAMP reaction in which the primer according to the present application is used. The degree of hybridization is influenced by various factors such as the degree of complementarity of any two nucleic acid molecules involved in it, their melting temperature (Tm) or stringency of the hybridization. Hybridization reaction conditions can also be determined in consideration of this. The stringency of the hybridization reaction is a condition that determines how easily any two nucleic acid molecules to be hybridized with each other can be combined.Complementarity, reaction temperature, ionic strength, and/or concentration of general substances included in the hybridization reaction solution And it may be determined according to the type, for example, J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed., 2012; and F. M. Ausubel, Ed., Short Protocols in Molecular Biology, Wiley; You can refer to those described in 5th Ed, 2002.

Specific binding or hybridization herein means that a specific nucleic acid molecule or primer does not substantially bind to a nucleic acid other than a target nucleic acid, but only binds to a target nucleic acid. The primer according to the present application can specifically bind to the target dengue virus nucleic acid or its complementary sequence according to the present application under high stringency conditions, and in template-based DNA synthesis, the primer sequence length, buffer solution, nucleic acid type, pH, magnesium concentration And it may be determined in consideration of the reaction temperature and the like.

The primer according to the present application specifically binds to the target nucleic acid of the dengue virus, and in one embodiment, binds to the target nucleic acid or its complementary sequence with 100% complementarity. It is designed to be able to combine all the parts where the bases do not match by using a mixed base.

The primers specifically binding to the target nucleic acid or its complementary sequence according to the present application are at least 15 nucleotides for F3 and B3, at least 30 nucleotides for FIP and BIP, and at least 15 nucleotides for LF and LB. It has at least 70% complementarity, in particular 80% complementarity, more 90% complementarity, even more particularly 95%, 96%, 97%, 98%, 99% or 100% complementarity with the corresponding portion of the target nucleic acid.

In one embodiment, the primers according to the present application include primers represented by SEQ ID NOs: 1 to 8 and substantially the same, and specifically binds to a target nucleic acid or a complementary sequence thereof. Primers of substantially the same sequence have 70% complementarity, in particular 80% complementarity, more particularly 90% complementarity, even more particularly 95%, 96%, 97%, 98%, 99% or It has 100% complementarity. Some of the primers included in the primer set according to the present application are designed to contain mixed bases at specific positions and bind to viruses of each serotype with 100% complementarity.

The primers according to the present application and compositions or kits and methods comprising the same can be used for the analysis of any biological sample in need of detection of dengue virus.

In one embodiment, the biological sample is typically a liquid or cell or tissue sample of a mammal, including humans. Samples that can be used herein include, but are limited to, for example whole blood, serum, and plasma. It also includes a fraction or derivative of the blood, cells or tissues. When using cells or tissues, cells themselves or lysates of cells or tissues may be used.

In one embodiment, it is derived from a subject in need of detection of dengue virus, such subject comprising a sample from a subject in need or expected to require detection of dengue virus, a mammal, particularly a primate, particularly a human.

In other embodiments, the biological sample may be a dried form, a liquid, or a tissue sample.

In addition, such a biological sample may be obtained directly from the subject by a conventional sample obtaining method requiring detection of dengue virus immediately before the test, or may be previously separated and stored.

In another embodiment, a tissue/cell or a lysate thereof obtained from a subject in need of dengue virus detection; Alternatively, a cell culture or a lysate thereof may be used in vitro, but is not limited thereto.

In one embodiment according to the present application, serum or a nucleic acid isolated therefrom is used.

In another embodiment, the biological sample may be a nucleic acid isolated from the liquid or tissue sample described above as a sample. Separation refers to separation from a sample containing nucleic acids, and includes separation from various purity levels.

The primers according to the present application and compositions or kits and methods comprising the same are used for analysis in a LAMP manner using any biological sample in which dengue virus is likely to be present.

In the reaction for LAMP analysis, a primer set according to the present application, dNTPs, buffers, magnesium and DNA polymerase, and biological samples requiring detection of dengue virus are used. In addition, it may further include a substance such as betaine or DMSO to enhance the reaction.

The primer set included in the reaction product is as described above, and 8 primer sets may be used.

Magnesium is used in the form of salts such as magnesium acetate, magnesium chloride or magnesium sulfate.

The buffer may be, for example, sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer, or Tricine buffer.

DNA polymerases that can be used in the reaction include polymerases derived from thermophilic microorganisms, in particular polymerases lacking the 5'->3' exonuclease function. Non-limiting examples include Bacillus stearothermophilus , Bst, DNA polymerase; Thermus thermophilus , Tth, DNA polymerase; Thermus aquaticus , Taq, DNA polymerase; Thermococcus litoralis DNA polymerase; Pyrococcus furiosus , Pfu, DNA polymerase; And Bacillus caldotenax DNA polymerase.

In addition, nucleotide analogs may also be used in place of dNTPs in the reactants. Nucleotide analogs are those that are modified or not found in nature and can be polymerized alone or together with the natural nucleotides in the process of template-derived DNA synthesis. Including an analog of a nucleotide base as an analog that can be polymerized through such Wason Creek base pairing is, for example, a substituted purine or pyrimidine, deazapurine, methylpurine, methylpyrimidine, aminopurine, aminopyrimidine, thio. Purine, thiopyrimidine, indole, pyrrole, 7-deazaguanine, 7-methylguanine, hypoxanthine, shadowcytosine, shadowisocytosine, isocytosine, isoguanine, 2-thiopyrimidine, 4-thiothymine , 6-thioguanine, nitropyrrole, nitroindole and 4-methylindole. Nucleotides comprising substituted deoxyribose analogs include substituted or unsubstituted arabinose, substituted or unsubstituted xylose, and substituted or unsubstituted pyranose. The nucleotides containing the phosphate ester analogs include phosphorothioate, phosphorodithioate, phosphoroamidate, phosphorocellonoate, phosphoroanilothioate, phosphoroanilideate, phosphoroamidate, boron. And alkylphosphonates such as phosphates, phospholiesters and methylphosphonates.

In particular, since the genome of the dengue virus is RNA, the synthesis of cDNA is required for the LAMP reaction. In the LAMP reaction according to the present application, cDNA synthesis and amplification are performed in one step, for this purpose, reverse transcriptase is included in one reaction product, and RT -LAMP reaction is carried out. A known reverse transcriptase may be used, and in one embodiment, an AMR reverse transcriptase is used, but is not limited thereto.

In another embodiment, after synthesizing cDNA from the extracted RNA by a separate reaction, the LAMP reaction may be performed using the cDNA as a template.

In one embodiment, the reactants are 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM of each LF and LB primer, 0.4 M betaine, 10 mM MgSO ₄ , 1.4 mM dNTPs, in a total of 25 μl. 1×ThermoPol reaction buffer (New England Biolabs), 16U Bst DNA polymerase (New England Biolabs), 0.625 U AMV reverse transcriptase (invitrogen), and 2 μl of dengue virus as a sample required to be detected, contains RNA.

In another embodiment, the reactants are 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM of each LF and LB primer, 0.4 M betaine, 10 mM MgSO ₄ , 1.4 mM dNTPs, in a total of 25 μl. 1×ThermoPol reaction buffer (New England Biolabs), 16U Bst DNA polymerase (New England Biolabs), and cDNA synthesized from RNA as a sample requiring detection of 2 μl of dengue virus are included.

The reaction is then reacted at a temperature suitable for the activity of the DNA polymerase. The reaction temperature can be determined without difficulty in consideration of the enzyme used in the reaction and the nucleic acid sequence of the target. The reaction is then reacted at a time suitable for amplification of the target nucleic acid. Those skilled in the art will be able to determine the reaction time in consideration of the reaction conditions, for example, it may be determined from 15 minutes to 60 minutes, but may vary depending on the amount of target nucleic acid contained in the sample, and may be determined. For example, the reaction time can be increased to increase the sensitivity.

The LAMP analysis according to the present application may be analyzed in a variety of analysis formats, and for example, a liquid phase reaction or some components may be performed in a state in which they are adsorbed to a solid substrate.

The primer according to the present application and the composition or kit and method comprising the same are used for analysis in the LAMP method using any biological sample in which dengue virus may be present, and the amplified product has a sequence corresponding to the molecule used as a template, It can be analyzed by a variety of methods known in the art.

After amplification, the amplification reaction product can be detected by various methods, for example, color change, turbidity change, fluorescence and/or electrophoresis of the reaction solution according to DNA synthesis, and the detected product is positive and/or It is compared with the product of the negative control sample and used for quantitative or qualitative analysis of dengue virus.

In one embodiment, the amplification product according to the present application may be detected as a color change of a reaction solution according to DNA synthesis. In this case, the reaction solution may contain an appropriate indicator, and a product of a positive and/or negative control sample using HNB (hydroxy naphthol blue) as a dye that changes color in response to the concentration of magnesium ions in the reaction solution. Compared with, it can quantitatively or qualitatively analyze dengue virus, and in particular, it can be detected with the naked eye, increasing its convenience.

In other embodiments, the amplification product may be detected by a direct or indirect method. In the direct method, a detectably labeled primer may be used, and whether a nucleic acid is amplified is detected through a signal emitted through the labeled primer after specific binding to the nucleic acid, and real-time detection is possible. In the indirect detection method, a labeled probe capable of binding to the amplified nucleic acid may be used. Detectably labeled herein is labeled with a substance capable of emitting a detectable signal using any suitable method, such as spectroscopic, optical, photochemical, biochemical, enzymatic, electrical and/or immunochemical methods. Means substance. Such substances include, for example, fluorescent moieties, chemiluminescent moieties, bioluminescent moieties, magnetic particles, enzymes, substrates, radioactive and chromophore substances.

In one embodiment, the label for detection is not limited thereto, but includes a compound capable of generating or eliminating detectable fluorescence, chemiluminescence, or bioluminescence signals such as light emission, light scattering, and light absorbing substances, See, for example, Garman A., Non-Radioactive Labeling, Academic Press 1997. Fluorescent substances are, for example, but not limited to, fluorescein (e.g., U.S. Patent 6,020,481), rhodamine (e.g. U.S. Patent 6,191,278), benzophenoxazine (e.g. U.S. Patent 6,140,500), donors and acceptors. Energy transfer fluorescent dyes (e.g. U.S. Patent 5,945,526) and cyanine (e.g. WO1997-45539), including lysamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham), Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, 6-FAM, Fluorescein Isothiocyanate , HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, Tetramethylrhodamine, and/or Texas Red as well as other detectables. It contains any fluorescent moiety capable of generating a signal. The fluorescent dye is 6-carboxyfluorescein; 2',4',1,4,-tetrachlorofluorescein; And 2′,4′,5′,7′,1,4-hexachlorofluorescein, but are not limited thereto. In one embodiment, SYBR-Green, 6-carboxyfluorescein ("FAM"), TET, ROX, VIC, or JOE is used as a fluorescent label. In one embodiment, a probe labeled with two fluorescent materials, a reporter fluorescent material and an erasing fluorescent material, is used, and in this case, a fluorescent material emitting a spectrum with a distinguishable wavelength is used. In addition, as a marker, a compound capable of enhancing, stabilizing, or affecting the binding of a nucleic acid, for example, an intercalator including ethyl bromide and SYBR-Green, a minor groove conjugate, and a crosslinkable functional group may be used. , Without limitation, Blackburn et al., eds. See “DNA and RNA Structure” in Nucleic Acids in Chemistry and Biology (1996).

In addition, when detection of non-specific amplification due to the occurrence of non-specific priming of the primer is necessary, a non-specific nucleic acid binding agent such as acid bromide or picogreen is used in a control reaction that does not contain a template, and non-specifically synthesized total amplification. The amount of product can be determined.

In another aspect, the present application also relates to a composition or kit for detecting dengue virus comprising a primer or primer set according to the present application as described above.

The composition according to the present application may include components used in the LAMP reaction as described above except for a template and a primer set according to the present application.

The kit according to the present application may provide the components used in the LAMP reaction as described above except for the template and the primer set according to the present application separately or in one tube. The kit according to the present application further includes a positive control, a negative control and instructions for use. A sample that does not contain nucleic acid as a negative control group, and a pattern control group may contain one or more nucleic acids to be detected.

The primers, kits and compositions according to the present application detect dengue virus at the nucleic acid level in a biological sample, and compare it with a control or reference group, and the presence or absence of a nucleic acid, an increase in the nucleic acid, or an infection with a dengue virus depending on the degree of change in the nucleic acid concentration. If you receive post-treatment, you can predict the effectiveness of the treatment.

In this aspect, the present application also relates to a method of detecting dengue virus in a biological sample, in order to provide necessary information for detection, diagnosis or prognosis of dengue virus.

Accordingly, in one embodiment, the method according to the present disclosure includes the steps of providing a sample derived from a subject in need of detection of the dengue virus; Performing a LAMP reaction using the above-described primer set: And if there is a difference by analyzing the LAMP reaction result and comparing it with a sample of a negative control group or a reference group, determining the sample as dengue virus infection do. The primer included in the method may be prepared by referring to the above. When there is a difference compared to the negative control group, the LAMP reaction in the sample is positive, and whether or not there is a positive difference can be determined by referring to Examples of the present application. In addition, it includes a case where there is a difference in the presence or absence of a nucleic acid, an increase in nucleic acid, and a degree of change in the nucleic acid concentration compared to a sample of a negative control group or a reference group, and those skilled in the art will be able to determine it with reference to Examples herein.

In another embodiment, the method according to the present application is performed using a primer set, composition or kit as described above, and in one embodiment, providing a sample derived from a subject in need of detection of dengue virus; Amplifying a target nucleic acid from the sample by performing LAMP using the primer set according to the present application; Then, comprising the step of comparing the amplification result with a sample of a control group, and when there is a change in the amount of the amplification product compared to the subject sample, the method for detecting dengue virus, comprising the step of determining that the amplification product is infected with dengue virus. About.

The method according to the present application can determine the presence of infection through qualitative or quantitative analysis, for example, when the amount is increased compared to the negative control, or is not detected in the negative control, but a positive reaction is obtained from the sample, or If more than a certain amount is detected, it may be judged as an infection. The judgment or judgment criteria may be easily determined in consideration of standards in the art.

Samples, reagents, amplification methods, and reaction conditions used in the method of the present application may be referred to as described above.

The present invention will be described in detail through the following examples, but this embodiment is only illustrative and does not limit the scope of the present application in any way.

Unless otherwise stated, the present invention can be carried out using conventional techniques within the skill level of those skilled in the art in cell biology, cell culture, molecular biology, gene transformation technology, microbiology, and DNA recombination technology. In addition, for a more detailed description of the general technology, see Molecular Biotechnology: (Bernard et al., ASM press 2014); Molecular Cloning: A Laboratory Manual, 4th Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2012); Short Protocols in Molecular Biology, 5th Ed. (Ausubel F. et al. eds., John Wiley & Sons 2002).

Example

Example 1. Securing Dengue Virus Standards and Preparation of RNA Samples

Dengue 1, 2, 3, and 4 virus standards were distributed from the Korea Centers for Disease Control and Prevention, and RNA of the standard was extracted using the QIAamp Viral RNA Mini Kit (Qiagene, Germany) according to the manufacturer's method. The extracted RNA was used as a template RNA in the RT-LAMP reaction after measuring the concentration and purity with a UV spectrometer. Template RNA was stored at -80°C until use.

Example 2. Preparation of dengue virus common primer

In this application, as RT-LAMP primers, primers capable of detecting the presence or absence of dengue virus irrespective of dengue 1, 2, 3, 4 types were designed. Dengue 1, 2, 3, 4 gene sequences were obtained from GenBank, and the strains used are shown in Table 1 below.

[Table 1]

In order to construct a primer capable of detecting all dengue types 1, 2, 3, and 4, the gene sequences of dengue types 1, 2, 3, and 4 were aligned using a ClustalX multiple sequence alignment program. As for the dengue common detection primer, a 3'UTR site in which the gene sequences of the dengue 1, 2, 3, and 4 types most matched was used to prepare a primer. As for the primers of the present application for detecting all four types of dengue virus, six primers of the conventional LAMP method were constructed as one set, not one set. With the addition of FIP and LF primers, all four types could be detected using one primer set. FIP1 and LF1 have 100% complementarity with types 1 and 3, and FIP2 and LF2 have 100% complementarity with types 2 and 4. In addition, it was designed to be able to combine all the parts where the bases do not match by using a mixed base.

It is optimal in terms of specificity and sensitivity, and is designed to be optimized for LAMP reaction. Primers were synthesized by requesting the outside, and the primer sequences are shown in Table 2.

[Table 2] Common Dengue Primers

Mixed Base mark in Table 2: R: A or G, Y: T or C, W: T or A, M: C or A

Example 3. Sensitivity and specificity analysis of dengue virus common primers

For the sensitivity analysis of the reaction using the primer set prepared in Example 2, 1, 2, 3, 4 type virus RNA was extracted, respectively, and then ^{serially diluted to 10 4} copies, 10 ³ copies, and 10 ² copies, followed by a one-step RT-LAMP reaction. Was performed.

In order to measure the specificity, the specificity analysis or cross-reactivity for 18 kinds of microorganisms (see Table 3) recommended by the FDA guidelines were also carried out, and the used genome concentrations are also listed in Table 3. It is recognized as a reliable diagnostic method and can be used in the field only if it does not show cross-reactivity to 18 kinds of microorganisms that are similar to the nucleotide sequence of dengue virus or cause symptoms similar to those of infection.

[Table 3]

In particular, since the genome of the dengue virus is RNA, it is necessary to synthesize cDNA for the LAMP reaction, and in the LAMP reaction according to the present application, cDNA synthesis and amplification are performed in one step, and for this purpose, reverse transcriptase is included in one reaction product.

The reactants were 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM of each LF and LB primer, 0.4 M betaine, 10 mM MgSO ₄ , 1.4 mM dNTPs, 1×ThermoPol reaction in a total of 25 μl. buffer (New England Biolabs), 16U Bst DNA polymerase (New England Biolabs), 0.625 U AMV reverse transcriptase (invitrogen), and samples required for detection of 2 μl of dengue virus were included. RT-LAMP was reacted at 58°C for 40 minutes and then reacted at 80°C for 2 minutes to terminate.

The common primers were able to detect all types 1, 2, 3, and 4 to the same degree (see Fig. 2a), and did not show cross-reactivity against 18 types of microorganisms recommended by the FDA guidelines, indicating that they are very specific to dengue virus ( 2b).

In addition, the sensitivity of the common primer was 1,000 copies. (See Fig. 3).

Example 4. Clinical sensitivity and specificity measurement of the primer set according to the present application

Clinical sensitivity and specificity analysis using the primer set of the present application was performed as follows.

RNA isolated from serum (Korea University Guro Hospital) was used as a sample. Figure 5 shows the status of registration of the specimens. A total of 252 specimens were used, and there were no specimens excluded from dropout and violation of the clinical trial protocol in this clinical trial.

4-1 Target group included in the analysis

The main analysis of this clinical trial was conducted on FA Set, and the secondary analysis was on PP Set. In this clinical trial, only FA set results were presented because the subjects of the FA set and PP set were identical. The FA set was to perform statistical analysis including the sample that was dropped out, and the PP set was to perform statistical analysis without including the sample that was dropped out. Only presented.

4-2 Efficacy evaluation result

As the primary efficacy endpoint of this clinical trial, if the gene measurement value of the dengue virus diagnosis exceeded the analysis threshold, it was judged as positive. In the diagnosis of dengue virus infection, the clinical sensitivity and clinical specificity were calculated, and a 95% confidence interval was presented.

In the FA group, the sensitivity of the dengue virus diagnosis was confirmed to be 99.21% (125/126 samples), and the clinical specificity was confirmed to be 100% (126/126 samples) (Table 4).

[Table 4] Clinical sensitivity and clinical specificity of the primer set according to the present application

Example 5. Full sensitivity analysis of dengue virus common primers compared to existing primer sets

For each dengue virus type, a set of existing primers capable of detecting dengue virus (Ref 1: Teoh et al., BMC Infectious Diseases (2013): 387; Ref2: Dauner et al., Diagnostic Microbiology and Infectious Disease 83 ( 2105): 30-36) and compared with the sensitivity were analyzed. Samples used for the reaction (dengue virus RNA_ of each serotype, reagents, and analysis conditions were the same as in Example 3.

The results are shown in Figure 4. As shown, the primer set according to the present application was found to be 100 times superior to the conventional primer set in terms of sensitivity.

Furthermore, as described in the table below, the set according to the present application includes a total of 8 primers, whereas the existing primers include 9 and 11, respectively, which increases the cost of manufacture as well as non-specific reactions due to the binding between the primers. There are drawbacks that can be done.

[Table 5] Comparison between the primer set of the present application and the existing primer set

When reacting for 40 minutes under the reaction conditions according to the present application, the sensitivity was 100 times different. This can be judged to be due to the fact that the primers of MMonitor according to the present application are designed to have 100% complementarity to the template, while the compared primers have about 90% complementarity. In addition, the number of primers is also 8, which is less than that of thesis primers, thus reducing non-specific binding between primers, showing higher sensitivity compared to thesis primers.

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concept of the present application defined in the following claims are also included in the scope of the present application. It belongs to.

All technical terms used in the present invention, unless otherwise defined, are used in the same meaning as those of ordinary skill in the art generally understand in the related field of the present invention. The contents of all publications referred to herein by reference are incorporated into the present invention.

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Claims (6)

A primer set for LAMP analysis for common detection of dengue virus of four serotypes of serotypes 1, 2, 3 and 4,
The primer set is the F3 primer of SEQ ID NO: 1, the B3 primer of SEQ ID NO: 2, the FIP primer of SEQ ID NO: 3 and SEQ ID NO: 4, the BIP primer of SEQ ID NO: 5, the LF primer of SEQ ID NO: 6 and SEQ ID NO: 7 and SEQ ID NO: 8 It contains the LB primer of,
The primer set does not exhibit cross-reactivity to the following microorganisms, a primer set:
West Nile virus;
Japanese encephalitis virus;
Saint Louis encephalitis virus;
Yellow fever virus;
Hepatitis A virus;
Hepatitis B virus;
Hepatitis C virus;
Epstein Barr virus;
Borrelia burgdorferi;
Leptospira species;
Cytomegalovirus;
Varicella zoster virus (VZV);
Herpes simplex virus;
Eastern equine encephalitis virus;
Chikungunya virus;
Influenza virus A;
Influenza virus B; And
Measles virus.
The method of claim 1,
One or more primers of the set are labeled with a detectable labeling substance, a primer set for LAMP analysis for common detection of serotypes of dengue virus.
A method for detecting the presence or absence of dengue virus of four serotypes of serotypes 1, 2, 3, and 4 in an in vitro comprising the following steps, the method comprising:
Providing a sample derived from a subject in need of detection of the dengue virus;
Providing a primer set according to claim 1;
Performing a LAMP reaction in a reactant including the primer set and the sample: And
Analyzing the result of the LAMP reaction and comparing it with a negative control sample, if there is a difference, determining the sample as dengue virus infection.
The method of claim 3,
The sample is serum or RNA isolated from the serum or cDNA synthesized from the RNA.
The method of claim 3,
The sample is RNA isolated from the subject, and the LAMP reaction is a reverse transcription LAMP reaction.
The method of claim 3,
The analysis of the LAMP reaction product is determined through measurement using at least one of color change measurement or turbidity of the reaction product.

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