WO2021116735A1 - Procédés et kits de détection du virus de la dengue - Google Patents

Procédés et kits de détection du virus de la dengue Download PDF

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WO2021116735A1
WO2021116735A1 PCT/IB2019/060680 IB2019060680W WO2021116735A1 WO 2021116735 A1 WO2021116735 A1 WO 2021116735A1 IB 2019060680 W IB2019060680 W IB 2019060680W WO 2021116735 A1 WO2021116735 A1 WO 2021116735A1
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seq
nucleotide sequence
primer
serotype
primer set
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PCT/IB2019/060680
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Yong-Li Pan
Hsien-Chang Chang
Shainn-Wei Wang
Guey-Chuen Perng
Ko-Lun Yen
Chun-Yee Lau
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National Cheng Kung University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • the disclosure relates to a kit for detecting dengue virus (DENV).
  • the disclosure also relates to a method of detecting the presence of DENV in a biological sample using the kit.
  • DENV Dengue virus
  • C nucleocapsid protein
  • prM membrane precursor protein
  • E envelope protein
  • NS non-structural proteins
  • DENV serotypes There are four antigenically-distinguishable DENV serotypes, namely, DENV- 1, DENV-2, DENV-3 and DENV-4, and each serotype contains phylogenet ically distinct genotypes. It is estimated that yearly over 300 million people are infected with DENV, particularly in tropical and subtropical areas, such as Africa, Asia, the Pacific, Australia, and the Americas.
  • DF dengue fever
  • DHF dengue hemorrhagic fever
  • DSS hemorrhagic shock syndrome
  • Symptoms of DENV infection includes high fever, severe headache, severe joint, muscle, and bone pain, rash persistent vomiting, severe abdominal pain, and hemorrhagic manifestations such as ascites, pleural effusions, bleeding of internal organs or hemorrhagic shock.
  • Hu S. F. et al. disclosed four serotype-specific primer sets (each including six primers) used in RT-LAMP to target different DENV serotypes, which were designed by inspection of multiple sequence alignments of the non-structural protein (NS) 2Aof DENV-1,NS4B of DENV-2, NS4Aof DENV-3 and the 3'-UTR of DENV-4, respectively.
  • the detection results show that no cross-reactions of the four serotypes were observed, indicating that the designed primer sets are suitable for serotyping DENV (Hu S. F. et al. (2015), BMC Microbiology, 15:265).
  • this disclosure provides a method of detecting the presence of dengue virus (DENV) in a biological sample that can alleviate at least one of the drawbacks of the prior art.
  • the method includes : subjecting nucleic acids in the biological sample to a nucleic acid amplification reaction with a reaction mixture that includes at least one primer set for amplifying a target nucleic acid of the dengue virus; and detecting the presence or absence of an amplification product obtainable from the nucleic acid amplification reaction, wherein the presence of the amplification product is indicative of the presence of the dengue virus in the biological sample.
  • the at least one primer set is selected from the group consisting of:
  • a species-specific primer set for amplifying a region of 3' UTR of the DENV including a fifth forward outer primer having a nucleotide sequence of SEQ ID NO: 25 and a fifth backward outer primer having a nucleotide sequence of SEQ ID NO: 26.
  • this disclosure provides a kit for detecting dengue virus (DENV) that can alleviate at least one of the drawbacks of the prior art.
  • the kit includes at least one of the abovementioned primer sets for amplifying a target nucleic acid of the dengue virus .
  • RT-LAMP reverse transcription loop-mediated isothermal amplification
  • nucleic acid refers to a sequence of nucleotides connected by phosphodiester linkages and may be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule in either single-stranded or double-stranded form.
  • the nucleic acid may comprise naturally occurring nucleotides and known analogues thereof, as well as nucleotides that are modified in the sugar and/or phosphate moieties.
  • nucleic acid as used herein is interchangeable with the terms “gene”, “cDNA”, “mRNA”, “oligo-nucleotide”, and “polynucleotide” in use.
  • DNA fragment refers to a DNA polymer, in the form of a separate segment or as a component of a larger DNA construct, which has been derived either from isolated DNA or synthesized chemically or enzymatically such as by methods disclosed elsewhere.
  • nucleic acid sequence in addition to the specific sequences described herein, also covers its complementary sequence, and the conservative analogs, related naturally occurring structural variants and/or synthetic non-naturally occurring analogs thereof.
  • nucleic acid sequence is represented, it will be understood that the nucleotides are in 5' to 3' order from left to right and that "A” denotes deoxyadenosine or an analog thereof, “C” denotes deoxycytidine or an analog thereof, “G” denotes deoxyguanosine or an analog thereof, and “T” denotes deoxythymidine or an analog thereof.
  • 3' refers to a region or position in a polynucleotide or oligonucleotide 3' (i.e., downstream) from another region or position in the same polynucleotide or oligonucleotide.
  • the term “5'” refers to a region or position in a polynucleotide or oligonucleotide 5' (i.e., upstream) from another region or position in the same polynucleotide or oligonucleotide .
  • nucleic acid molecules refer to the end of the nucleic acid molecule which contains a free hydroxyl or phosphate group attached to the 5'carbon of the terminal pentose sugar.
  • the term "primer” refers to an oligonucleotide of defined sequence that is designed to hybridize with a complementary, primer-specific portion of a target polynucleotide sequence and undergo primer extension.
  • the primer can function as the starting point for the enzymatic polymerization of nucleotides using a polymerase.
  • the primer should be long enough to prevent itself from annealing to sequences other than the complementary portion.
  • the primer is between 10 to 50 nucleotides in length.
  • the primer is between 13 to 30 nucleotides in length.
  • the primer used herein may also be used in nucleic acid amplification.
  • amplification may refer to an increase of the number of copies of a target sequence or a complementary sequence of the target sequence, such as a gene or fragment of a gene.
  • a "copy” or “amplicon” does not necessarily mean perfect sequence complementarity or identity to template sequence.
  • copies can include nucleotide analogs such as deoxyinosine , intentional sequence alterations (such as sequence alterations introduced through a primer including a sequence that is hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.
  • the products of an amplification reaction are called amplification products.
  • the nucleic acid amplification may be performed using any method known in the art, e.g. a method employing multiple heat cycles during the amplification, or a method performed at a constant temperature (i.e., isothermal amplification) .
  • An example of in vitro amplification requiring heat cycles is polymerase chain reaction (PCR), in which a sample (such as a biological sample from a subject) is contacted with a pair of oligonucleotide primers, under conditions that allow for hybridization of the primers to a nucleic acid molecule in the sample.
  • PCR polymerase chain reaction
  • the primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid molecule.
  • in vitro amplification techniques may include, but are not limited to, PCR, quantitative PCR (qPCR), reverse transcription PCR (RT-PCR) , quantitative RT-PCR (RT-qPCR) , nested polymerase chain reaction, hot-start polymerase chain reaction, multiplex polymerase chain reaction, ligase chain reaction (LCR), gap ligase chain reaction (gLCR), etc.
  • isothermal amplification may be performed at a constant temperature, or a major aspect of the amplification process occurs at a constant temperature, i.e., without significant changes in temperature. Thus, it is carried out substantially at about the same single temperature.
  • isothermal amplification is substantially isothermal, for example, may include small variations in temperature, such as changes in temperature of no more than about 1°C to 3°C during the amplification reaction.
  • target sequence and “target nucleic acid” can be interchangeably used, and refer to a particular nucleic acid sequence which is to be detected and/or amplified.
  • target sequences include nucleic acid sequences to which the primers complex in a PCR reaction.
  • Target sequences may also include a probe hybridizing region with which a detection probe will form a stable hybrid under desired conditions.
  • a target sequence may be single-stranded or double-stranded.
  • target sequences of interest may be within the NS5 gene or 3'-untranlated region (3 -UTR) of DENV.
  • a "species-specific primer” is capable of hybridizing with the target sequence of more than one serotype of DENV.
  • hybridization and “annealing” can be interchangeably used, and refer to the process in which complementary nucleic acid strands react to form a “hybrid” or “duplex” that is stabilized via hydrogen bonding between the bases of the nucleotide residues .
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogsteen binding, or in any other sequence specific manner.
  • duplex and “hybrid” can be interchangeably used, and refer to a structure formed as a result of hybridization of two complementary sequences of nucleic acids.
  • duplexes can be formed by the complementary binding of two DNA segments to each other, two RNA segments to each other, or of a DNA segment to an RNA segment.
  • Either or both members of such duplexes can contain modified nucleotides and/or nucleotide analogues as well as nucleoside analogues.
  • such duplexes are formed as the result of binding of one or more probes to a sample sequence .
  • a method of detecting the presence of dengue virus (DENV) in a biological sample includes: subjecting nucleic acids in the biological sample to a nucleic acid amplification reaction with a reaction mixture that includes at least one primer set for amplifying a target nucleic acid of the dengue virus; and detecting the presence or absence of an amplification product obtainable from the nucleic acid amplification reaction. The presence of the amplification product is indicative of the presence of the dengue virus in the biological sample.
  • DEV dengue virus
  • the at least one primer set for amplifying the target sequence of the dengue virus includes:
  • a first serotype-specific primer set for amplifying a region of NS5 gene of dengue virus serotype 1 which includes a first forward outer primer having a nucleotide sequence of SEQ ID NO: 1 and a first backward outer primer having a nucleotide sequence of SEQ ID NO: 2;
  • a second serotype-specific primer set for amplifying a region of NS5 gene of dengue virus serotype 2 which includes a second forward outer primer having a nucleotide sequence of SEQ ID NO: 7 and a second backward outer primer having a nucleotide sequence of SEQ ID NO: 8;
  • a third serotype-specific primer set for amplifying a region of NS5 gene of dengue virus serotype 3 which includes a third forward outer primer having a nucleotide sequence of SEQ ID NO: 13 and a third backward outer primer having a nucleotide sequence of SEQ ID NO: 14;
  • a fourth serotype-specific primer set for amplifying a region of NS5 gene of dengue virus serotype 4 (DENV-4)which includes a fourth forward outer primer having a nucleotide sequence of SEQ ID NO: 19 and a fourth backward outer primer having a nucleotide sequence of SEQ ID NO: 20; and
  • a species-specific primer set for amplifying a region of 3'UTR of the DENV which includes a fifth forward outer primer having a nucleotide sequence of SEQ ID NO: 25 and a fifth backward outer primer having a nucleotide sequence of SEQ ID NO: 26.
  • the method of this disclosure may assay different target sequences simultaneously on the same sample, particularly target sequences specific for the DENV (i.e., differentiate the DENV from other arboviruses of genera Flavivirus (e.g., JEV and ZIKV) and Alphavirus (e.g., CHIKV)) and different serotypes of the DENV to perform DENV serotype determination.
  • target sequences specific for the DENV i.e., differentiate the DENV from other arboviruses of genera Flavivirus (e.g., JEV and ZIKV) and Alphavirus (e.g., CHIKV)
  • the amplification of the different target sequences may be performed in parallel, in separate reaction mixtures or in the same reaction mixture and detected independently.
  • the nucleic acids e.g., viral genomic RNA of the DENV
  • the procedures and conditions for extracting nucleic acids are within the expertise and routine skills of those skilled in the art (for example, see Teoh B.T. et al. (2013), supra and Hu S. F. et al. (2015), supra).
  • the nucleic acid amplification reaction may be conducted using at least one of the following methodologies: polymerase chain reaction, quantitative polymerase chain reaction (qPCR), reverse transcription polymerase chain reaction (RT-PCR), reverse transcription quantitative polymerase chain reaction (RT-qPCR), nested PCR, hot-start PCR, multiplex PCR, in situ PCR, single cell PCR, touchdown PCR, ligase chain reaction (LCR), gap ligase chain reaction (gLCR) and isothermal amplification.
  • qPCR quantitative polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • RT-qPCR reverse transcription quantitative polymerase chain reaction
  • nested PCR hot-start PCR
  • multiplex PCR multiplex PCR
  • in situ PCR single cell PCR
  • touchdown PCR ligase chain reaction
  • gLCR gap ligase chain reaction
  • isothermal amplification isothermal amplification.
  • the nucleic acid amplification reaction is an isothermal amplification reaction using any of the various natural or engineered enzymes available for this purpose, e.g., DNA polymerases having strong strand-displacement activity in isothermal conditions, thereby obviating the need for thermal cycling.
  • DNA polymerases are well-known in the art, and may include DNA polymerase long fragment (LF) of thermophilic bacteria, such as Bacillus stearothermophilus (Bst), Bacillus Smithii (Bsm), Geobacillus sp .M (GspM) and Thermodesulfatator indicus (Tin), and engineered variants therefrom as well as Taq DNA polymerase variants.
  • LF DNA polymerase long fragment
  • Bst Bacillus stearothermophilus
  • Bsm Bacillus Smithii
  • GspM Geobacillus sp .M
  • Tin Thermodesulfatator indicus
  • Non-limiting examples of polymerase which can be used to perform the method of the disclosure include Bst DNA polymerase, Bst 2,0 DNA polymerase, Bst 2.0 WarrnStart DNA polymerase (New England Biolabs, Ipswich, Mass.), GspM LF DNA polymerase, GspSSD LF DNA polymerase, Tin exo-LF DNA polymerase and SD DNA polymerase Phi29 DNA polymerase, Bsu DNA polymerase, OmniAmpTM DNA polymerase (Lucigen, Middleton, Mich.), Taq DNA polymerase, Vent ® and Deep Vent ® DNA polymerases (New England Biolabs), 9°NmTM DNA polymerase (New England Biolabs), Klenow fragment of DNA polymerase I, PhiPRDl DNA polymerase, phage M2 DNA polymerase, T4 DNA polymerase, and T5 DNA polymerase, which are usually used at 50-75°C, more generally at 55 to 65°C.
  • about 1 to 20 U (such as about 1 to 15 U, about 2 to 12 U, about 10 to 20 U, about 2 to 10 U, or about 5 to 10 U) of DNA polymerase is included in the reaction.
  • the polymerase used has strand displacement activity and lacks 5'-3' exonuclease activity.
  • the DNA polymerase used is Est 2,0 WarmStartTM DNA polymerase, for example, about 8 U of Bst 2,0 WarmStartTM DNA polymerase per reaction.
  • Examples of the isothermal amplification may include, but are not limited to, strand-displacement amplification (SDA), rolling-circle amplification (RCA), cross-priming amplification (CPA), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), transcription-mediated amplification, loop-mediated amplification (LAMP), and competitive annealing mediated isothermal amplification (CAMP).
  • SDA strand-displacement amplification
  • RCA rolling-circle amplification
  • CPA cross-priming amplification
  • NASBA nucleic acid sequence-based amplification
  • RPA recombinase polymerase amplification
  • HDA helicase-dependent amplification
  • transcription-mediated amplification loop-mediated amplification
  • LAMP loop-mediated amplification
  • CAMP competitive annealing mediated isothermal amplification
  • RNA When the nucleic acid to be amplified or detected is RNA, a reverse transcription is conducted prior to the isothermal amplification of the target sequence.
  • the reverse transcription may be performed using any suitable reverse transcriptase (RT).
  • RT is well-known in the art and may include, but is not limited to, Avian Myeloblastosis Virus (AMV) RT and Moloney Murine Leukemia virus (MMLV) RT.
  • AMV Avian Myeloblastosis Virus
  • MMLV Moloney Murine Leukemia virus
  • the Reverse transcription and DNA amplification may be performed in the same reaction mixture that includes the DNA polymerase and RT enzymes applied.
  • the method of the disclosure may use a DNA polymerase having both strong displacement activity and RT activity, such as Pyrophage 3173 DNA polymerase .
  • the nucleic acid amplification reaction is conducted by RT-LAMP, where denaturation of the DNA template is not required and thus the LAMP reaction can be conducted under isothermal conditions (e.g., ranging from 60°C to 67°C).
  • amplification process becomes self-sustaining, and proceeds at a constant temperature in a continuous and exponential manner (rather than a cyclic manner, like PCR).
  • an additional pair of loop primers i.e., forward loop primer (LF) and backward loop primer (LB)
  • LF forward loop primer
  • LB backward loop primer
  • the first serotype-specific primer set for LAMP assay includes the first forward and backward outer primers, and a first forward inner primer having a nucleotide sequence of SEQ ID NO: 3 and a first backward inner primer having a nucleotide sequence of SEQ ID NO: 4.
  • the first serotype-specific primer set may further include one of a first forward loop primer having a nucleotide sequence of SEQ ID NO: 5, a first backward loop primer having a nucleotide sequence of SEQ ID NO: 6, and a combination thereof to accelerate the reaction of LAMP.
  • the second serotype-specific primer set for LAMP assay includes the second forward and backward outer primers, and a second forward inner primer having a nucleotide sequence of SEQ ID NO: 9 and a second backward inner primer having a nucleotide sequence of SEQ ID NO: 10.
  • the second serotype-specific primer set may further include one of a second forward loop primer having a nucleotide sequence of SEQ ID NO: 11, a second backward loop primer having a nucleotide sequence of SEQ ID NO: 12, and a combination thereof to accelerate the reaction of LAMP.
  • the third serotype-specific primer set for LAMP assay includes the third forward and backward outer primers, and a third forward inner primer having a nucleotide sequence of SEQ ID NO: 15 and a third backward inner primer having a nucleotide sequence of SEQ ID NO: 16.
  • the third serotype-specific primer set may also further include one of a third forward loop primer having a nucleotide sequence of SEQ ID NO: 17, a third backward loop primer having a nucleotide sequence of SEQ ID NO: 18, and a combination thereof to accelerate the reaction of LAMP.
  • the fourth serotype-specific primer set for LAMP assay includes the fourth forward and backward outer primers, and a fourth forward inner primer having a nucleotide sequence of SEQ ID NO: 21 and a fourth backward inner primer having a nucleotide sequence of SEQ ID NO: 22.
  • the fourth serotype-specific primer set may also further include one of a fourth forward loop primer having a nucleotide sequence of SEQ ID NO: 23, and a fourth backward loop primer having a nucleotide sequence of SEQ ID NO: 24, and a combination thereof.
  • the nucleic acid amplification reaction is conducted by CAMP.
  • CAMP The principle of the CAMP method is disclosed in Rui Mao, et al. (2016), The Royal Society of Chemistry, 143:639-642. Like LAMP, the CAMP method relies on auto-cycling DNA synthesis by a DNA polymerase with strand displacement activity, and may only require a pair of specially designed primers, i.e., a forward inner primer and a backward inner primer. Additional outer primers and loop primer (s) may be designed and selected for CAMP, which may improve the amplification efficiency and sensitivity by accelerating isothermal amplification and reducing detection time. The extra additions of outer primers, loop primers and both in the CAMP reaction are termed as O-CAMP, L-CAMP and OL-CAMP, respectively.
  • the species-specific primer set for CAMP includes a fifth forward inner primer having a nucleotide sequence of SEQ ID NO: 27 and a fifth backward inner primer having a nucleotide sequence of SEQ ID NO: 28.
  • the species-specific primer set for CAMP may further include the fifth forward and backward outer primers and/or a fifth loop primer having a nucleotide sequence of SEQ ID NO: 29.
  • the reaction mixture for the LAMP or CAMP reaction further includes a suitable buffer (such as a phosphate buffer or Tris buffer).
  • the buffer may also include additional components, such as potassium and/or sodium salts (such as KC1 or NaCl), magnesium and/or manganese salts (e.g., MgCl2, MgS04, MnCi2, and/or MnS0 4 ), and/or ammonium salts (e.g., (NH 4 ) 2 S0 4 )), detergents (e.g., TWEEN ® -20, TRIT0N ® -X100), or other additives (such as betaine or dime thylsulfoxide ).
  • potassium and/or sodium salts such as KC1 or NaCl
  • magnesium and/or manganese salts e.g., MgCl2, MgS04, MnCi2, and/or MnS0 4
  • ammonium salts e.g., (NH 4 ) 2 S
  • the buffer includes 50 mMKCl, 10 mM (NH 4 ) 2 S0 4 , 0.1% TWEEN ® -20, 4 mM MgS0 4 , and 0.8 M betaine.
  • Exemplary commercially available reaction buffers include 1 c Isothermal Amplification Buffer (New England Biolabs, Ipswich, Mass.), LoopAmp Reaction Mix (Eiken Chemical Co., Ltd., Tokyo, Japan), and ILLUMIGENE reaction buffer (Meridian Bioscience, Inc., Cincinnati, Ohio).
  • the reaction mixture also includes nucleotides or nucleotide analogs. In some examples, an equimolar mixture of dATP, dCTP, dGTP, and dTTP (referred to as dNTPs) is included.
  • a detectable label may be attached or conjugated to the primer (s) according to this disclosure using techniques well known to those skilled in the art, so as to quantitatively detect the target dengue virus.
  • the detectable label suitable for use in the present disclosure include, but are not limited to: a hapten label, such as biotin and digoxigenin; a chemiluminescent label, such as acridinium esters, thioesters, orsulfonamides , luminol, isoluminol, phenanthridinium esters, and the like; a fluorescent label, such as fluorescein, rhodamine, FAM, TET, HEX, JOE, TAMA, NTB, TAMRA, ROX, VIC, NED, cyanin dye, Texas Red, DABCYL , DABSYL, malachite green, AlexaFluor dye, LC-Red dye, PromoFluor dye, and derivatives thereof; a hapten label
  • the primers may be modified to have 15 to 35 nucleotides in length by adding at least one nucleotide residue at 5' and/or 3' terminal end thereof or by deleting at least one nucleotide residue from 5' and/or 3' terminal end thereof.
  • the amplification product may be detected using any of the various methods available for this purpose which are well-known in the art.
  • the detection method may be turbidity measurement, fluorescence detection, bioluminescence detection, gel electrophoresis, colorimetric detection, immunoenzymat ic detection, electrochemical detection, and combinations thereof.
  • the detection may be semi-quantitative or quantitative.
  • the detection may also be real-time detection, wherein the signal resulting from the presence of the amplification product is measured during the course of the nucleic acid amplification reaction to monitor the accumulation of specific amplification products.
  • Turbidity measurement is used when the amplification reaction, such as LAMP, produces large amounts of magnesium pyrophosphate (a white precipitate) and dsDNA, which allow visual inspection of results or with a turbidimeter.
  • Fluorescence detection may use DNA intercalating dyes, fluorescent molecular beacon probes or a fluorescence metal indicator (such as calcein).
  • Bioluminescence detection may be through measurement of bioluminescent output of the coupled conversion of inorganic pyrophosphate produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase.
  • Colorimetric detection may use a colored indicator for alkaline metal ions, such as hydroxy naphthol blue (HNB) (see Goto et al., BioTechniques, 2009 Mar, 46(3) :167-72) and/or a pH indicator with a capacity of prominent color alteration within the pH range of 7.5 ⁇ 1.1, such as phenol red (pH 6.8-8.2), neutral red (pH 6.8-8.0), cresol red (pH 7.2-8.8), etc.
  • HNB hydroxy naphthol blue
  • Electrochemical detection may use a pH meter for direct measurement of released hydrogen ions during the amplification reaction, such as LAMP, or integrated electrodes for measuring decreases in current resulting from increasing binding of electrochemical ly-active DNA-binding redox reporters, such as Methylene Blue, to amplification products.
  • a pH meter for direct measurement of released hydrogen ions during the amplification reaction, such as LAMP
  • integrated electrodes for measuring decreases in current resulting from increasing binding of electrochemical ly-active DNA-binding redox reporters, such as Methylene Blue, to amplification products.
  • Immunoenzymat ic detection includes enzyme-linked immunosorbent assays (ELISA) and lateral flow immunoassays based on the use of specific probes (see, e.g. Hashimoto M. et al., Malar J. r 2018 Jun 19, 17 (1):235; Sun Yu-Ling et al., J. Vet Med Sci. 2014 Apr, 76(4):509-516) .
  • ELISA enzyme-linked immunosorbent assays
  • lateral flow immunoassays based on the use of specific probes
  • the "biological sample” is a sample obtained from an organism (such as an animal subject) or from components (such as cells and tissues) of the organism.
  • the biological sample include, but are not limited to, a blood sample, a plasma sample, a serum sample, a corneal tissue sample, a tear sample, a saliva sample, a cerebrospinal fluid sample, a feces sample, a tissue biopsy, a surgical specimen, a urine sample, a fine needle aspirate, and combinations thereof .
  • a kit for detecting dengue virus includes at least one of the primer sets as described above.
  • the kit further includes the colored indicator for alkaline metal ions, the pH indicator or the combination thereof.
  • the kit may also further include a nucleic acid dye for monitoring the DNA amplification product.
  • the nucleic acid dye include, but are not limited to, ethidium bromide (EtBr), SYBR GREEN I, SYBR GREEN II, SYBR Orange, SYBR GOLD, propidium iodide (PI), SYTOX Blue and SYPRO Ruby, etc .
  • Vero cells used in the examples were purchased from American Type Culture Collection (ATCC, Manassas, Va ., USA).
  • BiomateTM 100 bp + 3 K DNA RTU Ladder (100-3 k) (Cat.No. BR332-500, BiomateTM) used in the examples were purchased from Rainbow Biotechnology Co., Ltd.
  • RNA of viruses were extracted using Direct-zolTM RNA MiniPrep (Cat. No. R2052, Unimed Healthcare Inc.) in accordance with the manufacturer's instructions.
  • Example 1 Serotype-specific primer sets for detecting DENV serotypes 1 to 4 NS5 protein is an RNA-dependent RNA polymerase involved in genome replication. NS5 genes of all four DENV serotypes were analyzed from GenBank database, maintained by the National Institutes of Health, USA.
  • the conserved region of NS5 gene in reference spans nucleotides 7839 to 8029 in the full length genome sequence of DENV-1 (Genbank accession number NC_0 01477.1), nucleotides 9769 to 10011 in the full length genome sequence of DENV-2 (Genbank accession number NC_001474 .2), nucleotides 7565 to 7881 in the full length genome sequence of DENV-3 (Genbank accession number NC_001475.2), or nucleotides 9549 to 9826 in the full length genome sequence of DENV-4 (Genbank accession number NC_002640.1).
  • the primer pairs and loop primer pairs for detecting and differentiating DENV serotypes 1 to 4 were designed based on the conserved regions of NS5 genes of respective DENV serotypes using PrimerExplorer V4 software.
  • the specificity of the primer pairs and the loop primer pairs were analyzed by BLAST analysis available in the GenBank of the NCBI website (http://www.nebi.nlm.nih.gov/BLAST).
  • a serotype-specific primer set 1 comprising two primer pairs (designated as DGI-Opti-B F3 / DGI-Opti-B B3 and DGI-Opti-B FIP / DGI-Opti-B BIP) and a loop primer pair (designated as DGI-Opti-B LF / DGI-Opti-B LB) for DENV-1
  • a serotype-sped fic primer set 2 comprising two primer pairs (designated as DGII-Opti-C F3 / DGII-Opti-C B3 and DGII-Opti-C FIP / DGII-Opti-C BIP) and a loop primer pair (designated as
  • DGII-Opti-C LF / DGII-Opti-C LB for DENV-2
  • a serotype-specific primer set 3 comprising two primer pairs (designated as DGIII-Opti F3 / DGIII-Opti B3 and DGIII-Opti FIP / DGIII-Opti BIP) and a loop primer pair (designated as DGIII-Opti LF / DGIII-Opti LB) for DENV-3
  • a serotype-specific primer set 4 comprising two primer pairs (designated as DGIV-Opti F3 /
  • DGIV-Opti B3 and DGIV-Opti FIP / DGIV-Opti BIP and a loop primer pair (designated as DGIV-Opti LF /
  • DGIV-Opti LB for DENV-4 were obtained for specific amplification of each DENV serotype.
  • the serotype-specific primer set 1 for detecting DENV-1 a the nucleotide residues corresponding to the nucleotides shown in the boxes b: the nucleotide residues corresponding to the underlined nucleotides.
  • the serotype-specific primer set 2 for detecting DENV-2 a the nucleotide residues corresponding to the nucleotides shown in the boxes b: the nucleotide residues corresponding to the underlined nucleotides.
  • the serotype-specific primer set 3 for detecting DENV-3 a the nucleotide residues corresponding to the nucleotides shown in the boxes b: the nucleotide residues corresponding to the underlined nucleotides.
  • the serotype-specific primer set 4 for detecting DENV-4 a the nucleotide residues corresponding to the nucleotides shown in the boxes b: the nucleotide residues corresponding to the underlined nucleotides.
  • the universal primer pairs and loop primers for detecting all the four DENV serotypes were designed based on the conserved regions of 3'-UTR of DENV serotypes 1-4 using PrimerExplorer V4 software.
  • the specificity of the universal primer pairs and the loop primers were analyzed by BLAST analysis available in the GenBank of the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST). Therefore, a species-specific primer set comprising two primer pairs (designated as CAMP-B F2 / CAMP-B B2 and CAMP-B NF / CAMP-B NR) and a loop primer (designated as CAMP-B LFr) were obtained.
  • the detailed information of the abovement ioned primer pairs and the loop primer is summarized in Table 6.
  • the species-specific primer set for detecting DENV a the nucleotide residues corresponding to the nucleotides shown in the boxes b: the nucleotide residues corresponding to the underlined nucleotides.
  • Example 3 Evaluation for detection and differentiation effect of serotype-specific primer sets according to this disclosure on DENV serotypes 1 to 4
  • R-LAMP reverse transcription loop-mediated isothermal amplification
  • the color change of the reaction mix in the reaction tube was visually observed, and the reaction mix changed color from red to yellow in response to the pH change during nucleic acid amplification.
  • the resultant product having a yellow color indicated that the amplification of RNA of DENV was successful.
  • the resultant products were subjected to 2% agarose gel electrophoresis to verify the presence of amp1icons.
  • Figures 1-4 respectively show detection and differentiation effect of the serotype-specific primer sets 1-4 on DENV serotypes 1-4.
  • the gel electrophoresis analysis demonstrated that using the serotype-specific primer set 1, a ladder-like banding pattern was observed in the corresponding target virus (namely DENV-1).
  • a RT-LAMP amplified product having a yellow color was visually observed in DENV-1.
  • No ladder-like banding patterns and color change were observed in the non-target viruses (namely DENV-2, DENV-3, DENV-4, and the non-DENV viruses) .
  • similar results were observed with respect to the serotype-specific primer sets 2-4 (see Figures 2-4).
  • R-CAMP reverse transcription competitive annealing mediated isothermal amplification
  • RNA of a respective one of JEV, ZIKV, DENV-1, DENV-2, DENV-3, and DENV-4 obtained from Example 3 was used as a template and subjected to the RT-CAMP using the species-specific primer set described in Table 6.
  • the RT-CAMP reaction conditions were similar to those described in Table 7, except that the isothermal-amplification reaction was conducted at 61°C for 90 minutes, and the final concentrations of the primers were as follows:CAMP-B F2 (0.2 mM) / CAMP-B B2 (0.2 mM), CAMP-B NF (1.6 mM) / CAMP-B NR (1.6 mM), and CAMP-B LFr (0.8 mM).
  • the resultant products were subjected to colorimetric assay and agarose gel electrophoresis analysis according to the method described in Example 3.
  • Figure 5 shows the universal detection effect of the species-specific primer set on DENV serotypes 1-4. It can be seen from Figure 5 that a ladder-like banding pattern and a RT-CAMP amplified product having a yellow color were observed in all of the four DENV serotypes. No ladder-like banding patterns and color change were observed in the non-target viruses (namely JEV, ZIKV, and CHIKV). These results indicate that the species-specific primer set of the present disclosure can be used to universally and efficiently detect DENV.
  • Example 5 Evaluation for effect of aforesaid five primer sets according to this disclosure on detecting DENV in plasma specimens Experimental materials :
  • Plasma specimens were obtained from 14 patients (11 men, average age of which was 53 years; 3 women, average age of which was 51 years) clinically diagnosed with DENV-2 infection enrolled in National Cheng Rung University Hospital (Tainan, Taiwan). The plasma specimens were collected with an approval from the Institutional Review Board (IRB)/the Committee of Medical Ethics and Human Experiments of National Cheng Rung University Hospital (Tainan, Taiwan). All procedures involving human subjects adhered to the Declaration of Helsinki.
  • IRS Institutional Review Board
  • the plasma specimen of each patient was cultured and passaged 1 time in Vero cells (culture conditions: 37°C and 5% CO2). The supernatant of the respective resultant culture was collected, followed by being subjected to extraction of RNA according to section 1 of "General Procedures" .
  • RNA total RNA (5 ng) of the plasma specimen of the respective patient was used as a template and subjected to RT-LAMP assay according to the method described in Example 3 for detection and differentiation of DENV serotypes 1-4.
  • RT-LAMP assay according to the method described in Example 3 for detection and differentiation of DENV serotypes 1-4.
  • the same experiments were performed using a known primer set described in B.T. Teoh et al. (2013), BMC Infect. Dis., doi :
  • the primers of the known primer set were synthesized by Genomics Biosci & Tech (Taipei, Taiwan). The detailed information of the known primer set is summarized in Table 8.
  • RNA 5 ng
  • RT-CAMP assay according to the method described in Example 4 for universal detection of DENV.
  • Table 9 shows the detection effects of the aforesaid six primer sets on DENV. It can be seen from Table 9 that all the plasma specimens were tested positive for DENV-2 by the RT-LAMP assay using the serotype-sped fic primer set 2 or the known primer set described in B.T. Teoh et al. (2013) . In addition, all the plasma specimens were tested positive for DENV by the RT-CAMP assay using the species-specific primer set. None of the serotype-specific primer sets 1, 3, and 4 reacted with RNA of DENV-2, demonstrating that these primer sets did not cross-react with DENV-2.
  • DGIV-Opti LB primer for detecting Dengue virus serotype 4 ⁇ 400> 24 catgtagaaa ccaggatgaa ctg 23

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Abstract

L'invention concerne un kit comprenant au moins l'un parmi quatre ensembles d'amorces spécifiques du sérotype respectivement destinés à être utilisés dans la détection des sérotypes 1 à 4 du virus de la dengue et un ensemble d'amorces spécifiques d'une espèce destiné à être utilisé dans la détection du virus de la dengue. L'invention concerne également un procédé de détection de la présence de virus de la dengue dans un échantillon biologique à l'aide d'au moins l'un des ensembles d'amorces.
PCT/IB2019/060680 2019-12-12 2019-12-12 Procédés et kits de détection du virus de la dengue WO2021116735A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040126387A1 (en) * 1999-12-01 2004-07-01 Callahan Johnny Dale Serotype and dengue group specific flurogenic probe based PCR (TaqMan) assays against the respective C and NS5 genomic and 3' non-coding regions of dengue virus
MY141230A (en) * 2005-07-28 2010-03-31 Univ Malaya Detection, serotyping and quantification of dengue virus
CN106868223A (zh) * 2017-04-19 2017-06-20 广州中医药大学 一种登革病毒ns5基因片段的不对称pcr扩增引物及其应用
KR20190041237A (ko) * 2017-10-12 2019-04-22 고려대학교 산학협력단 뎅기 바이러스 검출용 올리고뉴클레오티드 세트 및 이의 용도

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Publication number Priority date Publication date Assignee Title
US20040126387A1 (en) * 1999-12-01 2004-07-01 Callahan Johnny Dale Serotype and dengue group specific flurogenic probe based PCR (TaqMan) assays against the respective C and NS5 genomic and 3' non-coding regions of dengue virus
MY141230A (en) * 2005-07-28 2010-03-31 Univ Malaya Detection, serotyping and quantification of dengue virus
CN106868223A (zh) * 2017-04-19 2017-06-20 广州中医药大学 一种登革病毒ns5基因片段的不对称pcr扩增引物及其应用
KR20190041237A (ko) * 2017-10-12 2019-04-22 고려대학교 산학협력단 뎅기 바이러스 검출용 올리고뉴클레오티드 세트 및 이의 용도

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Title
CHIEN LI-JUNG; LIAO TSAI-LING; SHU PEI-YUN; HUANG JYH-HSIUNG; GUBLER DUANE J; CHANG GWONG-JEN J: "Development of real-time reverse transcriptase PCR assays to detect and serotype dengue viruses", JOURNAL OF CLINICAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 44, no. 4, 1 April 2006 (2006-04-01), US, pages 1295 - 1304, XP009139728, ISSN: 0095-1137, DOI: 10.1128/JCM.44.4.1295�1304.2006 *

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