WO2003014397A1 - Sonde destinee a la detection de virus enteriques, kit de detection et procede de detection de virus enteriques - Google Patents

Sonde destinee a la detection de virus enteriques, kit de detection et procede de detection de virus enteriques Download PDF

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WO2003014397A1
WO2003014397A1 PCT/KR2002/001530 KR0201530W WO03014397A1 WO 2003014397 A1 WO2003014397 A1 WO 2003014397A1 KR 0201530 W KR0201530 W KR 0201530W WO 03014397 A1 WO03014397 A1 WO 03014397A1
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probe
seq
enteric
virus
dna
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PCT/KR2002/001530
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English (en)
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Yong-Seok Jeong
Sung-Wook Yoon
Jeong-Mi Kim
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Biomedlab Corporation
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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 present invention relates to a probe(s) for detecting enteric viruses, a detection kit comprising the probe(s), and a detection method for enteric viruses using the same. More specifically, the present invention provides probes for detecting enteric viruses including pan-enteroviruses, adenoviruses, rotaviruses, astroviruses, Norwalk viruses, Norwalk-like viruses, caliciviruses, and small round-structured viruses (SRSV), a detection kit comprising the probes, and a detection method for enteric viruses using the same.
  • probes for detecting enteric viruses including pan-enteroviruses, adenoviruses, rotaviruses, astroviruses, Norwalk viruses, Norwalk-like viruses, caliciviruses, and small round-structured viruses (SRSV), a detection kit comprising the probes, and a detection method for enteric viruses using the same.
  • SRSV small round-structured viruses
  • enteric virus generally means a virus which primarily infects a human intestinal tract via mouth from the natural environment such as from water or soil, and then diffuses within a human body, thereby seriously influencing the individual involved.
  • enteric viruses about 100 kinds of enteric viruses are known, most of them (about 67) being human enteroviruses (Glass RI, Bresee J, Jiang B, Gentsch J, Ando T, Fankhauser R, Noel J, Parashar U, Rosen B, Monroe SS. Gastroenteritis viruses: an overview. Novartis Found Symp 238:5-19; discussion 19-25, 2001 ; Morens, D. M., M. A. Pallansch, and M. Moore. Polioviruses and other enteroviruses. In Belshe, (ed.) ( Text Book of Human Virology, Mosbey Year Books. 427-497, 1991).
  • Viral enteritis is caused by a virus infecting intestines, and symptoms
  • Diarrhea is less serious than influenza and tuberculosis, but it affects daily life and industrial behavior, thereby causing large economic losses.
  • viruses causing diarrhea are known to include rotaviruses, adenoviruses, caliciviruses, Norwalk viruses, Norwalk-like viruses, astroviruses, SRSV, reoviruses, etc. (F. Le Guyader, L. Haugarreau, L. Miossec, E. Dubois, and M. Pommepuy. Three-Year Study to Assess Human Enteric Viruses in Shellfish. Appl. Envir. Microbiol. 66:3241-3248, 2000).
  • the hepatitis A and E viruses which are classified as enteric viruses, also cause liver disease.
  • Viral diarrhea is usually transferred through physical contact with patients, and through food stuff contaminated by the virus. Contamination of food stuff can be caused by carefulness, for example, by a patient cooking without washing hands after using the restroom. Drinking water and shellfish can be infected by the virus contained in sewage including a patient's excreta.
  • Viral diarrhea occurs worldwide. Viruses often show epidermal characteristics, as with the rotavirus and astrovirus that are epidermal in winter. The adenovirus, however, is epidermal in all seasons. Nonbacterial collective diarrhea can be commonly caused by viruses occurring at collective feeding places such as schools, kindergartens, institutions for the aged, cruise ships, and dormitories. Major diseases that the enteric viruses cause via primary infection and secondary infection, and the detecting and identifying method of the viruses, are summarized in the following Table 1.
  • the diseases caused by enteroviruses have a tendency to be epidermal worldwide, with symptoms of nonbacterial encephalomeningitis, encephalomyocarditis, encephalitis, epidermal hemorrhagic keratoconjunctivitis, and foot-mouth disease causing eruptive, flare, as well as intestine-related diseases.
  • the methods of detection and identification for enteric viruses include i) a direct-detecting method of the enteric virus contained in feces and cerebrospinal fluid (CSF) of patients and concentrated water samples, ii) an antibody neutralization test after virus enrichment by inoculating the sample to a cultured cell, or iii) a detecting method consisting of the steps of amplifying the enriched virus through PCR or RT-PCR, and then hybridizing the PCR product with southern blot hybridization or sequencing.
  • CSF cerebrospinal fluid
  • the identification method using a specific antigen is accepted by the WHO as a standard method, but it has disadvantages in methodological and economical aspects in that before an antigen-antibody reaction, the virus must be isolated and amplified, and in that the method can only be used for restricted field due to cross-reactivity.
  • the nucleic acid-based detection method has been widely used recently, but it has a problem in that when a clinical sample is directly used for detection, it is not possible to determine whether the virus is infectious or not.
  • the virus in a sample must be replicated or amplified by firstly infecting a cultured cell before being hybridized with a specific probe. Then, the viral genetic materials replicated in the cultured cell can be detected by hybridizing with a specific probe directly, or after amplification of the viral genetic materials through PCR or RT-PCR (LA Jaykus, R De Leon, and MD Sobsey: A virion concentration method for detection of human enteric viruses in oysters by PCR and oligoprobe hybridization. Appl. Environ. Microbiol. 62:2074-2080. 1996).
  • the nucleic acid-based detection method When the nucleic acid-based detection method is applied for detecting the enteric virus, the nucleotide sequence must be analyzed for subsequent identification, and then compared with that of a reference sequence.
  • the nucleic acid-based detection method is more or less advantageous in economic and experimental aspects compared with the antibody neutralization method, the method requires professional manpower, expensive experimental devices, and a large space. Thus, an identification
  • the present invention provides probes for detecting enteric viruses which can be used for easy and rapid detecting of the enteric viruses without an antibody specific to enteric viruses or a sequencing analysis.
  • the present invention also provides a detection kit for enteric viruses comprising the probe(s) for an enteric virus.
  • the present invention also provides a preparation method of DNA chip for detecting an enteric virus.
  • the present invention also provides a DNA chip for detecting an enteric virus.
  • the present invention also provides a detection method of enteric virus with the probes for detecting an enteric virus.
  • the present invention provides a probe comprising a nucleotide sequence which can hybridize DNA of an enteric virus, which is selected from the group consisting of oligonucleotides having nucleotide sequences set forth in SEQ ID NO. 1 to SEQ ID NO. 15.
  • the present invention provides a process for preparing a DNA chip for detecting an enteric virus which comprises the steps of:
  • the present invention provides a DNA chip for detecting an enteric virus, comprising a probe or probe set selected from the group consisting of nucleotide sequences of SEQ ID NO. 1 to SEQ ID NO. 15.
  • the present invention provides a detection kit for an enteric virus, which comprises:
  • a DNA chip comprising a probe or probe set selected from the group consisting of nucleotide sequences of SEQ ID NO. 1 to SEQ ID NO. 15;
  • primers for amplifying DNA obtained from a clinical sample of an enteric virus and
  • (c) means for labeling the amplified DNA hybridized with the probes of the said DNA chip.
  • the detection kit for the enteric virus is used for identifying, detecting, or genotyping an enteric virus that is selected from the group consisting of human pan-enterovirus, human enteric adenovirus, human rotavirus, astrovirus, calicivirus, Norwalk virus,
  • the present invention provides a detection method for an enteric virus which comprises the steps of:
  • the present invention provides a detection method for an enteric virus which comprises the steps of:
  • the present invention provides the detection method, wherein in step a), three kinds of viral nucleotide sequences comprising human pan-enterovirus, human enteric adenovirus, and human rotavirus are simultaneously amplified from the sample through triplex PCR or RT-PCR with primers derived from 5'-UTP of human pan-enterovirus, a hexon gene of human enteric adenovirus, and a nucleotide sequence encoding capsid glycoprotein VP7 of human rotavirus, and then are hybridized with the probe or probe set.
  • the present invention provides the detection method, wherein in step a), seven kinds of viral nucleotide sequences comprising human pan-enterovirus, human enteric adenovirus, human rotavirus, astrovirus, calicivirus, Norwalk virus, Norwalk- like virus, and SRSV are simultaneously amplified in the sample multiplex PCR or RT-PCR with primers derived from 5'-UTP of human pan-enterovirus, a hexon gene of human enteric adenovirus, a nucleotide sequence encoding capsid glycoprotein VP7 of human rotavirus, a nucleotide sequence encoding a capsid protein of astrovirus, or nucleotide sequences encoding a capsid glycoprotein of calicivirus, Norwalk virus, Norwalk-like virus, and SRSV, and then are hybridized with the probe or probe set.
  • the present invention provides a primer for amplifying a nucleotide sequence of an enteric virus, which is selected from the group consisting of a nucleotide sequence of SEQ ID NO. 19, SEQ ID NO. 23, SEQ ID NO. 24, and SEQ ID NO. 29 to SEQ ID NO. 37.
  • Fig. 1 a and 1 b are examples of a detection kit for enteric viruses according to the present invention
  • Fig. 2 shows a result of detecting an enteric virus obtained from a water sample with the detection kit of the present invention.
  • Fig. 3a, 3b, and 3c show results of detecting a standard enterovirus with the detection kit of the present invention.
  • Fig. 4 shows a result of detecting a standard adenovirus with the detection kit of the present invention.
  • Fig. 5 shows a result of detecting a standard rotavirus with the detection kit of the present invention.
  • Figs. 6a, 6b, 6c, and 6d show results of detecting an enteric virus obtained from a clinical sample with the detection kit of the present invention.
  • Fig. 7 shows results of detecting standard enteric viruses including astrovirus, calicivirus, Norwalk virus, Norwalk-like virus, SRSV, and rotavirus with the detection kit of the present invention.
  • Inventors of the present invention invented probes that can complementarily bind to nucleic acid (DNA or RNA) of seven (7) groups in total of human enteric viruses consisting of three (3) groups which can be cultured and four (4) groups which cannot be cultured, including enterovirus, adenovirus, rotavirus, calicivirus, Norwalk virus, Norwalk-like virus, SRSV, and astrovirus, and the use thereof.
  • the enteroviruses which constitute 67 kinds among 100 kinds of enteric viruses are also called pan-enteroviruses, and they are divided into 3 kinds of polioviruses, 23 kinds of Coxsackie virus A, 6 kinds of Coxsackie virus B, 31 kinds of echoviruses, and 4 other kinds of enteroviruses called enterovirus 68, 69, 70, and 71.
  • Each enterovirus group shares a genotype- specific region which is located in a nucleic acid sequence at position 164- 526 bp of a highly conserved 5'-UTR(3, 16).
  • the oligonucleotide probes (Entero 1 , 2, and 3 as shown in Table 2) capable of detecting all the pan- enteroviruses are designed on the basis of the genetic characteristics conserved in subtypes, and can be used for detecting most of the pan- enteroviruses.
  • the probes specific to the enteric viruses prepared according to the present invention are shown in Table 2 and SEQ ID NO. 1 to 3.
  • the adenoviruses include human enteric adenovirus type 31 , human enteric adenovirus type 40, human enteric adenovirus type 41 , and other kinds of adenoviruses.
  • human enteric adenovirus type 31 human enteric adenovirus type 40, human enteric adenovirus type 41 , and other kinds of adenoviruses.
  • a nucleotide sequence located in 18858-19158 bp of a hexon gene which can be used for classifying the serotypes of the adenoviruses (M Puig, et al., Appl. Environ. Microbiol. 60:2963-2970, 1994; N. Jothikumar, et al., J. Virol. Method.
  • the probes for detecting adenoviruses can be designed to discriminate between enteric adenoviruses (Adeno 1 and Adeno 3 as shown in Table 2) and non-enteric adenoviruses (Adeno 2 as shown in Table 2).
  • the probes specific to the adenoviruses prepared according to the present invention are shown in SEQ ID NO. 4-6.
  • serotype G contains capsid glycoprotein VP7
  • serotype P contains VP4 which can be cleaved by proteinase.
  • 14 kinds of rotavirus serotype G have been reported thus far, of which 10 kinds of serotype G can infect humans, and serotypes G1 to G4 are distributed widely (Estes MK et al., Microbiol. Rev. 53:410-449, 1989; Gentsch JR, et al. J. Infect. Dis.174:S30-S36, 1996).
  • probes for detecting rotavirus are designed to detect the serotype G (Rota 1 , Rota 2, Rota 3, and Rota 4 as shown in Table 4).
  • the probes specific to the rotaviruses prepared according to the present invention are shown in Table 2 and SEQ ID NO. 7-10.
  • the astroviruses cause 9-26% of viral diarrhea in developed and developing countries each year (Bon, F. P. et al., J. Clin. Microbiol. 37:3055- 3058.1999; Svenungsson, B. A. et al., Clin. Infect. Dis. 30:770-778. 2000).
  • the probes for detecting 8 kinds of astroviruses are designed (Astro 1 as shown in Table 4).
  • calicivirus, Norwalk virus, Norwalk-like virus, and SRSV belonging to the caliciviridae classification have significant variations in genotype and phenotype (B.A.
  • capsid-coding gene which is obtained by sequencing caliciviridae with VECTOR NTI (InforMax®), probes for detecting all kinds of caliciviridae are designed (Calici, Calici 1 , Calici 2, and Calici 3 as shown in Table 4).
  • the underlined part in Table 2 represents a linker (spacer) sequence of a probe to increase the specificity of the probe.
  • the probes, or a DNA chip comprising the probes are useful for testing a clinical sample including enteric viruses, and monitoring water-borne enteric viruses in water.
  • the detection kit for enteric viruses comprises a DNA chip comprising the probe or probe set selected from the nucleotide sequences as set forth in SEQ ID NO. 1 to 15), primers for amplifying the enteric viruses, and means for labeling the amplified DNA to hybridize with the probes.
  • the probes used for the DNA chip can be a probe or probe set selected from the group consisting of nucleotide sequences of SEQ ID NO. 1 to 15.
  • the DNA chip can further comprise a probe for beta-globin as a position marker and for control of the hybridization reaction.
  • the primers for amplifying enteric viruses are derived from 5'-UTR of human pan-enterovirus, a hexon gene of human enteric adenovirus, a gene encoding capsid glycoprotein VP7 of human rotavirus, a gene encoding capsid protein of astrovirus, or a gene encoding capsid protein of calicivirus, Norwalk virus, or SRSV.
  • Preferred primers are shown in Table 3.
  • the labeling step of the amplified DNA can be performed by amplifying the gene of enteric virus with the addition of Cy5 (Cyanine 5)- modified dNTP during PCR amplification, or by amplifying the gene with Cy5- modified primers.
  • the biotin-binding materials can be used for the labeling means.
  • the biotin-binding materials include streptavidine-R-phycoerythrin.
  • any labeling means which is known to an ordinarily skilled person in the field can be applied to the present invention, and they include, as examples, Cy3, Alexa 488, Alexa 532, EDANS(5-(2'-aminoethyl)amino-1 -naphthalene sulfuric acid), tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC), x-rhodamine, and Texas red.
  • the DNA chip for detecting enteric viruses can be prepared by linking an amine group to the 5' terminal of the DNA probes of the present invention (as shown in SEQ ID NO.
  • Entero 1 , Entero 2, or Entero 3 for pan-enteroviruses is applied to a DNA chip, it is preferable to spot them on a solid surface by mixing Entero 1 or Entero 3 with Entero 2.
  • the Entero 1 , 2, or 3 is designed from 238-266 bp, 532-556 bp, or 357-383 bp in a 164-599 bp position of 5'-UTR, respectively.
  • the functionality of the probe is better for a short enterovirus gene.
  • Entero 1 or Entero 3 is used in a mixture with Entero 2 at the ratio of 1 :2-2:1 , wherein the Entero 1 or Entero 3 is used alone or together. More preferably, Entero 1 or Entero 3 can be used in a mixture with Entero 2 at the ratio of 1 :1. When the ratio of Entero 1 or Entero 3 with Entero 2 in a mixture is out of this range, the probe has a low chance to hybridize with the target, thereby making detection difficult.
  • Entero 1 can be mixed with Entero 3 at the ratio of 1 :2- 2:1 , preferably 1 :1.
  • Adeno 1 or Adeno 3 is separately aligned on a solid surface so as to not mix with Adeno 2.
  • Adeno 1 and Adeno 3 are designed for enteric adenoviruses, and Adeno 2 is designed for non-enteric adenoviruses.
  • the separate alignment of the probes can provide a method for differentiating between non-enteric adenoviruses and enteric adenoviruses.
  • the probe for beta-globin can be affixed on the solid surface as a position marker and for control of the hybridization reaction.
  • the affixing step of DNA probes to the aldehyde-derivatized solid surface can be performed via Schiffs base reaction between the amine and the aldehyde groups under the conditions of a temperature of 30 to 40°C and 70 to 100% humidity.
  • the aldehyde can be reduced' by reducing agents such as NaBH4.
  • concentration of probes which react with the aldehyde- derivatized solid surface ranges from 100 to 300 pmol/ ⁇ l. When the concentration of probes is out of the range, the probe may not bind to the aldehyde.
  • the solid can be glass, silicon dioxide, plastics, or ceramics. According to the present invention, it is possible to simultaneously detect various enteric viruses by simultaneously amplifying genes of various enteric viruses with suitable primers, and applying the amplified genes to the probes of the present invention.
  • the amplified product can be obtained by simultaneously amplifying 7 kinds of the nucleotide sequences including human pan-enterovirus, human enteric adenovirus, human rotavirus, astrovirus, calicivirus, Norwalk virus, Norwalk-like virus, and SRSV in a sample with primers derived from 5'-UTP of human pan- enterovirus, a hexon gene of human enteric adenovirus, a gene encoding a capsid glycoprotein VP7 of human rotavirus, a gene encoding a capsid protein of astrovirus, or a gene encoding capsid glycoprotein of calicivirus, a Norwalk virus, Norwalk-like virus, and SRSV through multiplex PCR or RT- PCR, hybridizing with the probes, and then detecting the hybridization.
  • the nucleotide sequences including human pan-enterovirus, human enteric adenovirus, human rotavirus, astrovirus,
  • a gene of an enteric virus in a sample is amplified by addition of Cy5- modified dNTP through PCR to label the amplified gene.
  • the gene labeled by Cy5 can be detected by analyzing the fluorescent signal with a confocal laser scanner.
  • human enteric adenovirus type 31 In addition to a PCR of pan-enterovirus, human enteric adenovirus type 31 , human enteric adenovirus type 40, human enteric adenovirus type
  • the enteric viral genes were separated from a clinical sample and a water sample as follows:
  • the clinical materials were preserved at 4°C.
  • the clinical materials were suspended in PBS (pH 7.4) at 10 v/v%. 300 / ⁇ of suspended clinical
  • the silica matrix was placed at room temperature for 10 minutes.
  • the precipitated materials were washed with a washing buffer solution (Bio 101 inc. washing solution of RNaid kit) 3 times, and then dried completely under vacuum.
  • the dried nucleic acids were solubilized in 25 ⁇ of distilled water for subsequent RT-PCR or PCR.
  • Water samples including untreated water, purified water, and domestic water were obtained from the laboratory of molecular virology of the Department of Biology in Kyunghee University in Seoul, Korea. To perform pre-culture PCR, the water samples were dripped off and concentrated. 200 ⁇ of the concentrated samples were reacted with proteinase K (0.5 ⁇ g/ ⁇ &,
  • the supernatant solution was transferred to a new tube, and the above reactions were repeated after the addition of a 1 ⁇ TE buffer.
  • the separated supernatant solution was eluted with chloroform, incubated at room temperature for 1 hour with the addition of isopropanol at the same volume to prevent the sodium phosphate from precipitating, and then centrifuged at 7000 ⁇ g for 30 minutes.
  • the resultant precipitates were dried completely, and solubilized in 10 ⁇ of sterile triple-distilled water for
  • ICC-PCR Integrated Cell Culture
  • BGM cell line Africa green monkey kidney cell, EPA in U.S.A.
  • TCVA total culturable virus assay
  • the supernatant was removed from the culture, and the culture cell was washed with 4 ⁇ PBS 3 times, cultured at room temperature for 15 minutes with the addition of an RNA extraction solution (50 mM sodium acetate, pH 5.2; 0.6%SDS; 1 mM EDTA; proteinase K 60 / €/ml), and then ceil lysis was
  • RNA contained in the cells was separated according to the same method as described above.
  • the separated RNA was precipitated and purified with the addition of 100% ethanol at 5 times its volume.
  • the RNA was again separated by treating the suspended RNA with the PCI solution, and then precipitated and purified with the addition of ethanol.
  • the purified RNA was used as a PCR template. ICC- PCR with the cell lysate was performed under the same conditions as with the pre-culture PCR. 2-2: Amplifying sample DNA
  • PCR or RT-PCR was performed as follows: cDNA synthesis was performed by using a 1 x random primer (Gibco BRL) according to the Gibco BRL's Superscript 11 reverse transcriptase manual. The reaction conditions were that cDNA complementary to viral RNA was synthesized at 25 °C for 20 minutes, and then at 42 ° C for 1 hour, it was heated at 95 ° C for 5 minutes to inactivate the reverse transcriptase, and then the reaction was performed under the following condition.
  • the multiplex PCR in this Example was intended to amplify the nucleotide sequences of enterovirus, rotavirus, and adenovirus.
  • the synthesized cDNA was amplified and labeled by using primers as shown in Table 2 with addition of Cy ⁇ (dUTP or dCTP).
  • the PCR was performed with 100 ⁇ i
  • PCR was performed by using a hot starter with 3 early cycles of
  • Example 2 a detection kit comprising the probes (shown in Table 2) was prepared.
  • Each probe for detecting enteric viruses such as mixed probes (6) of Entero 1 and 3 for detecting enterovirus were fixed, and Adeno 1 (4) and Adeno 2 (5) to differentiate the adenoviruses were fixed respectively onto an aldehyde-derivatized silylated slide (1) consisting of eight reaction chambers (2).
  • Rota 1 , 2, 3, or 4 for detecting rotavirus (7), and astro 1 for detecting astrovirus (9) were spotted.
  • Calici, Calici 1 , Calici 2, or Calici 3, or mixed probes thereof at the ratio of 1 :198 for detecting calicivirus, Norwalk virus, Norwalk-like virus, and SRSV were spotted.
  • the probe of beta-globin (3) as a position marker and control of cross-reactivity was fixed as shown in Figs. 1 a and 1 b.
  • Each probe as prepared above was used at a concentration of 200
  • This Example was performed by analyzing the amplified samples of Example 2 with the DNA chip of Example 3 to show the usefulness of the probes of the present invention.
  • the hybridization and detection reactions were performed by the following methods.
  • the hybridization reactions were performed with small amounts of a
  • PCR product 20 ⁇ of a triplex PCR product and 5 ⁇ of a beta-globin PCR product were diluted in 151& of distilled water, incubated with the addition of
  • cover slips were removed after termination of the cross-reaction.
  • the slips were washed by agitating in 3X SSPE for 2 minutes and 1X SSPE for 2 minutes at about 50 rpm, and air- dried.
  • Color signals were detected with a confocal laser scanner (GSI Lumonics. Inc.), and the results are shown in Fig. 2 to Fig. 7.
  • Adenovirus types 5 and 41 classified into adenovirus, rotavirus, astrovirus, and viruses classified to caliciviridae were obtained from the Laboratory of Molecular Virology of Kyunghee University, Seoul, Korea and the Laboratory of Enteric Viruses in the Department of Virology in the National Institute of Health (Seoul, Korea). The results are shown in Fig. 3 to Fig. 7.
  • probes for detecting the enteric viruses could be applied to a method of detecting various kinds of enteric viruses with accuracy and rapidity.
  • adenoviruses including human enteric adenovirus were only detected by sequencing the nucleic acid prior to the present invention.
  • the adenovirus can be detected easily and directly according to the present invention without sequencing.
  • the probes for detecting enteric viruses can provide a method of monitoring water-borne enteric viruses in water and clinical samples accurately and rapidly.

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Abstract

La présente invention concerne une sonde ou un ensemble de sondes destiné à la détection de virus entériques, un kit de détection de virus entériques comprenant la sonde et le procédé de détection des virus entériques utilisant ledit kit. Elle concerne plus particulièrement une sonde oligonucléotidique comportant une séquence oligonucléotidique telle qu'elle est montrée dans SEQ ID NO. 1-15. En outre, le kit diagnostique pour virus entériques comprend une puce à l'ADN avec au moins une sonde possédant des séquences oligonucléotidiques complémentaires à l'ADN du virus entérique, au moins une amorce pour amplifier un ADN échantillon par procédé PCR et un système pour étiqueter un ADN échantillon hybridé avec la sonde. L'invention, qu'elle soit appliquée à la détection de virus entériques dans un échantillon clinique ou utilisée pour la surveillance de la contamination de l'eau par les virus entériques d'origine hydrique, permet d'obtenir des résultats rapides et précis.
PCT/KR2002/001530 2001-08-09 2002-08-09 Sonde destinee a la detection de virus enteriques, kit de detection et procede de detection de virus enteriques WO2003014397A1 (fr)

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EP1609874A3 (fr) * 2004-05-21 2006-05-03 Bioanalisi Centro Sud S.n.c. di Perseu Sinibaldo e C. Système pour la recherche et identification d' agents pathogéniques.
WO2006097339A2 (fr) * 2005-03-18 2006-09-21 Glaxosmithkline Biologicals S.A. Procede et matieres associees
WO2007045062A3 (fr) * 2005-10-20 2007-08-02 Fiocruz Fundacao Oswaldo Cruz Vecteurs d'expression plasmidiques destines a l'expression d'epitopes ou de proteines recombinantes du rotavirus et de l'astrovirus et procede de production de matiere premiere
CN100412205C (zh) * 2005-10-26 2008-08-20 山东省医药生物技术研究中心 一种同时检测a、b、c三组人轮状病毒的膜基因芯片及制备方法与应用
CN100447253C (zh) * 2005-10-26 2008-12-31 山东省医药生物技术研究中心 一种同时检测a、b、c三组人轮状病毒的玻璃基因芯片及制备方法与应用
CN102206713A (zh) * 2011-04-06 2011-10-05 浙江大学 三重荧光定量rt-pcr检测试剂盒及用途
WO2013177468A2 (fr) 2012-05-24 2013-11-28 Isis Pharmaceuticals, Inc. Procédés et compositions de modulation de l'expression de l'apolipoprotéine(a)
WO2014077646A1 (fr) * 2012-11-19 2014-05-22 가톨릭대학교 산학협력단 Amorce pour détecter des virus transmis par l'eau et kit de détection la comprenant
WO2014179625A1 (fr) 2013-05-01 2014-11-06 Isis Pharmaceuticals, Inc. Compositions et procédés de modulation de l'expression de l'apolipoprotéine (a)
US9574193B2 (en) 2012-05-17 2017-02-21 Ionis Pharmaceuticals, Inc. Methods and compositions for modulating apolipoprotein (a) expression
WO2017079739A1 (fr) 2015-11-06 2017-05-11 Ionis Pharmaceuticals, Inc. Modulation de l'expression de l'apolipoprotéine (a)
CN110938710A (zh) * 2019-12-19 2020-03-31 武汉中帜生物科技股份有限公司 一种轮状病毒和肠道腺病毒联合检测胶体金层析试剂盒及其应用
WO2020095274A1 (fr) 2018-11-09 2020-05-14 Novartis Ag Procédé de réduction du risque d'un événement cardiovasculaire avec des composés antisens conjugués ciblant l'apo(a)
US11634711B2 (en) 2012-05-17 2023-04-25 Ionis Pharmaceuticals, Inc. Methods and compositions for modulating apolipoprotein (a) expression
CN116286803A (zh) * 2023-02-07 2023-06-23 广州凯普医药科技有限公司 一种同步检测22种手足口病毒的检测引物探针组合及基因芯片

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WO2006097339A2 (fr) * 2005-03-18 2006-09-21 Glaxosmithkline Biologicals S.A. Procede et matieres associees
WO2006097339A3 (fr) * 2005-03-18 2007-02-22 Glaxosmithkline Biolog Sa Procede et matieres associees
WO2007045062A3 (fr) * 2005-10-20 2007-08-02 Fiocruz Fundacao Oswaldo Cruz Vecteurs d'expression plasmidiques destines a l'expression d'epitopes ou de proteines recombinantes du rotavirus et de l'astrovirus et procede de production de matiere premiere
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CN100447253C (zh) * 2005-10-26 2008-12-31 山东省医药生物技术研究中心 一种同时检测a、b、c三组人轮状病毒的玻璃基因芯片及制备方法与应用
CN102206713A (zh) * 2011-04-06 2011-10-05 浙江大学 三重荧光定量rt-pcr检测试剂盒及用途
US9574193B2 (en) 2012-05-17 2017-02-21 Ionis Pharmaceuticals, Inc. Methods and compositions for modulating apolipoprotein (a) expression
US11859180B2 (en) 2012-05-17 2024-01-02 Ionis Pharmaceuticals, Inc. Antisense oligonucleotide compositions
US11634711B2 (en) 2012-05-17 2023-04-25 Ionis Pharmaceuticals, Inc. Methods and compositions for modulating apolipoprotein (a) expression
EP3822352A2 (fr) 2012-05-24 2021-05-19 Ionis Pharmaceuticals, Inc. Procédés et compositions de modulation de l'expression de l'apolipoprotéine(a)
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WO2014077646A1 (fr) * 2012-11-19 2014-05-22 가톨릭대학교 산학협력단 Amorce pour détecter des virus transmis par l'eau et kit de détection la comprenant
WO2014179625A1 (fr) 2013-05-01 2014-11-06 Isis Pharmaceuticals, Inc. Compositions et procédés de modulation de l'expression de l'apolipoprotéine (a)
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WO2017079739A1 (fr) 2015-11-06 2017-05-11 Ionis Pharmaceuticals, Inc. Modulation de l'expression de l'apolipoprotéine (a)
WO2020095274A1 (fr) 2018-11-09 2020-05-14 Novartis Ag Procédé de réduction du risque d'un événement cardiovasculaire avec des composés antisens conjugués ciblant l'apo(a)
CN110938710A (zh) * 2019-12-19 2020-03-31 武汉中帜生物科技股份有限公司 一种轮状病毒和肠道腺病毒联合检测胶体金层析试剂盒及其应用
CN110938710B (zh) * 2019-12-19 2023-09-05 武汉中帜生物科技股份有限公司 一种轮状病毒和肠道腺病毒联合检测胶体金层析试剂盒及其应用
CN116286803A (zh) * 2023-02-07 2023-06-23 广州凯普医药科技有限公司 一种同步检测22种手足口病毒的检测引物探针组合及基因芯片
CN116286803B (zh) * 2023-02-07 2023-11-24 广州凯普医药科技有限公司 一种同步检测22种手足口病毒的检测引物探针组合及基因芯片

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