WO2010055184A1 - Méthode et trousse permettant de détecter le virus de l'hépatite e - Google Patents

Méthode et trousse permettant de détecter le virus de l'hépatite e Download PDF

Info

Publication number
WO2010055184A1
WO2010055184A1 PCT/ES2009/070492 ES2009070492W WO2010055184A1 WO 2010055184 A1 WO2010055184 A1 WO 2010055184A1 ES 2009070492 W ES2009070492 W ES 2009070492W WO 2010055184 A1 WO2010055184 A1 WO 2010055184A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
hev
primers
virus
pcr
Prior art date
Application number
PCT/ES2009/070492
Other languages
English (en)
Spanish (es)
Inventor
Marta Fogeda Moreno
José Manuel ECHEVARRÍA MAYO
Ana AVELLÓN CALVO
Francisco POZO SÁNCHEZ
Jesús Enrique ROYUELA CASAMAYOR
Original Assignee
Instituto De Salud Carlos Iii
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Instituto De Salud Carlos Iii filed Critical Instituto De Salud Carlos Iii
Publication of WO2010055184A1 publication Critical patent/WO2010055184A1/fr

Links

Classifications

    • 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
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention a combination of primers and a method for the molecular detection of the different circulating strains of hepatitis E virus (HEV) in humans and animal reservoirs distributed geographically throughout the world are described.
  • the amplification of the HEV genome is performed by nested RT-PCRs where the primers used amplify two fragments of the ORF1 (Open Reading Frame) and ORF2 regions of the genome of this virus.
  • the present invention includes a kit with the combination of the mentioned primers and other components.
  • Hepatitis E is a disease caused by the VHE virus. This virus has not been able to recognize specifically until recent dates. Even when it is really a virus that was well established in the human populations of the Old World in the past, the truth is that HEV is today an agent that only circulates significantly in the most economically depressed regions , in which it constitutes a frequent cause of epidemic outbreaks of acute viral hepatitis. HEV infection is endemic in central and southeast Asia, and has caused epidemics in the Middle East, in North and West Africa, and in Mexico. The route of fecal-oral transmission is the predominant one, and the majority of known epidemics have been related to the consumption of water contaminated with fecal matter (Ippagunta et al. (2007) J. Med. Virol.
  • HEV is a zoonotic agent that circulates in some species of farm animals, and that is transmitted to humans with a frequency that is difficult to specify and, due to circumstances, still little known.
  • Laboratory tests used to diagnose HEV infection include electronic immunomicroscopy (IME), serological tests for the identification of anti-HEV of the IgM and IgG classes, and molecular techniques for the detection of viral RNA in feces. and in serum.
  • the IME allows to detect viral particles directly in the feces of the infected patient. This technique is very specific, but it is expensive, laborious and not very sensitive, so it is not used for diagnostic purposes.
  • Acute HEV infection can also be diagnosed by detection of viral RNA in feces and serum by genomic amplification procedures (patents WO9919732, WO2001046696, JP2005102689 and CN1300771). These tests yield positive results between 6 and 40 days, in this type of samples, after exposure to the virus, some days before the elevation of serum transaminase levels and Ia is observed. appearance of specific antibodies. Sometimes, these molecular diagnostic techniques yield positive results in cases that are not detected by serological diagnosis, which opens up the possibility that the latter may not be adequate to detect infections due to certain variants of HEV.
  • genomic amplification techniques facilitate, in addition to the etiological diagnosis, a useful material to obtain other information that serology cannot provide, such as the identification of the viral genotype involved in the infection and the possibility of carrying out phylogenetic studies that compare between yes the strains from different cases.
  • a combination of primers and a method for the molecular detection of the different circulating strains of hepatitis E virus (HEV) in humans and animal reservoirs distributed geographically throughout the world are described.
  • the amplification of the HEV genome is performed by nested RT-PCRs where the primers used amplify two fragments of the ORF1 (Open Reading Frame) and ORF2 regions of the genome of this virus.
  • the design of the primers has been based on the creation of degenerate primers of the ORF1 / ORF2 regions of the HEV genome.
  • the present invention therefore allows to determine the prevalence of the virus HEV in a representative sample of the general population, determine the clinical spectrum of acute infection in patients from rural and urban areas, evaluate the performance of serological diagnosis and molecular diagnosis in the identification of such cases, study the presence of HEV in pig farms and in environmental samples and obtain sequences of all fragments and conduct studies aimed at assessing their phylogenetic relationships.
  • a first aspect of the present invention is the use of the direct primers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 and the reverse primers SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 to detect hepatitis E virus (HEV) in an isolated biological sample.
  • HEV hepatitis E virus
  • a primer is a sequence of a specific oligonucleotide complementary to a target nucleotide sequence that is capable of hybridizing with the target nucleotide sequence and serving as a starting point for a nucleotide polymerization catalyzed by RNA polymerase, DNA polymerase or reverse transcriptase.
  • a previous step can be the retrotranscription of the sequence located upstream of the reverse primer by means of the use of a retrotranscriptase enzyme.
  • the primers that are part of the described uses have been designed following criteria detailed in the examples. First, all the virus strains described from 1987 to the current date were chosen. After the regions of the viral genome that allowed amplification in all types of known viruses were selected. The two regions selected were the ORF1 and the ORF2 of the HEV genome.
  • the ORF1 codes for the non-structural proteins of this virus (methyltransferase, RNA polymerase, protease and RNA helicase), the primers of the present invention are designed within the methyltransferase region, which allows the construction of robust phylogenetic trees with the identification of the viral genotype involved in the infection.
  • the ORF2 region codes for the viral capsid protein, which plays a very important role in the evasion of the immune system and in the formation of the virion.
  • ORF2 an exhaustive analysis of the entire genome of this viral agent (7200 base pairs) was performed, always looking for a highly conserved area to cover all the natural variants of the agent (ORF2) and a conserved but variable enough area (ORF1) that would allow genotypically characterize circulating strains.
  • the sequences of the primers have degenerate nucleotides in specific positions that allow the detection of the different circulating strains of HEV in humans and animal reservoirs distributed geographically throughout the world.
  • primers with degenerations in other different nucleotide sites could be used so that they hybridize in the same nucleotide sequence to the primers proposed in the present invention or a different one within the ORF described in the present invention, which would allow amplification fragments of HEV DNA.
  • HEV is a non-enveloped icosahedral symmetry RNA virus, approximately 32-34 nm in diameter. That genome is a single-stranded RNA of positive polarity, with a size of 7.2 kb. HEV has been classified within the Hepeviridae family, according to the latest report of the International Committee on Viral Taxonomy (2004), and is the only member of the genus Hepevirus. Through these primers it is possible to detect strains of the VHE virus that belong to any of the four known genotypes: genotype 1 is subdivided into five subtypes and includes epidemic strains characteristic of some endemic regions, such as the prototype strain isolated in Burma and the related strains of Africa and other regions of Asia.
  • Genotype 2 has two subtypes, and includes the prototype isolated in Mexico and several strains detected from Nigeria.
  • Genotype 3 is subdivided into ten subtypes, has a wide geographical distribution, and includes strains of human or swine origin isolated from native cases in Europe, USA. UU, Argentina and New Zealand.
  • genotype 4 is subdivided into seven subtypes, and includes strains that come, above all, from the Far East.
  • the primers of the present invention it is possible to detect HEV in endemic areas such as central and southeast Asia, north and west Africa and in Mexico. In non-endemic countries such as Spain, they can be used to identify those non-affiliated cases (of unknown origin) and to be able to characterize the origin of the strain causing the infection, that is, to know if it is imported or native.
  • This use facilitates, in addition to the etiological diagnosis, a useful material to obtain other information that the serology cannot provide, such as the viral genotype involved in the infection and the possibility of carrying out phylogenetic studies that compare strains from different cases.
  • the present invention allows the molecular characterization of the circulating strains, aiding in the investigation of the epidemic outbreaks produced by this virus.
  • These primers can, therefore, be used in the study of hepatic pathologies of unknown etiology (acute non-affiliated hepatitis), in epidemiological studies, and in the field of animal health.
  • Another aspect of the present invention is a method for the detection of hepatitis E virus (HEV) comprising: to. obtain a biological sample and isolate its nucleic acids, b. Simultaneously amplify two fragments of the ORF1 and ORF2 regions of the nucleic acid of section (a) by means of the primer pair SEQ ID NO: 1 / SEQ ID NO: 2 and the primer pair SEQ ID NO: 5 / SEQ ID NO: 6, c. Simultaneously amplify two internal fragments of the fragments obtained in section (b) by means of the pair of primers SEQ ID NO: 3 / SEQ ID NO: 4 and the pair of primers SEQ ID NO: 7 / SEQ ID NO: 8 d. determine the deviation of sections (b) and (c) with respect to controls.
  • HEV hepatitis E virus
  • the biological sample is isolated from the mammalian body to carry out the rest of the steps of the in vitro method.
  • the sample can come from a physiological fluid such as blood, plasma, serum, urine, faecal samples and / or any cellular tissue from an organism.
  • a physiological fluid such as blood, plasma, serum, urine, faecal samples and / or any cellular tissue from an organism.
  • the sample is serum or feces.
  • amplifications are carried out comprising at least the back transcription of the corresponding fragment of the VHE RNA followed by a polymerase chain reaction. (PCR) that is, an RT-PCR.
  • PCR polymerase chain reaction
  • a quantitative or real-time RT-PCR can also be carried out, or any other technique in which the combination of primers described is required to carry out the amplification.
  • the first step is to amplify two fragments simultaneously using primer pairs SEQ ID NO: 1 / SEQ ID NO: 2 (direct primer / reverse primer) and SEQ ID NO: 5 / SEQ ID NO: 6 (direct primer / reverse primer).
  • a DNA sequence coding (cDNA) from the region is synthesized upstream of the hybridization site of the reverse primer with the RNA by means of the retrotranscription reaction (RT).
  • the cDNA synthesis is performed by means of any enzyme (retrotranscriptase; RT or reverse transcriptase) that uses the RNA isolated from the biological sample and the reverse primers SEQ ID NO: 2 and SEQ ID NO: 6, which will act as initiators to obtain the single chain or single stranded cDNA complementary to the RNA sequence of the VHE virus.
  • double-stranded DNA fragments obtained by PCR are obtained using the cDNA described and the primer pairs SEQ ID NO: 1 / SEQ ID NO: 2 and SEQ ID NO: 5 as template. / SEQ ID NO: 6. This PCR is called RT-PCR.
  • dNTPs nucleotides that is, dATP, dTTP, dCTP and dGTP, thermo-resistant DNA polymerase, magnesium chloride in a given concentration as well as buffers that create the appropriate physico-chemical conditions for the operation of all the components and thus the amplification is achieved (see examples).
  • dATP dATP
  • dTTP dTTP
  • dCTP dCTP
  • dGTP thermo-resistant DNA polymerase
  • magnesium chloride magnesium chloride
  • the template DNA used is the product of the PCR reaction obtained in the first step of
  • SEQ ID NO: 7 / SEQ ID NO: 8 direct primer / reverse primer. With these primers two subfragments are amplified taking as a template the two amplified fragments in the first PCR reaction.
  • SEQ ID NO: 3 / SEQ ID NO: 4 amplifies a sub-fragment of the fragment of
  • ORF1 of a size of 171 base pairs and SEQ ID NO: 7 / SEQ ID NO: 8 amplifies a subfragment of the ORF2 fragment of 220 base pairs.
  • This type of PCR is known with the term nested PCR where the product of an amplification is used as a template to perform a second amplification with primers that are located within the first amplified sequence.
  • the controls are those amplifications that come both from samples in which the HEV virus is absent (negative control) and from samples in which it is present in a certain way (positive control).
  • the measurement of the deviation of the amplifications obtained from the problem samples with respect to the controls can be carried out by comparing the presence or absence of bands, as well as the measurement of the band intensity or the measurement of the expression can be taken to carried out by real-time or quantitative PCR. This deviation can be attributed to the presence or absence of the VHE virus in the analyzed samples.
  • the isolated biological sample is obtained from a mammal.
  • the mammal is selected from the list comprising human or non-human.
  • Non-human mammals are selected from the list comprising pig, wild boar, mongoose or primate.
  • VHE virus is transmitted not only by water (contaminated by the feces of these animals) but also by the consumption of food contaminated with this viral agent.
  • it may be of special interest to carry out epidemiological studies and / or basic experimentation studies in other non-domestic animals such as mongoose or primates as possible zoonotic reservoirs of HEV.
  • VHE virus In addition to the detection of the VHE virus in Spanish pig farms, it has also been found in wastewater from urban environments. On the other hand, in this class of virus the Ia is frequent appearance of viral variants with the ability to cross the barrier between species and infect new hosts that can cause diseases.
  • the method includes an internal control to evaluate the presence of amplification inhibitors.
  • This control consists of a DNA based on the sequence of a plasmid of known sequence.
  • the primers are included for the amplification of a fragment that allows to verify, by the amplification conditions described above, that the amplification reaction has not been inhibited.
  • Another preferred embodiment is the method that also allows to determine the genotype of the VHE virus by means of the nested RT-PCR of the ORF1 region.
  • the sequencing of the amplified fragments is carried out by means of the nested RT-PCR as described in the present invention, of the ORF1 region.
  • the sequences obtained can be assigned to a given genotype by comparing them with the VHE sequences of the available databases.
  • Another preferred embodiment is the method described in the preceding paragraphs as a tool for monitoring the evolution of HEV infection, where serial samples of affected mammals are taken. The analysis of the presence or absence of the expected fragments or of the ascent or decrease of the quantification of the amplified DNA allows to evaluate whether the response to the treatment is negative or positive.
  • the monitoring procedure comprises a series of steps that begin by taking serial samples.
  • Serial sampling is understood as the extraction of the biological samples mentioned in the present invention. The sampling is carried out at different times since the treatment is administered, so that the amplification of the fragments of the ORF1 and ORF2 of the VHE virus in each of the samples from the same patient, indicate the efficacy thereof.
  • a decrease in the deviation of the amplification values with respect to a control the latter represented, for example, by amplification values in the same individual, prior to the treatment, assumes that the treatment is taking effect in the sense of decreasing The amount of HEV virus causing hepatitis E. This example would not be limited only to the use of this type of control.
  • kits for determining the HEV genotype in a biological sample isolated by nested RT-PCR of the ORF1 region comprising, at least, primers SEQ ID NO: 1 / SEQ ID NO: 2 and SEQ ID NO: 3 / SEQ ID NO: 4.
  • kits for the detection of the VHE virus in an isolated biological sample by nested RT-PCR comprising, at least, the direct primers SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO : 5 and SEQ ID NO: 7 and reverse primers SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
  • a combination of primers with degenerations at other different nucleotide sites could be used so that the primers hybridized in the same or other different nucleotide sequence within the ORFs described in the present invention that allowed amplifying fragments of HEV RNA.
  • the kit could include the reagents necessary for amplification by RT-PCR, DNA polymerase, retrotranscriptase, nucleotides, or other components. It could also include instructions for carrying out the amplification by means of the appropriate conditions; alignment temperature, hybridization, elongation, number of amplification cycles, etc.
  • the kit for the detection of the VHE virus is used for monitoring the response to a hepatitis E treatment, as described in a previous paragraph.
  • kits also comprise an internal control necessary to evaluate the presence of amplification inhibitors.
  • This control consists of a DNA based on the sequence of a plasmid of known sequence.
  • the primers are included for the amplification of a fragment that allows to verify, by the amplification conditions described above, that the amplification reaction has not been inhibited.
  • primers SEQ ID NO: 9 (direct) and SEQ ID NO: 9 (direct) are included for the amplification of a fragment that allows to verify, by the amplification conditions described above, that the amplification reaction has not been inhibited.
  • FIG 1. Shows the results of the sensitivity of nested PCRs for the ORF1 and ORF2 regions of HEV.
  • ORF1 171 base pair band.
  • ORF2 220 bp band.
  • Serial dilutions of 10 in 10 of the reference strain of HEV SAR-55 (genotype 1, 10 4 5 PID50 / ml).
  • FIG. 2. shows the results obtained from the phylogenetic analysis performed with 13 sequences obtained from the PCR products of the ORF1 region.
  • the phylogenetic tree was constructed with the sequences obtained from the amplification products of the ORF1 region of HEV (nucleotides 26-197) together with the sequences deposited in the GeneBank of the same viral fragment, of genotypes 1, 2, 3 and 4.
  • FIG 3. Shows the results of the specificity of the method.
  • ORF1 and B ORF2.
  • CN negative control
  • C + positive control of ORF1 for HEV (171 base pairs); and positive control of ORF2 for HEV (220 bp).
  • Cl internal control (350 bp).
  • HCV hepatitis C virus
  • HBV hepatitis B virus
  • EXAMPLE 1 Design of the primers of the invention.
  • ORF1 encodes the non-structural polyprotein, and allows the genotypic characterization of the detected HEV strain
  • ORF2 region which encodes the structural protein, confirms the microbiological detection of this pathogen.
  • each of the regions chosen was subsequently analyzed with additional software, locating areas of approximately 20 nucleotides in length, whose efficacy as primers for amplification was also empirically estimated.
  • nucleotides degenerated into specific positions (in some cases, more than one nucleotide in a certain position). These degenerations allow us to identify the different circulating strains of HEV in humans and animal reservoirs distributed geographically throughout the world.
  • the specificity of each of the regions identified as valid for primers was further verified by comparison with public access sequence databases (GenBanK) by means of public access software (Blast).
  • an internal control consisting of a chimeric DNA based on the sequence of a plasmid (Pozo F et al., (2007) J Clin Virol 40: 224-228) was included in the reaction mixture. as well as the primers necessary for the amplification of a 350 base pair fragment, which gives us a quality control to evaluate the presence of amplification inhibitors in each of the samples tested.
  • the primers for amplifying the internal control are the following: SEQ ID NO: 9 (direct) and SEQ ID NO: 10 (reverse) for the first PCR amplification, SEQ ID NO: 11 (direct) and SEQ ID NO: 12 (reverse) for the second PCR amplification.
  • liver damage All patients at the time of admission had symptoms and signs of acute hepatitis when they were treated in their corresponding hospitals. Other possible causes of liver damage were ruled out in these patients (hepatic drug poisoning, autoimmune hepatitis, infection by other liver viruses A, B and C, cytomegalovirus infection and Epstein-Barr infection).
  • a new design of primers was developed to be used in the polymerase chain reaction (PCR) in order to detect the HEV genome.
  • the objective of this new design is to be able to detect all the new circulating variants of HEV both in humans and in the different reservoirs detected.
  • the design of the method was based on the creation of degenerate primers of the ORF1 / ORF2 regions of the HEV genome.
  • the nucleotide sequence of the primers is shown in Table 1.
  • the HEV RNA was extracted from a total of 100 ⁇ l of serum with the MagNA Puré automatic extractor (ROCHE, Mannheim, Germany). The RNA obtained was resuspended in an elution buffer at a final volume of 50 ⁇ l and subsequently stored at -8O 0 C until use. RT-PCR was performed with 5 ⁇ l of RNA extract in a final volume of 50 ⁇ l.
  • MagNA Puré automatic extractor ROCHE, Mannheim, Germany
  • the reaction mixture contains 25 ⁇ l of Master Mix (Taq polymerase: 50U / ml; dNTPs: 40OuM; MgCI2: 3mM; Promega, Co., Madison, Wisconsin, USA), 1 ⁇ l of the enzyme AMV Reverse Transcripse (Promega Co .), and 50 pmol of each primer.
  • the PCR reaction consists of 39 cycles of denaturation at 94 0 C for 35 sec, banding at 51 0 C for 45 sec (ORF1 test), and 48 0 C for 45 sec (ORF2 test), and an extension at 72 0 C for 1 min, with a final elongation at 72 0 C for 7 min.
  • the amplification of the 2 to round PCR was performed with 2 ⁇ l of PCR product of the first round amplification, and consisted of 29 cycles of denaturation at 94 0 C for 35 sec, annealing at 48 0 C for 45 sec (ORF1 test), and at 5O 0 C for 45 sec (ORF2 test), extension at 72 0 C for 1 min, with a final incubation at 72 0 C for 7 min and 50 pmol of each primer.
  • Table 1 Degenerated primers used for amplification by nested RT-PCR.
  • ORF1 F ORF1 Direct 1 to PCR SEQ ID NO: 1 9-29
  • ORF1 R - Reverse 1 to PCR SEQ ID NO: 2 627-643
  • ORF1 RN - Reverso 2 to PCR SEQ ID NO: 4 178-197
  • ORF2F ORF2 Direct 1 to PCR SEQ ID NO: 5 6265-6285
  • the position of the nucleotides is numbered according to the strain of the Mexico virus.
  • the sequence SEQ ID NO: 4 contains a nucleotide n which is an Inosine.
  • the phylogenetic tree (FIG 2) was performed with the MEGA version 4.0 program (Tamura K et al., (2007) Mol Biol Evol 24: 1596-9), and distances with the Neighbor-Joining (NJ) Kimura 2 method -parameters with 1,000 replicas.
  • hepatitis E virus HEV
  • This methodology allowed to identify 13 cases of acute hepatitis produced by this viral agent, out of a total of 95 samples tested.
  • Table 2 shows the results obtained from the comparison of the serological techniques and the method designed for the detection of the viral RNA of HEV in the ORF1 / ORF2 regions.
  • 13 were positive for HEV by PCR in both regions of the genome (ORF1 / ORF2).
  • Five cases (1-5) belonged to the retrospective study and the remaining eight correspond to the prospective study.
  • Antibodies (Anti-HEV IgM) were analyzed in all samples, with only 11 of them.
  • One of the patients studied was an immigrant of African origin who was treated in the health services of Spain, specifically in the city of Ceuta. The remaining 12 patients were Spanish and residents in this country.
  • Genotype 1 strains corresponded to the immigrant from African origin and two travelers who came from India and Bangladesh.
  • Genotype 3 strains corresponded to samples from six patients who had no history of travel to endemic areas of HEV in their clinical history. The only genotype 4 found corresponded to the patient who came from Vietnam.
  • the results obtained from the molecular studies carried out revealed the existence of seven imported cases of acute hepatitis due to HEV. Of these seven imported cases, six of them were genotype 1 and corresponded to patients with a history of travel to endemic areas of this genotype (India), and a patient of African origin residing in Ceuta. The other imported case belonged to genotype 4, and it was a Madrid patient who had recently been to Vietnam. The finding of genotype 3 in samples of six Spanish patients with acute hepatitis, who had no history of travel to endemic areas by this viral agent, confirmed that the origin of these infections was native.
  • Genotype 3 has been found in humans and pigs, as well as in several species of wild animals in Europe and North America (mainly Wild Boar) (from Deus N et al., (2008). Vet Microbiol: 129: 163-170 ; Kaci S et a /., (2008). Vet Microbiol: 128: 380-385) and in urban waters (Clemente-Casares P et al., (2003). Emer ⁇ Infect Dis: 9: 448-454).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Communicable Diseases (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une combinaison d'amorces et une méthode de détection moléculaire des différentes souches en circulation du virus de l'hépatite E (VHE) chez des humains et dans des réservoirs animaux lesquelles sont réparties géographiquement dans le monde entier. L'amplification du génome du VHE s'effectue par RT-PCR imbriquées au cours desquelles les amorces utilisées amplifient deux fragments des régions ORF1 (Open Reading Frame) et ORF2 du génome de ce virus. En outre, la présente invention concerne une trousse comprenant la combinaison des amorces susmentionnées et d'autres composants.
PCT/ES2009/070492 2008-11-11 2009-11-10 Méthode et trousse permettant de détecter le virus de l'hépatite e WO2010055184A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200803220A ES2338853B1 (es) 2008-11-11 2008-11-11 Metodo y kit para la deteccion del virus de la hepatitis e (vhe).
ESP200803220 2008-11-11

Publications (1)

Publication Number Publication Date
WO2010055184A1 true WO2010055184A1 (fr) 2010-05-20

Family

ID=42121365

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2009/070492 WO2010055184A1 (fr) 2008-11-11 2009-11-10 Méthode et trousse permettant de détecter le virus de l'hépatite e

Country Status (2)

Country Link
ES (1) ES2338853B1 (fr)
WO (1) WO2010055184A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013156432A1 (fr) * 2012-04-18 2013-10-24 F. Hoffmann-La Roche Ag Recherche du virus hev
CN110878378A (zh) * 2019-12-02 2020-03-13 昆明理工大学 用于检测体液中戊型肝炎病毒的引物组合及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1300771A (zh) * 1999-12-23 2001-06-27 中国药品生物制品检定所 新型戊型肝炎病毒基因序列及其应用
WO2001068921A2 (fr) * 2000-03-14 2001-09-20 Investigen Compositions et procedes de detection simultanee de multiples entites biologiques
US6432408B1 (en) * 1997-07-18 2002-08-13 The United States Of America As Represented By The Department Of Health And Human Services Swine hepatitis E virus and uses thereof
US20030049601A1 (en) * 1997-10-15 2003-03-13 George G. Schlauder Methods and compositions for detecting hepatitis e virus
EP1400588A1 (fr) * 2001-06-25 2004-03-24 Kabushiki Kaisha Toshiba Sonde polynucleotidique et amorce originaire du virus de l'hepatite e des japonais, puces pourvues de cette sonde, kits dotes de cette sonde et procede pour detecter le virus de l'hepatite e au moyen de cette sonde
JP2005102689A (ja) * 2003-09-08 2005-04-21 Mitsubishi Kagaku Bio-Clinical Laboratories Inc Ｅ型肝炎ウイルス様中空粒子

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6432408B1 (en) * 1997-07-18 2002-08-13 The United States Of America As Represented By The Department Of Health And Human Services Swine hepatitis E virus and uses thereof
US20030049601A1 (en) * 1997-10-15 2003-03-13 George G. Schlauder Methods and compositions for detecting hepatitis e virus
CN1300771A (zh) * 1999-12-23 2001-06-27 中国药品生物制品检定所 新型戊型肝炎病毒基因序列及其应用
WO2001068921A2 (fr) * 2000-03-14 2001-09-20 Investigen Compositions et procedes de detection simultanee de multiples entites biologiques
EP1400588A1 (fr) * 2001-06-25 2004-03-24 Kabushiki Kaisha Toshiba Sonde polynucleotidique et amorce originaire du virus de l'hepatite e des japonais, puces pourvues de cette sonde, kits dotes de cette sonde et procede pour detecter le virus de l'hepatite e au moyen de cette sonde
JP2005102689A (ja) * 2003-09-08 2005-04-21 Mitsubishi Kagaku Bio-Clinical Laboratories Inc Ｅ型肝炎ウイルス様中空粒子

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GEORGE G. SCHLAUDER: "Novel hepatitis E virus (HEV) isolates from Europe: Evidence for additional Genotypes of HEV", JOURNAL OF MEDICAL VIROLOGY, vol. 57, 1999, pages 243 - 251 *
JAMES C. ERKER: "A hepatitis virus E variant from the US: molecular characterization and transmission in cynomolgus macaques", JOURNAL OF GENERAL VIROLOGY, vol. 80, 1999, pages 681 - 690, XP002957112 *
XIANG-JIN MENG: "A novel virus in swine is closely related to the human hepatitis E virus", PROC. NATL. ACAD. SCI., vol. 94, 1997, pages 9860 - 9865, XP002088357 *
YOUCHUN WANG ET AL.: "A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis", JOURNAL OF GENERAL VIROLOGY, vol. 80, 1999, pages 169 - 177 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013156432A1 (fr) * 2012-04-18 2013-10-24 F. Hoffmann-La Roche Ag Recherche du virus hev
US9702017B2 (en) 2012-04-18 2017-07-11 Roche Molecular Systems, Inc. HEV assay
US10689718B2 (en) 2012-04-18 2020-06-23 Roche Molecular Systems, Inc. HEV Assay
CN110878378A (zh) * 2019-12-02 2020-03-13 昆明理工大学 用于检测体液中戊型肝炎病毒的引物组合及其应用

Also Published As

Publication number Publication date
ES2338853A1 (es) 2010-05-12
ES2338853B1 (es) 2011-09-14

Similar Documents

Publication Publication Date Title
Benetka et al. Prevalence of feline coronavirus types I and II in cats with histopathologically verified feline infectious peritonitis
Simons et al. A mRNA PCR for the diagnosis of feline infectious peritonitis
ES2895262T3 (es) Cuantificación del genoma de anelovirus como biomarcador de la inmunosupresión
Martella et al. Genotyping canine distemper virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks
Moreau et al. Serendipitous identification of natural intergenotypic recombinants of hepatitis C in Ireland
ES2534362T3 (es) Medios y métodos para distinguir el FECV y el FIPV
Tong et al. Accurate genotyping of hepatitis C virus through nucleotide sequencing and identification of new HCV subtypes in China population
CN101696454A (zh) 用于可视化检测猪瘟病毒野毒株的rt-lamp引物
EP2707496A1 (fr) Procédé de détermination rapide de virus à l'aide de diagnostics moléculaires basés sur des acides nucléiques, et trousse pour sa mise en oeuvre
ES2338853B1 (es) Metodo y kit para la deteccion del virus de la hepatitis e (vhe).
Rauf et al. Prevalence of hepatitis B and C virus in the general population of Hill Surang area, Azad Jammu and Kashmir, Pakistan
Tanaka et al. African origin of GB virus C/hepatitis G virus
RU2422536C1 (ru) НАБОР СИНТЕТИЧЕСКИХ ОЛИГОНУКЛЕОТИДОВ ДЛЯ ВЫЯВЛЕНИЯ ДНК В КРОВИ И ДРУГИХ БИОМАТЕРИАЛАХ ВОЗБУДИТЕЛЯ ЛАТЕНТНОЙ ВИРУСНОЙ ИНФЕКЦИИ - ВИРУСА Torque teno virus СЕМЕЙСТВА Circoviridae МЕТОДОМ ПОЛИМЕРАЗНОЙ ЦЕПНОЙ РЕАКЦИИ
Rodrigues et al. Genomic characterization of a novel pegivirus species from free-ranging bottlenose dolphins (Tursiops truncatus) in the Indian River Lagoon, Florida
JP4080995B2 (ja) 日本人からのｅ型肝炎ウイルスに由来するポリヌクレオチドプローブおよびプライマー、それらを有するチップ、それらを有するキット、並びにそれらによるｅ型肝炎ウイルスを検出する方法
ES2676245T3 (es) Cebadores y procedimientos para detectar variantes del virus de la hepatitis C (VHC) humano en una muestra aislada
EP3916111A1 (fr) Ensembles d'amorces universelles pour la détection de flavivirus et leur utilisation
CN105861753A (zh) 检测寨卡病毒的巢式rt-pcr方法、引物及试剂盒
JPWO2006009260A1 (ja) Ｅ型肝炎ウイルスの検出方法
Chowdhury et al. Detection of classical swine fever virus by RT-PCR in West Bengal, India
KR101924093B1 (ko) E형 간염 바이러스 유전자형 3 및 4 진단용 프라이머 세트 및 이를 이용한 진단 방법
Müller et al. Hepatitis C virus genotypes in Hungarian and Austrian patients with chronic hepatitis C
JP4127722B2 (ja) 日本人からのｅ型肝炎ウイルスに由来するポリヌクレオチドプローブおよびプライマー、それらを有するチップ、それらを有するキット、並びにそれらによるｅ型肝炎ウイルスを検出する方法
KR20070116377A (ko) 유전자칩, 이를 이용한 돼지콜레라 바이러스 또는소바이러스성설사증 바이러스 유전자의 검사방법 및 그를포함하는 진단키트
EA201000306A1 (ru) Способ идентификации генотипа и подтипа вируса гепатита с на биологическом микрочипе

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09825792

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09825792

Country of ref document: EP

Kind code of ref document: A1