TWI261618B - Method for detecting and differentiating enteroviruses and the primers and probes therefor - Google Patents

Abstract

The present invention discloses pairs of oligonucleotide primers for use in detecting the presence or absence of an enterovirus in a sample. Also disclosed are synthetic nucleotide sequences capable of specifically hybridizing to a sense strand of an enterovirus nucleic acid or a nucleic acid corresponding to the sense strand. The present invention provides a method of detecting the presence or absence of an enterovirus nucleic acid in a sample as well as kit for detecting an enterovirus in a sample. The present invention particularly provides a method and a kit for detecting and differentiating enterovirus type 71 and/or coxsackievirus A16 in a sample.

Description

1261618 A7 _____ B7 V. INSTRUCTIONS (,) Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives Background of the Invention Enterovirus belongs to the picornavirus family and e). The Virology family represents a very large family of RNA viruses in terms of number of members, but is one of the smallest in terms of virus particle size and complexity. The virion of enterovirus consists of 60 subunits, each subunit consisting of four proteins (VP1-VP4) in a icosahedral symmetrical manner around a genome formed by a single positive sense RNA. The enterovirus temporarily inhabits the human digestive tract and can also be isolated from the posterior segment of the intestine or from the throat. Enteroviruses of human origin include poliovirus (blood type 1 to 3), soxavirus group A (CAV, blood type 1 to 22 and 24) and soxavirus group B (CBV, blood type 1) To 6), intestinal cytopathic diseases of autism virus (blood type 1 to 9, 11 to 27 and 29 to 33) and enterovirus (blood type 68-71). Human enterovirus infection can cause a wide range of sputum and nerves, skin and mucous membranes, acute symptoms of heart and muscle, eye, respiratory and gastrointestinal diseases, as well as fever without obvious characteristics, a wide range of infant diseases and diabetes. In non-polioviruses, infections caused by some unclassified blood stasis types are particularly prevalent in some countries and regions in summer and early autumn, such as enterovirus types 70 and 71. Enterovirus type 71 has been isolated from patients with meningitis, encephalitis, and polio-like myelitis, and remains the leading cause of the world's deadly central nervous system disease. Furthermore, this type of virus causes human hand, foot and mouth disease outbreaks in some areas, such as in Japan, Sweden and Taiwan. The general diagnosis of enterovirus usually involves viral harvesting and blood sputum testing. The isolation of the virus may include the isolation of the virus from the throat water, the throat swab, the stool, the rectal cotton swab, and sometimes the virus from the cerebrospinal fluid (in the case of aseptic meningitis). Inoculate the isolated virus sample into tissue culture or suckling mice (the latter please read the back)

Page IIIII Book 4 This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1261618 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. Invention Description (>) Dedicated to Keshaqi Virus ) breeding and identification. This virus harvesting process takes one to two weeks to complete. In addition, qualified technicians are required to participate in the process. In the blood sputum test, a neutralization test is usually performed to detect a specific neutralizing antibody against a virus. For some intestinal cytopathic solitary viruses, the hemagglutination inhibition test may indicate a blood-stasis-specific infection. Blood sputum antibodies can also be detected and titrated by immunofluorescence using an infected cell culture as an antigen on a coverslip. However, hemorrhagic trials are difficult to assess because there are many types of blood stasis, unless the antigen used is isolated by a particular individual or isolated in the event of a typical clinical outbreak. In all blood tests, those skilled in the art must determine from the result readings which are cut-off values. At the same time, the antigens used in these assays typically require special techniques and a period of time to prepare to reduce the incidence of heterogeneous or cross-reactive reactions. Viral harvesting and blood tests do not provide a quick diagnostic tool. However, acute symptoms associated with enterovirus can develop very rapidly, and in some cases may develop into fatality within a few days. Therefore, there is a strong demand for a method for rapidly and easily detecting enterovirus in practice. SUMMARY OF THE INVENTION The present invention relates to novel oligonucleoside pairs for detecting the presence or absence of enteroviruses in a sample. The individual primer pairs of the present invention include a first primer and a second primer which facilitate rapid diagnosis of diseases or symptoms associated with enteroviruses via nucleic acid amplification assays such as polymerase chain reaction. The present inventors have surprisingly found that some nucleotide sequences are associated with enteric disease. This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 297 297 mm) _ ϋ n I nn an ϋ one: eJ n · ϋ ϋ I _1 in I (Please read the note on the back? Write this page) 1261618 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperative Printed 5, Inventions (h) Conservative areas in toxic nucleic acids. Accordingly, the present invention further provides a synthetic nucleotide sequence which is capable of hybridizing specifically to a sense strand of enterovirus nucleic acid or a nucleic acid associated with the sense strand. In another aspect, the invention relates to a method of detecting the presence or absence of enterovirus nucleic acid in a sample. The method of the present invention comprises (a) contacting the sample with one of the oligonucleoside primer pairs of the present invention in an amplification method; and (b) determining the presence or absence of the enterovirus by detecting the presence or absence of the amplification product; . Preferably, in the method of the present invention, the sample is simultaneously contacted with a second pair of oligonucleotide primers of the present invention in addition to the first pair of primers in the amplification method. The primers in the second pair are different from the ones in the first pair. The primers in the second pair may have a first primer or a second primer that is different from the corresponding primer in the first pair. Preferably, both of the second pair of primers are different from the first pair of primers. Further, the sample may simultaneously contact a third pair of oligonucleotide primers of the present invention other than the first pair and the second pair of primers in the amplification method. Similarly, the primers in the third pair are different from the ones in the first pair or the second pair. In another aspect, the method of the invention can further comprise contacting the product of step (a) with a second oligonucleoside primer pair of the invention in an amplification method. The second pair of primers is selected for use in an amplification method for an amplification sequence which is identical to or is included in the sequence obtainable by the amplification method using the first pair of primers. In another aspect of the method of the invention, the amplified product detected in step (b) can be specifically hybridized with at least one synthetic nucleotide sequence of the invention. In another aspect, the present invention relates to detecting and identifying intestines in a specimen. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). I Μ··· Μ·· η·· · I Μ·· · SMI ΒΜ» * ΗΒΗ II · ι^ (Please read the note on the back to write this page) 1261618 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperative Printed 5, Invention Description (4) Virus Type 71 Methods. The method comprises (a) contacting the sample with at least one pair of oligonucleoside primers of the invention in an amplification method; (b) determining the presence or absence of enterovirus 71 by detecting the presence or absence of the amplification product; And (c) performing the specific hybridization reaction of the amplified product detected in the step (b) with the synthetic nucleotide sequence of the present invention comprising the sequence of SEQ ID NO: 12 or SEQ ID Ν 0··13. The detected amplification product is subjected to a specific hybridization reaction with the synthetic nucleotide sequence of the present invention comprising the sequences of SEQ ID NO: 12 and SEQ ID NO: 13. In another aspect, the present invention relates to A method for detecting and identifying a Crohn's virus A16 in a sample, the method comprising: (a) contacting the sample with at least one pair of oligonucleoside primers of the invention in an amplification method; (b) detecting The presence or absence of the amplification product to determine the presence or absence of the Crox virus A16 type; and (c) the amplification product detected in step (b) and the sequence of the invention comprising SEQ ID NO: 14 or SEQ ID NO : 15 synthetic nucleotide sequence for specific hybridization reaction. Preferably, the detected increase in yield The system is subjected to a specific hybridization reaction with the synthetic nucleotide sequence of the present invention comprising the sequences SEQ ID NO: 14 and SEQ ID Ν 0··15. In another aspect, the present invention relates to a detection in a sample and A method of identifying Enterovirus 71 and/or Croxvirus Α16. The method comprises (a) contacting the sample with at least one pair of oligonucleoside primers of the invention in an amplification method; (b) detecting Detecting the presence or absence of the amplification product to determine the presence or absence of Enterovirus 71 and/or Croxvirus A16; and (c) including the amplification product detected in step (b) with at least one of the present invention The synthetic nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13 and at least one containing sequence 6 (please read the back of the note first).

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J · This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1261618 A7 B7 V. Inventive Note (<) Synthetic nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 15. Perform a specific hybridization reaction. Preferably, the detected amplification product is carried out with the synthetic nucleotide sequence of the invention comprising the sequences SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15. Specific hybridization reaction. In another aspect, the invention provides a kit for detecting enterovirus in a sample comprising at least one pair of oligonucleotide primers of the invention. Preferably, the kit of the invention comprises more than one pair of oligonucleotide primers of the invention. In another aspect of the invention, the kit can further comprise at least one synthetic nucleotide sequence of the invention. In another aspect, the invention provides a kit for detecting and identifying an enterovirus 71 type in a sample comprising at least one pair of oligonucleotide primers of the invention and at least one comprising the sequence of SEQ ID NO: 12 or SEQ ID NO: A synthetic nucleotide sequence of 13. Preferably, the kit of the invention comprises more than one pair of oligonucleotide primers of the invention. In a preferred specific aspect of the invention, the kit comprises a synthetic nucleotide sequence comprising the sequence SEQ ID NO: 12 or SEQ ID NO: 13. In another aspect, the invention provides a kit for detecting and identifying a Croxvirus A16 type in a sample comprising at least one pair of oligonucleotide primers of the invention and at least one comprising the sequence SEQ ID NO: 14 or SEQ ID NO: A synthetic nucleotide sequence of 15. Preferably, the kit of the invention comprises more than one pair of oligonucleotide primers of the invention. In a preferred specific aspect of the invention, the kit comprises a synthetic nucleotide sequence comprising the sequence SEQ ID NO: 14 or SEQ ID NO: 15. In another aspect, the present invention provides for detecting and identifying enterovirus in a sample. 7 The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the back note before writing this page) Order --------- Monument Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 1261618 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (") Figure 4 is enterovirus 71, The nucleic acid of the Crohn's virus A16 type and the intestinal cytopathic autism virus type 3 is amplified by multiplex PCR, and is combined with the enterovirus common probe (p, p2, p3) and enterovirus. Type 71, the result of hybridization of the Croxvirus A16-specific probe (71-2, 71-3, 16-1, 16-2). The primers used in the polymerase chain reaction were paired with f5/rl and f7/r4, and the position of the probe attached to the nylon membrane probe was as follows: this 6x6 lattice cross was equally divided into four 3x3 aliquots, of which the upper left block belongs to Enterovirus common probe block, each row is 3 repeats of 3 probes, the upper right block is enterovirus 71 type region, which is staggered by two specific probes, and the lower left block is saxag virus The A16 type region is staggered by two specific probes. DETAILED DESCRIPTION OF THE PREFERRED DETAILS The proper noun "specific hybridization" or "specific hybridization" means hybridization between an oligonucleotide and a target sequence. The proper noun "specificity" or "speciality" Sexually π means the specificity exhibited by the complementary hybridization, which allows for a slight mismatch between the oligonucleotide and the target sequence without adversely affecting the detection of hybridization signal binding (annealing) The proper noun "sample" means a sample including any biological substance containing nucleic acid. Preferably, the proper noun ''sample'' means biological examination including whole blood 'blood sputum' urine, saliva, cerebrospinal fluid, semen, tears, throat swab, rectal cotton swab, excrement, etc. body. The proper noun ''amplification method'' means a test or method for amplifying a nucleic acid sequence. For example, the "amplification method" includes a polymerase chain reaction (PCR) test, a ligase chain reaction (LCR) test, and an increase in Qfl-replicase. Law, In Vitro Transcription Method ' 9 I---- (Please read the note on the back first? Matters ^$ write this page) I: Order --- 4 This paper scale applies to Chinese national standards (CNS A4 size (210 X 297 mm) 1261618 A7 B7 V. Description of invention (?) In vitro reverse transcription and self-sustaining sequence replication. The proper noun "highly stringent conditions" means that the selected hybridization conditions are about 5 ° C lower than the thermal melting point (Tm) for a particular sequence and a given ionic strength at pH 。. At the point of hot melt (at a given ionic strength and pH )), 50% of the target sequence can hybridize to the fully matched probe. Typically, under highly stringent conditions, the salt concentration is about 0.2 M at pH 7 and the temperature is at least above 60 °C. As used herein, a salt-based designation includes the symbols (i.e., A, G, T, and C) representing natural sources of purines and pyrimidines that are found in natural DNA molecules, as well as those skilled in the art of synthesizing oligonucleotides. Known to represent the code of the mixed base (such as R, Y, S, H). For the code used in the present specification, A (or a) means adenine, G (or g) means guano, T (or t) means thymine, C (or c) means cytosine, R (or r) Represents adenine or guano, Y (or y) represents thymine or cytosine, S (or s) represents guanosine or cytosine, and Η (or h) represents adenine, thymine or cytosine. The invention facilitates the detection of nucleic acids derived from enteroviruses. The present invention provides a rapid and sensitive method for detecting the presence or absence of nucleic acids derived from enteroviruses. In a preferred embodiment of the invention, the method of the invention is based on a polymerase chain reaction (PCR) assay. In another aspect, the invention provides a very specific pair of PCR primers that can be used to detect and/or confirm a particular enterovirus blood sputum type, such as enterovirus 71 and oxavirus A16. In another aspect, the invention provides a specific nucleotide sequence which is capable of undergoing a specific hybridization reaction with a nucleic acid fragment obtainable by the amplification method using the primer of the present invention. Some amplification methods are available, but a PCR based method is preferred. The PCR method is well known in the relevant art, so it is only used here as a simple 10 (please read the note on the back first to write this page)

I* ΜΒΒ ΗΜ I ΜΒ» · II Ml· MM MM AIBIM 4 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed This paper scale applies China National Standard (CNS) A4 specification (210 297 297 mm) 1261618 A7 B7 V. Invention Description (4). For an overview of the PCR method and procedure, see, for example, uS Patent Nos. 4,683,195 and 4,683,202; 4,965,188; and the PCR procedure of the editor of Innis Temple "A Guide to Methods and Application" (Academic Press, Inc., San Diego, CA. 1990). PCR reagents and method programs are also commercially available. Since enterovirus is an RNA virus, the first step in the amplification method is to make a DNA copy (cDNA) for the region to be replicated. The reverse transcription method can be carried out in a separate manner or by homogenous reverse transcription-polymerase chain reaction (RT-PCR), which is an improved method for polymerase chain reaction for amplifying RNA. Methods suitable for PCR amplification of enterovirus nucleic acids are described in "Diagnostic Molecular Biology: Principles and Applications" by Romero and Rotbart, pp. 401-406, edited by Persing et al. (Mayo Foundation, Rochester, MN 1993); Rotbart et al. Patent No. 5,075,212 and Egger et al, J. Clin. Microbiol, 33: 1442-1447 (1995). The present invention provides novel pairs of oligonucleotide primers for detecting the presence or absence of enterovirus in a specimen. The individual primer pairs of the present invention include a first primer and a second primer which facilitate rapid diagnosis of a disease or symptom associated with enterovirus via a nucleic acid amplification assay (e.g., polymerase chain reaction). The primers of the present invention are searched to encode significant or antisense strands of a particular conservative region. A single primer pair or a combination of a plurality of primer pairs results in an array amplification product that can be used to detect enteroviruses present in the sample. In a preferred combination of the invention, the first primer comprises a sequence selected from the group consisting of: TTGTRCGCCTGTTTTA (SEQ ID NO: 1), 11 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 χ 297 f) (Please read the note on the back ^ fill out this page) Pack ------- book ·-- 1 - Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 1261618 Α7 Β7 5, invention description ( , ): CAAGCACTTCTGTHHCCCCGG (SEQ ID NO: 2), 〇: TACTTCGAGAARCCYAGTA (SEQ ID NO: 3), f5: AAGAGYCTATTGAGCTA (SEQ ID NO: 4) and f7: GGI TGG TRS TGG AAR TTI CC (SEQ ID NO: 5). The second primer comprises a sequence R1 selected from the group consisting of: CACYGGATGGCCAATCCAA (SEQ ID NO: 6), R2: ATTGTCACCATAAGCAGCCA (SEQ ID NO: 7), and R4: AR RTTIAT CCA YTG RTGIGG (SEQ ID NO: 8) . The sequence of the primer of the present invention is related to the conventional enterovirus cDNA sequence shown in Figure 1 in the box. When these primers are designed, base modifications are performed on the relevant viral CDNA sequences to increase the efficiency of primer binding and to expand the range of sequence discrimination detectable using these primers. The primers comprising the sequences of the present invention SEQ ID NOs: 5 and 8 are related to the coding region of the enterovirus genome. Primers relating to the coding region of the enterovirus genome are intended to encompass primers comprising degenerate sequences associated with the sequences SEQ ID NOs: 5 and 8. The above listed sequences SEQ ID NOs: 5 and 8 are schematically depicted in a suitable triplet based on the codons associated with the sequences. In the present invention, a more preferred combination comprises one or more pairs of the following primers: F1 (SEQ ID NO: l) / rl (SEQ ID NO: 6); f2 (SEQ ID NO: 2) / rl (SEQ ID NO) : 6); F3 (SEQ ID NO: 3) / rl (SEQ ID NO: 6); f5 (SEQ ID NO: 4) / rl (SEQ ID NO: 6); F1 (SEQ ID NO: l) / r2 (SEQ ID NO: 7); C (SEQ ID NO: 2) / r2 (SEQ ID NO: 7); F3 (SEQ ID NO: 3) / r2 (SEQ ID NO: 7); f5 (SEQ ID NO: 4) /r2 (SEQ ID NO: 7); and F7 (SEQ ID NO: 5) / r2 (SEQ ID NO: 7).

In a preferred aspect of the invention, the primer pair f7 (SEQ ID NO: 5) / r4 (SEQ ID ΝΟ · 8) is particularly suitable for use in an amplification method for detection of enterovirus 71 (EV71) and 12 paper scales for Chinese countries. Standard (CNS) A4 specification (210 X 297 mm) (Please read the note on the back 3⁄4 to fill out this page) β 订--------- Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1261618 A7 B7 V. INSTRUCTIONS (0) Croxvirus A16 (Cox A16). According to Fig. 1, those skilled in the art will know how to select at least one pair of primers of the present invention suitable for detecting and/or amplifying a desired fragment of the enterovirus genome, and thus easily determine the pair of primers of the present invention to be used. As shown in Figure 1, when the first primer comprises the sequence SEQ ID NO: 5 or its degenerate sequence then the second primer should comprise the sequence SEQ ID NO: 8 or its degenerate sequence. In order to increase the target nucleic acid sequence in the sample via PCR, the sequence must be accessible to the elements in the amplification system. Typically, this contact is confirmed by detaching the nucleic acid from the sample. Techniques for extracting nucleic acids from biological samples, such as ribonucleotides, are known in the related art. Alternatively, if the assay system is relatively susceptible to disruption, the nucleic acid will need to be purified prior to amplification by PCR techniques. That is, if the specimen includes cells (especially peripheral blood lymphocytes or monocytes), the dissolution and dispersion of intracellular components can be easily achieved by suspending the cells in a low-tension solution. Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumer Cooperatives. The first step in each PCR cycle is to separate the double strands of nucleic acid from the extension of the primer. When the double strands are separated, the next step in the PCR is to hybridize (i.e., bind) the separated nucleic acid strands to primers flanking the target sequence. The primer is then extended to form a complementary copy of the target sequence. For successful PCR amplification, the primers are designed in such a way that each primer is hybridized along the double-stranded sequence, so that the extension product synthesized from one primer is separated from the template and used as a template for extension of another primer. The period of denaturation, hybridization, and elongation is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid. In the better specific case of the PCR method, the completion of the stock separation is based on the 13-sheet paper scale applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ' 1261618 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed A7 B7 V. INSTRUCTIONS (S) The reaction is heated to a high temperature for a sufficient period of time to cause denaturation of the double strands without causing irreversible denaturation of the polymerase. In the PCR, the template-dependent extension of the primer is based on the presence of a suitable amount of metal ions in the presence of a suitable amount of four deoxyribonucleoside triphosphates (typically dATP, dGTP, dCTP and dTTP). And catalyzed by the incubation liquid of the pH buffering system. Suitable polymeric materials are those known to catalyze stencil-dependent DNA synthesis. In the present invention, the initial template for the elongation of the primer is typically RNA. Reverse transcriptases (RTs) suitable for the synthesis of cDNA from RNA templates are conventional. PCR is most commonly carried out using automated methods using thermal stability enzymes. In this method, the temperature of the reaction mixture is automatically circulated through the denaturation range, the primer adhesion range, and the extended reaction range. As described above, the preferred specific fact of the present invention contains an RT-PCR amplification reaction. However, those skilled in the art will recognize that the increase in the target sequence in the sample can be accomplished by any conventional method, such as ligase chain reaction (LCR), Qfi-replicase amplification, in vitro transcription, and daughter-holding sequence replication, All of the above can provide sufficient increase. The size of the amplified fragment amplification product ") produced by the method of the invention is typically sufficient to determine the presence or absence of enterovirus. Thus, in some specific instances of the invention, the size of the amplified fragments produced in the specimen (e. g., gel electrophoresis analysis) can be used to determine the presence or absence of enterovirus in the specimen. This is typically augmented by introducing the control group containing the known nucleic acid with the same primer used for the amplification of the target sample. After the amplified sequence was electrophoresed on an agarose gel and labeled with ethidium bromide according to the conventional method, the sample and the 14-sheet paper size were applied to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). ' -- π 装 -------- set --- ------. (Please read the notes on the back 3⁄4 fill out this page)

I 1261618 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (, 'Ί) according to the strip type of the group. Differences in the results of the specimens compared with the control group or additional bands indicate the presence of enterovirus. To determine the correctness of the PCR amplification results, more than one pair of primers can be used in an amplification method. In the method of the present invention, pairs of primers different from each other can be used simultaneously or sequentially. In the case where one or more primers of the present invention are simultaneously used in one amplification method, the difference between a pair of primers and another pair of primers may be the first primer or the second primer, or the first primer or the second primer There are different. In another case, if two pairs of primers of the present invention are continuously used in an amplification method, the second pair of primers can be used for the sequence of amplifications, which is equivalent to the sequence that can be obtained by using the first pair of primers in the amplification method or Available within the sequence range. According to Fig. 1, the practitioner can carry out the method of the invention by easily determining whether to use one or more of the primers simultaneously or sequentially. Alternatively, the amplification products of the invention can be detected using oligonucleotide probes specific for the target nucleic acid. The probe is typically selected from a region of the enterovirus genome that has a specific target sequence. The sequence-specific probe hybridization reaction is a well-known method for detecting a desired nucleic acid in a sample. Under sufficient stringent hybridization reaction conditions, the probe only undergoes a specific hybridization reaction with a substantially complementary sequence. The stringency of hybridization reaction conditions can be relaxed to tolerate varying degrees of sequence inconsistency. Detection of the amplified product uses this sequence-specific hybridization reaction to determine that the correct amplified target is detected, thereby reducing the chance of false positives due to the presence of similar sequences from other organisms or other contaminating sequences. The present inventors have surprisingly found that some nucleotide sequences are associated with conserved regions of enterovirus nucleic acid. Therefore, the present invention further provides such synthetic nucleosides 15 The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) > --- (Please read the notes on the back first 填写1 fill out this page) f 1261618 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (A) (antiligand) or antibody binding. Alternatively, the probe can be directly conjugated to a calibration such as a fluorescent generating group, a chemiluminescent agent or an enzyme. The choice of calibration method depends on the sensitivity required, the ease with which the probe is conjugated, the stability requirements, and the instrument available. The probes and primers of the present invention can be synthesized and calibrated using conventional methods. Oligonucleotides as probes and primers can be described in accordance with Beaucage, SL and Caruthers, 1981·Η· (1981, Tetrahedron Letts, 22(20): 1859-1862). Methods Chemical synthesis was performed using an automated synthesizer as described by Needham-Van Devanter, DR et al., 1984, Nucleic Acid Res., 12: 6159-6168. Purification of oligonucleotides can be carried out by natural acrylamide gel electrophoresis or via anion-exchange resin HPLC, as described by Pearson, JD. and Regnier, FE, 1983, J. Chrom., 255: 137-149. Narrator. The above primers and tests are used to detect enteroviruses in specimens, to diagnose diseases and symptoms associated with enteroviruses, and to determine (or cut off) the association between specific symptoms or combinations of symptoms and the presence of specific enteric viruses. Diagnostic applications can be supplemented and confirmed with a history and characteristics of the individual being tested. Enterovirus diseases, syndromes and symptoms diagnosed by the method of the present invention include all diseases, syndromes and symptoms which have been reported in this and the scientific literature, including aseptic meningitis, enterovirus diabetes, enterovirus conjunctivitis, Acute weakness g 庳, acute benign pericarditis, rash, endothelial rash, dilated cardiomyopathy, □ foot disease, chronic fatigue symptoms, fever and upper respiratory tract infection. The detection of enterovirus infection and its association with syndromes makes it possible to develop vaccines and treatments. 18 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ^ nn ϋ ϋ ϋ ϋ · ^1 I I ϋ ϋ 1_1 11 n · ϋ I (Please read the phonetic on the back first? This page)

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1261618 A7 B7 V. INSTRUCTIONS (J) The present invention also provides kits that are multi-container units and include ingredients that facilitate the performance of the methods of the present invention. An advantageous kit can include a probe for detecting the desired target nucleic acid. In some cases, the probe can be attached to a suitable support membrane. The kit will also include the primers provided by the present invention. Other optional components of the kit include, for example, reverse transcriptase or polymerase, matrix nucleoside triphosphates, for calibration purposes (eg, when the calibration is biotin, avidin-enzyme is present) Conjugates and enzyme matrices and chromogens) and suitable buffers for reverse transcription, PCR, or hybridization reactions. In addition to the above ingredients, the kit may also include instructions for performing the methods of the invention. The documents cited in the description of the present invention are incorporated herein by reference. Other features and advantages of the present invention will be apparent from the following description of the specific facts and claims. EXAMPLES The following examples are intended to illustrate the invention. The examples are not intended to limit the scope of the invention in any way, but are intended to indicate how to practice the materials and methods of the invention. Example 1: Detection of Enteroviruses Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Printed 1.1. 59 enterovirus samples used in the virus (see Table 1) or isolated from clinically collected samples. Kind of Center (ATCC). The enterovirus was identified by a neutralization test using confluent immune blood clots, and then the blood type was confirmed by a single-type neutralizing multi-drug antibody. As shown by the results, 59 samples included different enterovirus blood sputum types (see Table 1). Virus in MRC5 monolayer cell culture 19 This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1261618 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (J)

1.2. Extracting RNA

Viral RNAs are obtained by singulating total nucleic acids from centrifuged pellets of infected monolayer cell cultures. About 4-6 x 10 6 cells (per ml) were dissolved in lysis buffer (5 mM NaOH, 20 mM Tris HCl (pH 7.5) '50 mM EDTA, 1% sodium dodecyl sulfate), and extracted three times with phenol-chloroform. It was extracted once with chloroform and then precipitated from 2.5 M ammonium acetate containing ethanol. The nucleic acid pellet was washed with 75% ethanol, dried and suspended in 100 μl of sterile distilled water. : RNA preparations were stored at -80 °C. 1.3. PCR Amplification Assay The first step in the amplification assay is the synthesis of a DNA copy (cDNA) of the portion of the enterovirus RNA gene to be amplified (ie, the reverse transcription step). Reverse transcription by polymerase chain reaction on 20 μl of reaction mixture (Ιμΐ of individual total RNA, 20 mM Tr*is-HCl (pH 8.4), 50 mM Κα, 5 mM MgCl 2 , 10 mM DTT, each 0.5 mM It is carried out in dATP, dCTP, dGTP and dTTP, and 50 ng of random hexamed body. The samples were incubated at 65 ° C for 5 minutes and then placed on ice for 1 minute. 50 units of the reversed enzyme was added and the mixture was incubated at 42 ° C for 50 minutes. The reaction was terminated by incubation at 70 °C for 15 minutes. The resulting cDNA product was stored at -20 °C. DNA amplification via PCR was applied to the 25 1 reaction mixture [12 1 cDNA, 20 mM Tris-HCl (pH 8.0), 0·1 mM EDTA, 1 mM DTT, 1.0% triton X-100, 50% glycerol, 1.5 mM MgCl2 was carried out in 150 M of each of dATP, dCTP, dGTP and dTTP, each of 10 pmol of each primer and 2·5 U of Taq DNA polymerase. Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) in the increase of 20 paper scales (please read the notes on the back and fill out this page)

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1261618 A7 _ B7_ V. INSTRUCTIONS () The primer pairs used in the application are primers containing the following sequences: fl(SEQ ID NO: l)/rl(SEQ ID NO: 6) » G(SEQ ID NO: 2) /rl (SEQ ID NO: 6), C (SEQ ID NO: 3) / rl (SEQ ID NO: 6); f5 (SEQ ID NO: 4) / rl (SEQ ID NO: 6), fl (SEQ ID NO: l) / r2 (SEQ ID NO: 7), C (SEQ ID NO: 2) / r2 (SEQ ID NO: 7), fi (SEQ ID NO: 3) / r2 (SEQ ID NO: 7) R F5 (SEQ ID NO: 4) / r2 (SEQ ID NO: 7). The primers rl and r2 are labeled with biotin at the 5' end for subsequent detection steps. Each amplification reaction was carried out for 40 temperature cycles (denaturation at 94 ° C for 4 minutes, adhesion at 55 ° C for 1 minute, and extension at 72 ° C for 1 minute). 1.4. Isolation of DNA product A small amount of PCR product (10 μl each) was electrophoresed on a 1.5% agarose gel in 0.5 μM buffer (0.045 Μ Tris-borate, 0.001 M EDTA) at 100 volts for 30 minutes. . After electrophoresis, the gel was stained with ethidium bromide and observed under a UV light source. All virus samples showed positive results using the amplification of the primers of the present invention under UV light source. The PCR amplification data for the 59 virus samples using the primer pair f5/rl is shown in Table 1. Table 1 Number of bloody samples shows the number of positive PCR results EV71 17 17 CA7 2 2 CA9 1 1 CA10 1 1 CA 11 1 1 CA16 14 14 CA 24 1 1 CB 1 1 1 CB2 1 1 CB3 1 1 21 This paper The scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1261618 A7 B7 V. Invention description (/) CB4 1 1 CB5 5 5 CB6 1 1 Echo 1 1 1 Echo 2 1 1 Echo 3 1 1 Echo 4 1 1 Echo 5 1 1 Echo 6 1 1 Echo 7 3 3 Echo 9 1 1 Echo 11 1 1 Echo 14 1 1 Total 59 59 Electrophoretic analysis of the amplification of enterovirus 71 nucleic acid by polymerase chain reaction using a combination of different primers Shown in Figure 2. The pair of primers used in each of the a to h lines are: a: fl/rl, b: f2/rl, c: f3/rl, d: f5/rl, e: fl/r2, f: f2/r2, g: f3 /rl,h:f5/r2. m is a nucleic acid size marker, and 1 to 5 are: 1:600 bp, 2: 500 bp, 3: 400 bp, 4: 300 bp, 5: 200 bp. The above results prove that the primer of the present invention can be widely used for detection. The presence or absence of nucleic acid of enterovirus, thereby diagnosing the presence or absence of enterovirus. 1.5. Hybridization Analysis Three enterovirus-specific probes (pl, p2 and p3) were used to detect amplified DNA fragments comprising the sequences SEQ ID ΝΟ_·9, SEQ ID NO: 10 and SEQ ID, respectively. Hey: 11. The probes were heated to denature (95 ° C, 5 minutes) and quenched rapidly on ice for 2 minutes, and then these probes (each ΙΟμΜ) were fixed to a nylon membrane (from Boehringer Manngeim). Three probes (each 1 1) are applied and exposed 22 This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back side and then fill out this page)

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Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 1261618 A7 V. Invention description (^) Under ultraviolet radiation (254nm, 0.15 J/cm2). The biotin-labeled PCR products (each 8 μl) were denatured by heating at 95 ° C for 5 minutes and quenched on ice for 2 minutes, and a hybridization reaction solution [5x standard citrate (SSC), 0.1% (w/) was added. v) N-laurosyl myristic acid '0.1% (w/v) sodium dodecyl sulfate (SDS), lx blocking buffer (from Boehringer Manngeim, 20 ml/100 cm2). The hybridization reaction solution containing the PCR product is then incubated with a nylon membrane and a calibrated probe. After 5 hours of incubation: 1 hour after incubation, the membranes were rinsed five times with rinsing buffer (2xSSC and 0.1% SDS) at room temperature, followed by the addition of streptavidin alkaline phosphatase (2μ1/20ιη1 lx resistance). Broken buffer). The membrane was left at room temperature for 30 minutes and rinsed five times with maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, Ph 7.5) at room temperature. The membrane was equilibrated in detection buffer (〇.1!4丁1^-HC1; 0.1M NaCl; 50Mm MgCl2, pH 9.5) for 5 minutes and mixed with freshly prepared color-matrix solution (45μ1 NBT solution 35μ1 X- Phosphoric acid solution), and added to the test solution with 1 ml of detection buffer for 1 minute in the dark. The membrane was finally rinsed with lxTE buffer to stop the reaction and allowed to air dry at room temperature. The hybridization reaction signal is detected and recorded. The results are shown in Table 2. (Please read the precautions on the back and fill out this page.) Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Printed Table 2 The number of blood samples received by the sample shows the number of samples of the detectable hybridization signal. P2 P3 CA16 6 6 6 6 CA21 1 1 1 1 CA 24 1 1 1 1 23 This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1261618 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative prints A7 B7 V. INSTRUCTIONS (,) CB 1 1 1 1 1 CB2 2 2 2 2 CB3 1 1 1 1 CB4 1 1 1 1 CB5 1 1 1 1 CB6 1 1 1 1 Echo 3 3 3 3 3 Echo 5 1 1 1 1 Echo 6 1 1 1 1 Echo 9 2 2 2 2 Echo 11 3 3 3 3 Echo 14 1 1 1 1 Echo 21 3 3 3 3 Echo 24 1 1 1 1 Echo 30 2 2 2 2 Echo 31 2 2 2 2 EV71 7 7 7 7 Polio 1 2 2 2 2 Polio 2 2 2 2 2 Polio 3 2 2 2 2 Enterovirus 71 and oxacin A16 nucleic acids are amplified by polymerase chain reaction and enterovirus-conjugated probes ( The results of the hybridization of pi, p2, p3) are shown in Fig. 3. The primers used in the polymerase chain reaction were paired with f5/rl, and the positions of the probes fixed in the nylon membrane probe were as follows: the uppermost column and the lowermost column were the hybridization control group, and the probes used were porcine abortion and respiratory syndrome virus (porcine). Reproductive and respiratory syndrome virus (PRRSV) A nucleic acid sequence of ORF 7 that has a nucleic acid sequence complementary to the probe in the hybridization solution, so that the two columns will generate a signal when the hybridization reaction is successfully completed. Point 2 to 4 of the first row on the left: probe pi, point 2 to 4 in the middle row: probe p2, point 2 to 4 in the first row on the right: probe p3. 1 and 2: Enterovirus 71 24 This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

1261618 A7 B7 V. INSTRUCTION DESCRIPTION (#) The type and the Croxvirus A16 have signals at the hybrid control point and the enterovirus common probe position. 3: For the control group containing no nucleic acid, only the hybridization control probe has a signal. Example 2: Detection and identification of Enterovirus 71 (EV71) and Croxvirus A16 (Cox A16) 2.1. PCR amplification assay The RNA extraction and reverse transcription steps were carried out under the same conditions as described in Example 1. The PCR primer used in this assay is a primer pair comprising the sequence f7 (SEQ ID NO: 5) / r4 (SEQ ID NO: 8). The PCR reaction mixture and temperature cycle conditions were adjusted as described in Example 1. 2.2. Hybridization reaction assay The probe for detecting EV71 is a probe containing the sequences of SEQ ID NO: 12 (71-2) and SEQ ID NO: 13 (71-3). The probe for detecting Cox A16 is a probe containing the sequences SEQ ID NO: 14 (16-1) and SEQ ID NO: 15 (16-2). The hybridization reaction assay was carried out as described in Example 1. Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives show that the method of using the primers for f7/r4 and probes 71-2 and 71-3 can specifically detect and identify EV71 and non-EV71 enteroviruses. The results show that the method of the present invention using primer pair f7/r4 and probes 16-1 and 16-2 can specifically detect and identify Cox A16 and non-Cox A16 enterovirus. Example 3: Detection and identification of EV71, Cox A16, non-EV71 enterovirus and non-Cox A16 enterovirus sets 25-point scale applicable to China National Standard (CNS) A4 specification (210 X 297 shy) ~ ' 1261618 A7 ____ B7____ V. INSTRUCTIONS (/) The EV71 and CoxA16 are simultaneously detected using the kit of the present invention. The kit provides materials and procedures to enable the practitioner to perform a variety of PCR tests. The viral RNA extraction and reverse transcription steps were carried out under the same conditions as described in Example 1. Diversified PCR in 25 1 reaction mixture (12 1 cDNA per unit, 50 mM THs-HCl (pH 8.3), 70 mM KC b 2.5 mMMgCl2, each 200 M DATP, dCTP, dGTP and dTTP, 10 pmol primer f5 /rl, 20 pmol of primer f7/r4 and 3U of Taq DNA polymerase). Each amplification reaction was carried out for 4 Torr (4 minutes at 94 °C, 1 minute at 55 °C, and 1 minute at 72 °C). After the amplification of the diversified PCR, the obtained PCR product and the membrane of the immobilized probe (pi, p2 and p3 are directed against enterovirus, 71-2 and 71-3 are specific to EV71, and 16-1 and 16_2 are specific to Cox A16). ) Co-cultivation. All conditions of the hybridization reaction test were the same as those described in Example 1. The results show that the kit (providing a system including diversified PCR and diverse hybridization detection) can detect and identify EV71, Cox A16, non-EV71 enterovirus and non-c〇x A16 enterovirus. The hybridization reaction test data using the different probes of the present invention are shown in Table 3. 3 The Ministry of Economic Affairs, Intellectual Property Office, Staff and Consumer Cooperatives, the blood-type type of enterovirus-printed samples, the number of samples showing the detectable hybridization reaction signal, the number of _K, the i-promoting probe pi p2 p3 71- 2 71-3 16-1 16-2 CA16 6 6 6 6 0 0 6 6 CA21 1 1 1 1 0 0 0 0 CA 24 1 1 1 1 0 0 0 0 CB 1 1 1 1 1 0 0 0 0 CB2 2 2 2 2 0 0 0 0 26 This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 public) 1261618 A7 B7 V. Invention description ( CB3 0 CB4 0 0 0 0 CB5 0 0 0 0 CB6 0

Echo 3 0 0

Echo 5 0

Echo 6 0 0 0

Echo 9 2 2 2 2

Echo 11

Echo 14 0

Echo 21 0

Echo 24

Echo 30 2 2 2 2

Echo 31 2 2 2 2 EV71 7 7 7

Polio 1 2 2 2 2

Polio 2 2 2 2 2

Polio 3 2 2 2 2 Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, Printing Enterovirus 71, Crohn's virus A16 and ECHO virus type 3 nucleic acids were amplified by multiplex PCR, respectively. Hybridization results with enterovirus common probes (pi, p2, p3) and enterovirus 71, and oxacinvirus A16 specific probes (71-2, 71-3, 16-1 and 16-2) Shown in Figure 4. The primers used in the polymerase chain reaction were paired with f5/rl and f7/r4, and the positions of the nylon membrane probes were fixed as follows: This is a 6x6 point probe array, which is equally divided by a 6x6 dot matrix. There are 4 3x3 aliquots, wherein the upper left block belongs to the enterovirus common probe block, each row has 3 replicates of 3 probes, and the upper right block is EV71 type region, which consists of two specific ones. The sexual probes are staggered, and the lower left block is a Crohn's virus A16 type region, which is staggered by two specific probes. 1: Common and enterovirus sputum probe positions are all 5 tigers, 2: common type and saxag virus A16 type probe position is only 27, the paper scale is applicable to China National Standard (CNS) A4 specification ( 210 X 297 mm)

1261618 A7 ___B7__ V. Invention Description (A); 3: Only common area probes have signals. It is apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. Although the present invention has been described with respect to specific specific facts, it must be understood that the present invention should not be unduly limited to the specific details. In fact, the various modifications that are apparent to those skilled in the art are also within the scope of the following claims. Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives. 28 This paper scale applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm).

Claims (1)

Ab BS CS D8 126 Netting 6. Patent Application 1. An oligonucleotide primer pair for detecting the presence or absence of enterovirus in a sample, wherein the first primer in the pair contains one of the following sequences: SEQ ID NO: 1: TTGTRCGCCTGTTTTA ^ SEQ ID NO: 2: CAAGCACTTCTGTHHCCCCGG ^ SEQ ID NO: 3: TACTTCGAGAARCCYAGTA, SEQ ID N〇: 4: AAGAGYCTA Butyl GAGCTA, or SEQ ID NO: 5: GGITGGTRSTGGAARTTICC ^ and the pair of primers The second primer comprises any of the following sequences: SEQ ID NO: 6: CACYGGATGGCCAATCCAA ^ SEQ ID NO: 7: ATTGTCACCATAAGCAGCCA, or SEQ ID NO: 8: ARRTTIATCCAYTGRTGIGG ^ with the condition that when the first primer comprises the sequence SEQ ID NO: 5, The second primer then comprises the sequence SEQ ID NO: 8. 2. A primer pair according to item 1 of the scope of the patent application, comprising a sequence selected from the group consisting of: SEQ ID N〇: 1 / SEQ ID NO: 6; SEQ ID NO: 2 / SEQ ID NO: 6; SEQ ID NO: 3 / SEQ ID NO: 6; SEQ ID NO: 4 / SEQ ID NO: 6; SEQ ID NO: 1 / SEQ ID NO: 7; SEQ ID NO: 2 / SEQ ID NO: 7; N0:3/SEQ ID NO:7; SEQ ID N〇:4/SEQ ID N〇:7; and SEQ ID N〇:5/SEQ ID N〇:8. 3. According to the primer pair of claim 1 of the patent application, it is SEQ ID NO: 5 / SEQ ID N 〇: 8. 4. According to the introduction of the third paragraph of the patent application, it is beneficial to detect enterovirus type 71 (EV71) or saxch virus A16 (Cox A16). 5. A synthetic nucleotide sequence comprising any one selected from the group consisting of: the standard of the paper system (CNS) A4 specification (2] 0 X 297 乂茇) ---- ------------------------------ (Please read the back of the > ± meanings 1 {. write this page) Order 1261618 - C8 D8 VI. Patent application scope: SEQ]DN〇:9: Ding CC Ding CCGGCCCC Ding GAA Ding GCGGC Ding AA D, C, -------------------- ------------ (Please read the note on the back first to write this page) SEQ]D TG Ding CG Ding AACGSGCAAS Ding CYGYRGCGGAACCGAC, SEQ]D Ν〇:Π: TAC 丁丁丁GGG丁丁丁CCG butyl G butyl butyl butyl butyl butylate, SEQ] DN 〇:] 2: C butyl butyl A AGCAGAC butyl CAACCCGG butyl GCTGA butyl G, SEQ ID N 〇:] 3: butyl GGCA butyl CCAATA butyl CACAA butyl AACAG butyl G, SEQ ID Ν〇··] 4: C-butyl CGGCAC butyl A CGCAGGAGGGACCGGGAA and SEQ ID NO: 5: CC butyl ACGCCAC butyl ACACAGCC butyl GG butyl CAGG butyl butyl G, which can be associated with the significance of enterovirus nucleic acid The nucleic acid of the sense strand is subjected to specific hybridization. 6. A method for detecting the presence or absence of enterovirus nucleic acid in a sample, comprising the steps of: (a) introducing the sample with an oligonucleotide primer according to item 1 of the scope of the patent application in an amplification method Contact; and line (b) determine the presence or absence of enterovirus by detecting the presence or absence of an amplification product. 7. The method of claim 6, wherein in step (a), the sample is simultaneously contacted with a second pair of oligonucleotide primers according to item 1 of the scope of the patent application in an amplification method, and the The two pairs of primers are different from the first pair of primers. 8. The method of claim 7, wherein the first one of the second pair of primers is different from the first one of the first pair of primers. 9. The method of claim 7, wherein the second primer in the second pair of primers is different from the second primer in the first pair of primers. 1 This paper scale applies to China National Standard (CNS) A4 specification (2] 0 X 297 mm > 1261618 BS C3 D8 VI. Patent application scope 10. According to the method of claim 7 of the patent scope, in the second pair The two primers in the primer are different from the two primers in the first pair of primers. 11. The method of claim 7, wherein in step (a), the specimen is simultaneously and thirdly in an amplification method Contacting an oligonucleotide primer according to item 1 of the patent application scope, and the third pair of primers is different from the first pair and the second pair of primers. 12. According to the method of claim 6 of the scope of the patent application, Further comprising the step of contacting the product of step (a) in an amplification method with a second pair of oligonucleotide primers according to claim 1 of the scope of the patent application, wherein the second pair of primers can be used for the sequence of amplification, which is equivalent to the use of A sequence obtainable by the pair of primers in the amplification method or within the range of the obtainable sequence. 13. According to the method of claim 6, wherein the amplified product to be detected is further in accordance with at least one patent application scope Synthetic core of item 5 The acid sequence is subjected to a specific hybridization reaction, provided that when the second primer comprising the sequence SEQ ID NO: 8 is not used for amplification, the nucleotide sequence should not comprise any of the sequences SEQ ID Nos: 12-15; when the sequence SEQ ID NO is included When the first primer of 5 is not used for amplification, the nucleotide sequence should not contain any sequence SEQ ID Nos: 9-11 c. 14. The method according to claim 13 wherein the hybridization reaction is highly stringent 15. A method for detecting and identifying a EV71 in a sample, comprising: (a) the sample in an amplification method and the oligonucleotide according to item 1 of the scope of the patent application The primer pair is in contact with the condition that the second primer contains the sequence SEQ_ 3 This paper scale applies to the g S standard (CNS) A4 specification (2] 〇 x 297 2 PCT) (Please read the back note first) Line I Ik 1261618 i C.8 D8 VI. Patent application scope ID N0:8; (b) Determine the presence or absence of enterovirus 71 by detecting the presence or absence of the amplification product; and (c) Detecting the amplified product with at least one of the sequences comprising SEQ ID NO: 12 or SEQ ID NO: The synthetic nucleotide sequence of 13 is subjected to a specific hybridization reaction. The method of claim 15, wherein in the step (4), the amplification product comprises a sequence of SEQ ID Ν0·.12 and SEQ ID 分别0, respectively. The synthetic nucleotide sequence of _13 is subjected to a specific hybridization reaction. _ 17. A method for detecting and identifying a saxachvirus Α16 type in a sample, comprising: (a) detecting the sample in an amplification method Contact with an oligonucleotide primer pair according to item 1 of the scope of the patent application, provided that the second primer comprises the sequence of SEQ ID N〇: 8; (b) determining the presence or absence of the growth product by detecting the presence or absence of the amplification product And the presence or absence of the virus A16; and (c) the specificity of the detected amplification product with at least one synthetic nucleotide sequence comprising the sequence SEQ ID N〇: 14 or SEQ 1DN〇: Hybridization reaction. 18. The method of claim 17, wherein in step (4), the amplification product is subjected to a specific hybridization reaction with a synthetic nucleotide sequence comprising the sequences SEQ ID N: 14 and SEQ ID NO: 15 respectively. . 19. A method for detecting and identifying EV71 and/or Croxvirus A16 in a sample, comprising: (Please read the back note first. Write this page) • 1T: The paper size Appropriate with the China National Standard (CNS) A4 specification (210 X 297 mm) 1261618 g cs D8 ! --- f, the scope of patent application (a) in a method of increasing the number of samples and the scope of patent application One of the oligonucleotide primer pairs is contacted, provided that the second primer contains the sequence SEQ ID NO: 8; (b) determines the enterovirus 71 and/or oxavirus by detecting the presence or absence of the amplification product. The presence or absence of type A1; and (0) the detected amplification product and at least one synthetic nucleotide sequence comprising the sequence of SEQ ID N: 12 or SEQ ID N: 13 and at least one inclusion The synthetic nucleotide sequence of SEQ ID NO: NChl4 or SEQ ID NO: 15 is subjected to a specific hybridization reaction. 20. The method according to claim 19, wherein in the step (4), the amplified product line and the sequence comprising the sequence respectively ID NO: 12 'SEQ ID NO: 13, synthetic nucleotides of SEQ ID NO: 14 and SEQ ID NO: A specific hybridization reaction is carried out. 21. A kit for detecting enteroviruses in a sample, comprising at least one pair of nucleotide primers according to item 1 of the scope of the patent application, the condition of which is 'different' - the primer contains Sequence SEQ ID NO: 5, the second primer comprises the sequence SEQ ID NO: 8. 22. The kit according to claim 21 of the patent application 'which contains more than one pair of oligonucleotide primers according to claim 1 23. The kit according to claim 21 of the scope of the patent application 'which contains at least one synthetic nucleotide sequence according to item 5 of the patent application', provided that the second primer comprising the sequence SEQ ID NO: 8 is not used In the case of amplification, the nucleotide sequence shall not contain any sequence SEQ 1D Nos: 12-15; when the first primer comprising the sequence SEQ ID N〇: 5 is not used in 5 paper scale applications, the SS home standard (CNS) A4 size (2]0 X 297 ^茇) (Please read the notes on the back first to write this page) 1261618 g C3 D8 VI. Patent application scope. When increasing, the nucleotide sequence should not contain any sequence SEQ ID N〇s: 9-11 ο 24·--Detecting and identifying the EV71 in the sample a kit comprising: at least one pair of oligonucleotide primers according to claim 1 of the patent application, provided that the second primer comprises the sequence SEQ ID Ν0:8; and at least one comprises the sequence SEQ ID Ν0··12 or SEQ A synthetic nucleotide sequence of ID Ν0··13. 25. A kit for detecting and identifying a saxagvirus Α16 type in a sample, comprising: at least one pair of oligonucleotide primers according to claim 1 of the patent application, the condition that the second primer comprises a sequence SEQ ID Ν0:8; and at least one synthetic nucleotide sequence comprising the sequence SEQ ID Ν0:14 or SEQ ID Ν0:15. 26. A method for detecting and identifying a guineavirus type 71 and a keshaqi virus Α16 type in a sample, comprising: at least one pair of oligonucleotide primers according to claim 1 of the patent application, the condition being a second primer Is a sequence comprising SEQ ID Ν0:8; at least one synthetic nucleotide sequence comprising the sequence SEQ ID Ν0:12 or SEQ ID Nan and at least one synthetic nucleoside comprising the sequence SEQ ID ΝΟ: ι4 $ SEQ ID Ν0:15 Acid sequence. 27. A method for detecting and identifying a guineavirus type 71 or a saxag, a disease _ Α 16 type, comprising: at least one pair of oligonucleotide primers according to claim 1 of the patent application, g The second primer contains the sequence SEQ ID NO: 8; the paper scale applies to the Chinese National Standard (CNS) A4 specification (210 297 297 Y PCT) A8 BS C8 D8 1261618 6. At least one of the patent claims includes the sequence SEQ ID N0: 12 or a synthetic nucleotide sequence of SEQ ID NO: 13 and at least one synthetic nucleotide sequence comprising the sequence of SEQ ID NO: 14 or SEQ ID Ν 0··15. Wood paper scale applies to China National Standard (CNS) A4 specification (210 X 297V PCT)