CN101864496A - Detection kit for acute hemorrhagic conjunctivitis and detection method thereof - Google Patents
Detection kit for acute hemorrhagic conjunctivitis and detection method thereof Download PDFInfo
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- CN101864496A CN101864496A CN 201010145746 CN201010145746A CN101864496A CN 101864496 A CN101864496 A CN 101864496A CN 201010145746 CN201010145746 CN 201010145746 CN 201010145746 A CN201010145746 A CN 201010145746A CN 101864496 A CN101864496 A CN 101864496A
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Abstract
The invention relates to a detection kit for an enterovirus 70-type (EV70) and Coxackie A24-type virus variants (CA24v) which are relevant to acute hemorrhagic conjunctivitis and a detection method thereof. The kit comprises a RT-PCR (reverse transcription-polymerase chain reaction) reaction buffer solution, enterovirus 70-type (EV70) and Coxackie A24-type virus variant (CA24v) specific forward and reverse primer and fluorescent probe mixed liquor necessary for PCR amplification, a RT-PCR reaction enzyme system, DEPC H2O (diethyl pyrocarbonate) and EV70 and CA24v positive controls as well as negative controls. The invention has stable and reliable result and simple and rapid operation and can rapidly detect the acute hemorrhagic conjunctivitis caused by the enterovirus 70-type and Coxackie A24-type virus variants, and thereby, the early anti-epidemic work can be completed.
Description
Technical field
The present invention relates to the external diagnosis reagent technical field, be specifically related to a kind of Enterovirus 70 type (EV70) relevant and the detection kit and the detection method thereof of COxsackie A24 C-type virus C variant (CA24v) with acute hemorrhagic conjunctivitis.
Background technology
Acute hemorrhagic conjunctivitis is commonly called as pink eye disease, is one of Class C transmissible disease of " People's Republic of China's law on the prevention and control of infectious diseases " regulation report.This disease sickness rate height is propagated rapidly, and it is wide to involve scope, and infectivity is extremely strong, and latent period is very short, behind the contagion source in 12～48 hours eyes promptly fall ill, bring considerable influence for people's production and life.The general susceptible of crowd, it is many than children's number of the infected to be grown up.Breaking out with popular of acute hemorrhagic conjunctivitis all reported in a lot of in the world areas,
The acute hemorrhagic conjunctivitis epidemic situation of worldwide breaking out, main pathogen are EV70 and CA24, and acute hemorrhagic conjunctivitis was proved first by CA24v (CA24 virus variant) and is caused in Singapore in 1970 then.In China, acute hemorrhagic conjunctivitis once had big twice popular, was respectively 1971 and 1988, its former EV70 and CA24v of being that cause a disease.Because the extremely strong infectivity of this disease is broken out the postoperative infection large contingent, bring tremendous loss for economy and medical resource.
Enterovirus EV70 and Coxsackie virus all belong to Picornaviridae, and virus is spherical in shape, and the electron microscope observation diameter is 23～30nm, and viral capsid is the three-dimensional symmetry of icosahedro.The method of general identifying virus mainly contains following several method: (1) electron microscope is identified, the negative staining of virosome and ultrathin section(ing).This method can directly be utilized the microscopic examination virion, but the operator is required than higher; (2) identified by immunofluorescence, this method are to utilize the fluorescein specific marker to observe painted fluorescence, judge pathogenic agent according to antibodies specific.Immunofluorescence technique has fast, special advantage.(3) means such as serological method evaluation also are the conventional meanses that virus is identified, thereby judge the kind and the hypotype of virus by serological identification.Except above several method, present most of laboratories all adopt the polymerase chain reaction to carry out the evaluation of virus.EV70 and CA24v virus all are the RNA of strand, therefore adopt reverse transcriptase polymerase chain reaction to carry out the amplification of viral nucleic acid.
The real-time fluorescence PCR technology is a kind of direct detection nucleic acid and the Protocols in Molecular Biology that carries out nucleic acid quantification, has highly sensitively, and specificity is good, and is simple, advantages such as cheapness.It adds fluorophor a kind of in the PCR reaction system, utilize fluorescent signal accumulation monitoring PCR reaction process in real time, by the result data analysis starting template is carried out quantitatively.The present invention adopts the two-color fluorescence PCR method in the real-time fluorescence PCR technology, promptly two purpose viral nucleic acids (EV70 and CA24v) is increased, and according to the Two Colour Fluorescence of mark, judges the situation of viral nucleic acid amplification.The test kit that the present invention relates to can apply to the detection of the acute hemorrhagic conjunctivitis that caused by EV70 and CA24v widely.
Summary of the invention
One of technical problem to be solved by this invention provides a kind of test kit that the Enterovirus 70 type (EV70) relevant with acute hemorrhagic conjunctivitis and COxsackie A24 C-type virus C variant (CA24v) are detected, described test kit is highly sensitive, specificity is good, is convenient to detect.
On the present invention basis that the nucleotide sequence of all known EV70 and CA24v virus strain is compared on to GENBANK, seek the specificity conserved regions of EV70 and CA24v nucleotide sequence, and at target nucleotide primer and the probe of conserved regions design EV70 and CA24v.These primer probes are containing hot resistant DNA polymerase, high-quality deoxyribonucleoside triphosphate (dNTPs) and Mg
2+Deng the PCR reaction buffer in, realize the cyclic amplification of external nucleic acid by fluorescent PCR amplification instrument.
EV70 provided by the present invention and CA24v virus strain detection kit comprise following composition:
(1) RT-PCR reaction buffer: by Tris-HCl, MgCl
2, the KCl salt buffer forms;
(2) primer probe mixed solution: essential special forward and reverse primer and fluorescent probe during for pcr amplification, sequence is selected from the high conservative zone of EV70 and CA24v; Fluorescent probe is double-colored mark;
(3) RT-PCR reaction enzymes system: form by dNTPs, warm start Taq enzyme and MMLV enzyme and stablizer;
(4)DEPC?H
2O；
(5) EV70 and CA24v positive quality control product: be MS2 carrier pseudovirus;
(6) the negative quality control product of EV70 and CA24v: be physiological saline.
The RT-PCR reaction buffer preferably by Tris-HCl (50mmol/L, pH8.0), MgCl
2(8mmol/L), KCl (250mmol/L) forms.
The forward of EV70 target polynucleotide amplification and the sequence of reverse primer are respectively 5 '-CAACAAGTACCTCATCCATTCAAAAG-3 ' (SEQ ID NO:1) and 5 '-GTAAACAATTCCATCTTCCTCCT-3 ' (SEQ ID NO:2), the described EV70 oligonucleotide probe that is used for fluorescent PCR amplification and detection architecture, its sequence is 5 '-TTCTTCATCTGGACTTTGAACACCAGAGAACT-3 ' (SEQ ID NO:3), and the two ends of probe are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively;
Being used for the forward of CA24v target polynucleotide amplification and the sequence of reverse primer is respectively 5 '-GAGTGCTTGCCCAGATTTTAG-3 ' (SEQ ID NO:4) and 5 '-ATAACGGTGTCAATGGTCTCCTC-3 ' (SEQID NO:5), the CA24v oligonucleotide probe that is used for fluorescent PCR amplification and detection architecture, its sequence is 5 '-CGTGCGTCTGTTGAGAGACACCAAC-3 ' (SEQ ID NO:6), and the two ends of probe are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively.
Form the primer concentration described in (2) and all be preferably 0.2mmol/L, described concentration and probe concentration all is preferably 0.1mmol/L.
RT-PCR reaction enzymes system is made up of warm start Taq enzyme, MMLV, dNTPs, can adopt commercially available warm start enzyme, MMLV, dNTPs product, as Qiagen company product, wherein the consumption of everyone part reagent warm start Taq enzyme is that 5U, MMLV consumption are that 10U, dNTPs are 10mmol.
Carry out sample detection carrying out the simultaneously detection of two special quality control product to be checked in the test kit of the present invention, only when EV70, CA24v positive quality control product tests positive, negative quality control product detects when being negative, and the detected result of sample to be checked is just effective.
Another technical problem to be solved by this invention provides a kind of detection method that detects acute hemorrhagic conjunctivitis nucleic acid, comprises the steps:
(1) sample pre-treatment;
(2) nucleic acid extraction;
(3) carry out real-time fluorescence quantitative PCR amplification and detection according to test kit of the present invention:
A. reagent is prepared: primer probe mixed solution, RT-PCR reaction solution, the RT-PCR reaction enzymes of getting respective amount in proportion is to be distributed into the PCR reaction tubes by 30 μ l/ pipes behind the abundant mixing, and be standby.
B. application of sample: in the PCR reaction tubes, add positive and negative quality control product, sample rna solution 20 μ l after extracting respectively, the tight pipe lid of lid is put into the instrument sample cell.
The c.PCR amplification;
(4) interpretation of result.
The condition optimization of pcr amplification of the present invention is: 50 ℃ 15 minutes, 95 ℃ 15 minutes; 94 ℃ 15 seconds, 55 ℃ 45 seconds, 40 circulations (55 ℃ of 45 seconds annealing stages are collected fluorescent signal).
The present invention compared with prior art, has following advantage: 1. can detect to EV70 and CA24v viral nucleic acid level of amplification, the state that can reflect interior EV70 of patient's body and CA24v virus infection simultaneously, help the early diagnosis and therapy of disease, and can be used for monitoring therapeuticing effect and assess patient prognosis; 2. the fluorescent PCR technology has higher specificity at this viral nucleic acid specific sequence design primer, probe, has easy and simple to handle, cheap advantage, is applicable to that more Most patients detects; 3. stopped pipe detects and does not need the PCR aftertreatment, has avoided because false positive and the environmental pollution that the crossed contamination between sample causes.
Test kit of the present invention is when amplification sample to be checked, and testing process is finished automatically by commercially available quantitative real time PCR Instrument, and is simple to operate, consuming time few, and has reduced the generation of polluting to greatest extent.Detected result can be used for because a plurality of area researches such as detection, pharmacological agent and prognosis of the acute hemorrhagic conjunctivitis that EV70 and CA24v virus cause.
Description of drawings
The reaction conditions of pcr amplification among Fig. 1 embodiment 1;
The amplification curve of the negative quality control product of test kit among Fig. 2 embodiment 2;
Do not have S type amplification curve among the figure, illustrate and detect the amplification that does not have EV70 and CA24v viral nucleic acid in the sample;
The amplification curve of test kit positive quality control product among Fig. 3 embodiment 2;
The amplification curve of FAM passage is the S type among the figure, illustrates to detect the amplification that the EV70 viral nucleic acid is arranged in the sample;
The amplification curve of test kit positive quality control product among Fig. 4 embodiment 2;
The amplification curve of HEX passage is the S type among the figure, illustrates to detect the amplification that the CA24v viral nucleic acid is arranged in the sample;
The amplification curve of 3 routine negative sample among Fig. 5 embodiment 2;
Do not have S type amplification curve among the figure, the amplification that detects no EV70 and CA24v viral nucleic acid in the sample is described;
The amplification curve of the positive eye of 1 example conjunctival sac tear sample among Fig. 6 embodiment 2;
The amplification curve of FAM mark is the S type among the figure, illustrates to detect the amplification that the EV70 viral nucleic acid is arranged in the sample.There is the EV70 virus infection in the expression sample;
The amplification curve of the positive eye of 1 example conjunctiva swab sample among Fig. 7 embodiment;
The HEX amplification curve is the S type among the figure, illustrates to detect the amplification that the CA24v viral nucleic acid is arranged in the sample.There is the CA24v virus infection in the expression sample;
The amplification curve of 1 routine positive sample among Fig. 8 embodiment 2;
The amplification curve of FAM passage and HEX passage is the S type among the figure, illustrates to detect the amplification that EV70 and CA24v viral nucleic acid are arranged in the sample.There is the polyinfection of EV70 and CA24v virus in expression.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Acute hemorrhagic conjunctivitis detection kit and use thereof
1, preparation comprises the test kit of following moiety:
Primer probe mixed solution (25 μ l/ pipe) 1 pipe: the forward of EV70 target polynucleotide amplification and the sequence of reverse primer are respectively 5 '-CAACAAGTACCTCATCCATTCAAAAG-3 ' (SEQ ID NO:1) and 5 '-GTAAACAATTCCATCTTCCTCCT-3 ' (SEQ ID NO:2), the EV70 oligonucleotide probe, its sequence is 5 '-TTCTTCATCTGGACTTTGAACACCAGAGAACT-3 ' (SEQ ID NO:3), and the two ends of probe are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively; Being used for the forward of CA24v target polynucleotide amplification and the sequence of reverse primer is respectively 5 '-GAGTGCTTGCCCAGATTTTAG-3 ' (SEQ ID NO:4) and 5 '-ATAACGGTGTCAATGGTCTCCTC-3 ' (SEQ ID NO:5), the CA24v oligonucleotide probe that is used for fluorescent PCR amplification and detection architecture, its sequence is 5 '-CGTGCGTCTGTTGAGAGACACCAAC-3 ' (SEQ ID NO:6), and the two ends of probe are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively;
RT-PCR reaction buffer (250 μ l/ pipe) 1 pipe: by Tris-HCl (50mmol/L, pH8.0), MgCl
2(8mmol/L), KCl (250mmol/L) forms;
RT-PCR reaction enzymes system (75 μ l/ pipe) 1 pipe: Taq enzyme, MMLV, dNTPs form, and wherein the consumption of everyone part reagent warm start Taq enzyme is that 5U, MMLV consumption are that 10U, dNTPs are 10mmol;
Positive quality control product (200 μ l/ pipe) 1 pipe: MS2 carrier pseudovirus;
Negative quality control product (200 μ l/ pipe) 1 pipe: physiological saline;
DEPC H
2O (2000 μ l/ pipe) 1 pipe.
2, the collection of sample, storage and transport
2.1 be suitable for the sample type: eye conjunctival sac tear, eye conjunctiva swab or scrape and get thing.
2.2 collection of specimens, preserve and transport: sample should be onset 1～3 day with interior collection.Concrete grammar: embrocate the upper and lower papebral conjunctiva of upset and wipe away with the disinfecting silk or cotton swab and get tear and drop into immediately in the small test tube that sterile saline or Eagle liquid or 0.5% lactoalbumin hydrolysate Hanks liquid 2mL are housed, post label, put take to the laboratory in the curling stone or low temperature (20 ℃～-70 ℃) frozen.The long-distance employing dry ice that transports of sample.
3, nucleic acid extraction:
Use QIAamp Viral RNA Mini Kit to extract test kit.Carry out according to QIAamp Viral RNA Mini Kit specification sheets, collect 50 μ l RNA solution at last, directly detect or be stored in-20 ℃.
4, real-time fluorescence quantitative PCR amplification and detection
4.1 reagent is prepared: primer probe mixed solution, RT-PCR reaction solution, the RT-PCR reaction enzymes of getting respective amount in proportion are that (primer probe mixed solution 1 μ l+PCR reaction solution 10 μ l/ person-portion+RT-PCR enzymes are 3 μ l/ person-portion+DEPC H
2O 16 μ l), fully be distributed into the PCR reaction tubes by 30 μ l/ pipes behind the mixing, standby.
4.2 application of sample: in the PCR reaction tubes, add positive and negative quality control product, sample rna solution 20 μ l after extracting respectively, the tight pipe lid of lid is put into the instrument sample cell.
4.3 editor: (ABI Prism 7500 quantitative real time PCR Instruments)
Open the Setup window, positive and negative quality control product and sample to be measured are set in proper order, and the sample title is set in the Name hurdle by correspondence.Choose all that sample well is set, double-click, select Add Detector, selecting Reporter is that FAM and Quencher are BHQ1, and selecting Reporter again is that HEX and Quencher close window behind the BHQ1.In PassiveReference, select (none).Open the instrument window cycling condition be set: 50 ℃ 15 minutes, 95 ℃ 15 minutes; 94 ℃ 15 seconds, 55 ℃ 45 seconds, 40 circulations.(seeing accompanying drawing 1).After finishing, all settings preserve file, operation.
4.4 interpretation of result:
Reaction finishes the back and preserves the detection data file.Under Results, open Amp plot window.Select the purpose sample position of analysis.Change Baseline numerical value into start:3, stop:10, and open manual and set Threshold:1.5 ± 100000.Numerical value is opened Graph settings window on the double-click Rn coordinate, changes Log among the Post Run Settings into Linear, opens Analysis preferences window behind the OK, selects the automatic analytical results of Analyze under the Analysis menu.
Use the acute hemorrhagic conjunctivitis detection kit and detect clinical sample
Choose 3 examples and be accredited as EV70 and CA24v virus feminine gender through serological method, 3 example process serological methods are accredited as the sample of EV70 and CA24v virus-positive, the sample nucleic acid extraction, and pcr amplification and interpretation of result are carried out with reference to embodiment 1, carry out the moon simultaneously, the detection of positive quality control product.
Detected result is explained: the amplification curve of negative quality control product is irregular or do not have a CT value, the amplification curve of (seeing accompanying drawing 2) positive quality control product is that obvious S type curve (is seen accompanying drawing 3,4), in test kit Quality Control scope, illustrate that the amplification that detection architecture can detect EV70 and CA24v viral nucleic acid effectively (sees accompanying drawing 5,6,7,8). the yin and yang attribute quality control product all meets the Quality Control requirement of test kit in together with experiment.
Detected result and the serological identification result of 6 routine samples fits like a glove in this test, illustrates to utilize that EV70 and CA24v viral nucleic acid level of amplification are feasible in this test kit detection sample.This test kit is easy and simple to handle, and detection time is short, can realize high throughput testing, and it is cheap in addition, can be born by most of acute hemorrhagic conjunctivitis patient.
Sequence table
＜110〉Shanghai City Entry-Exit Inspection and Quarantine Bureau, Da
＜120〉a kind of acute hemorrhagic conjunctivitis detection kit and detection method thereof
<160>6
<170>PatentIn?version?3.5
<210>1
<211>26
<212>DNA
＜213〉EV70PCR amplifying specific forward primer sequence
<400>1
caacaagtac?ctcatccatt?caaaag 26
<210>2
<211>23
<212>DNA
＜213〉EV70PCR amplifying specific reverse primer sequence
<400>2
gtaaacaatt?ccatcttcct?cct 23
<210>3
<211>32
<212>DNA
＜213〉EV70 fluorescent probe sequence
<400>3
ttcttcatct?ggactttgaa?caccagagaa?ct 32
<210>4
<211>21
<212>DNA
＜213〉CA24vPCR amplifying specific forward primer sequence
<400>4
gagtgcttgc?ccagatttta?g 21
<210>5
<211>23
<212>DNA
＜213〉CA24vPCR amplifying specific reverse primer sequence
<400>5
ataacggtgt?caatggtctc?ctc 23
<210>6
<211>25
<212>DNA
＜213〉CA24v fluorescent probe sequence
<400>6
cgtgcgtctg?ttgagagaca?ccaac 25
Claims (10)
1. acute hemorrhagic conjunctivitis kit for detecting nucleic acid is characterized in that: comprise following composition:
(1) RT-PCR reaction buffer: by Tris-HCl, MgCl
2, the KCl salt buffer forms;
(2) primer probe mixed solution: essential Enterovirus 70 type (EV70) and forward and reverse primer of COxsackie A24 C-type virus C variant (CA24v) specificity and fluorescent probe during for pcr amplification, sequence is selected from the high conservative zone of EV70 and CA24v, and fluorescent probe is double-colored mark;
(3) RT-PCR reaction enzymes system: form by dNTPs, warm start Taq enzyme and MMLV enzyme and stablizer;
(4)DEPC?H
2O；
(5) EV70 and CA24v positive quality control product: be MS2 carrier pseudovirus;
(6) the negative quality control product of EV70 and CA24v: be physiological saline.
2. according to the described acute hemorrhagic conjunctivitis kit for detecting nucleic acid of claim 1, it is characterized in that: Tris-HCl is 50mmol/L, pH8.0 in the described RT-PCR reaction buffer, MgCl
2Be 8mmol/L, KCl is 250mmol/L.
3. according to the described acute hemorrhagic conjunctivitis kit for detecting nucleic acid of claim 1, it is characterized in that: described EV70PCR amplifying specific forward primer sequence is 5 '-CAACAAGTACCTCATCCATTCAAAAG-3 ' (SEQ ID NO:1), and the reverse primer sequence is 5 '-GTAAACAATTCCATCTTCCTCCT-3 ' (SEQ ID NO:2).
4. according to the described acute hemorrhagic conjunctivitis kit for detecting nucleic acid of claim 1, it is characterized in that: described EV70 fluorescent probe sequence is 5 '-TTCTTCATCTGGACTTTGAACACCAGAGAACT-3 ' (SEQ ID NO:3), and the two ends of probe are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively.
5. according to the described acute hemorrhagic conjunctivitis kit for detecting nucleic acid of claim 1, it is characterized in that: described CA24vPCR amplifying specific forward primer sequence is 5 '-GAGTGCTTGCCCAGATTTTAG-3 ' (SEQ ID NO:4), and the reverse primer sequence is 5 '-ATAACGGTGTCAATGGTCTCCTC-3 ' (SEQ ID NO:5).
6. according to the described acute hemorrhagic conjunctivitis kit for detecting nucleic acid of claim 1, it is characterized in that: described CA24v fluorescent probe sequence is 5 '-CGTGCGTCTGTTGAGAGACACCAAC-3 ' (SEQ ID NO:6), and the two ends of probe are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively.
7. according to the described acute hemorrhagic conjunctivitis kit for detecting nucleic acid of claim 1, it is characterized in that: described pcr amplification primer substrate concentration is 0.2mmol/L, and described concentration and probe concentration is 0.1mmol/L.
8. according to the described acute hemorrhagic conjunctivitis kit for detecting nucleic acid of claim 1, it is characterized in that: the consumption of everyone part reagent warm start Taq enzyme is that 5U, MMLV consumption are that 10U, dNTPs are 10mmol in the described RT-PCR reaction enzymes system.
9. a detection method that detects acute hemorrhagic conjunctivitis nucleic acid is characterized in that comprising the steps:
(1) sample pre-treatment;
(2) nucleic acid extraction;
(3) test kit according to claim 1 carries out real-time fluorescence quantitative PCR amplification and detection:
A. reagent is prepared: primer probe mixed solution, RT-PCR reaction solution, the RT-PCR reaction enzymes of getting respective amount in proportion is to be distributed into the PCR reaction tubes by 30 μ l/ pipes behind the abundant mixing, and be standby;
B. application of sample: in the PCR reaction tubes, add positive and negative quality control product, sample rna solution 20 μ l after extracting respectively, the tight pipe lid of lid is put into the instrument sample cell;
The c.PCR amplification;
(4) interpretation of result.
10. according to the described acute hemorrhagic conjunctivitis nucleic acid detection method of claim 9, it is characterized in that: described pcr amplification condition is: 50 ℃ 15 minutes, 95 ℃ 15 minutes; 94 ℃ 15 seconds, 55 ℃ 45 seconds, 40 circulations.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101580882A (en) * | 2008-05-14 | 2009-11-18 | 中山大学达安基因股份有限公司 | Real-time fluorescence quantitative PCR detection kit for human enterovirus 71 |
| CN101638693A (en) * | 2008-07-30 | 2010-02-03 | 中山大学达安基因股份有限公司 | Kit for detecting human enteric viruses by real-time fluorescent quantitative PCR |
-
2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101580882A (en) * | 2008-05-14 | 2009-11-18 | 中山大学达安基因股份有限公司 | Real-time fluorescence quantitative PCR detection kit for human enterovirus 71 |
| CN101638693A (en) * | 2008-07-30 | 2010-02-03 | 中山大学达安基因股份有限公司 | Kit for detecting human enteric viruses by real-time fluorescent quantitative PCR |
Non-Patent Citations (3)
| Title |
|---|
| 《Journal of Virological Methods》 20090731 XL Xiao et al. Simultaneous detection of enterovirus 70 and coxsackievirus A24 variant by multiplex real-time RT-PCR using an internal control Pages 23-28 1-10 , * |
| 《中华实验和临床病毒学杂志》 19991231 王君君等 急性出血性结膜炎的临床表现及病毒学检测 第279，283页 1-10 , * |
| 《中国病原生物学杂志》 20110630 李洁等 实时荧光定量RT-PCR 检测急性出血性结膜炎CoxA24v病毒 第411-413，423页 1-10 , * |
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