CN105238880A - Enterovirus real-time fluorescent quantitative detection kit - Google Patents

Enterovirus real-time fluorescent quantitative detection kit Download PDF

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Publication number
CN105238880A
CN105238880A CN201510715793.XA CN201510715793A CN105238880A CN 105238880 A CN105238880 A CN 105238880A CN 201510715793 A CN201510715793 A CN 201510715793A CN 105238880 A CN105238880 A CN 105238880A
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enterovirus
fluorophor
probe
detection kit
fluorescent quantitative
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杨浩
邓艳华
胡鹏
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Guangdong Peng Peng biological Co., Ltd.
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Fei Peng Biological Co Ltd
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/6851Quantitative amplification

Abstract

The invention discloses an enterovirus real-time fluorescent quantitative detection kit, which is used for distinguishing EV71 enterovirus from CA16 enterovirus, wherein the detection kit comprises a forward primer EV71F1 and a reverse primer EV71R1 for amplifying the EV71 enterovirus, a probe EV71P1 for detecting the EV71 enterovirus, a forward primer CA16F2 and a reverse primer CA16R2 for amplifying the CA16 enterovirus, a probe CA16P2 for detecting the CA16 enterovirus and a real-time fluorescent quantitative PCR (polymerase chain reaction) buffer solution, wherein a first fluorescence group and a first fluorescence quencher are respectively bonded to two ends of the probe EV71P1; a second fluorescence group and a second fluorescence quencher are respectively bonded to two ends of the probe CA16P2; and the first fluorescence group is different from the second fluorescence group. The enterovirus real-time fluorescent quantitative detection kit, when being used, can distinguish the EV71 enterovirus from the CA16 enterovirus by respectively detecting signals of the first fluorescence group and signals of the second fluorescence group.

Description

Enterovirus real time fluorescent quantitative detection kit
Technical field
The present invention relates to diagnostic techniques field, particularly relate to a kind of enterovirus real time fluorescent quantitative detection kit.
Background technology
Hand foot mouth disease is the transmissible disease caused by enterovirus, and causing the enterovirus of hand foot mouth disease has kind more than 20 (type), wherein with enterovirns type 71 (EV71) and coxsackie virus A 16-type (CoxA16, CA16) the most common.The two caused hand foot mouth disease is difficult to distinguish clinically, with CA16 type unlike, EV71 type not only can cause hand foot mouth disease, what is more important its can cause serious nervus centralis complication, as encephalitis, meningitis, acute flaccid paralysis etc., even cause death.The main symptomatic treatment of the effective medicine of current shortage.
The latent period of the diseases such as the hand foot mouth disease that enterovirus causes, viral encephalitis is generally 3 days ~ 7 days, does not have obvious forerunner's illness, and shows as most patient's sudden onset.So early diagnosis, early treatment just seem particularly important.Detection method traditional is at present, throat swab or stool sample are delivered to test in laboratory virus, but Viral diagnosis needs just can go out result, and there is complex operation for 2 weeks ~ 4 weeks, and susceptibility is low, does not reach " four early " requirement for disease prevention far away.
Along with developing rapidly of Protocols in Molecular Biology, using polymerase chain reaction technology is that the detection of pathogenic micro-organism provides new direction, the real-time fluorescence quantitative PCR particularly risen in recent years.The method by realizing the Real-Time Monitoring to product amount in PCR process to the detection of fluorescent signal, and can accurately calculate original template amount.This method except quantitatively accurately, detect fast, its maximum advantage is that have employed stopped pipe detects, and avoids crossed contamination, extensively by scientific research with clinical to accept.
Do not report the enterovirus real time fluorescent quantitative detection kit for distinguishing EV71 type enterovirus and CA16 type enterovirus at present temporarily.
Summary of the invention
Based on this, be necessary to provide a kind of enterovirus real time fluorescent quantitative detection kit that can be used in distinguishing EV71 type enterovirus and CA16 type enterovirus.
A kind of enterovirus real time fluorescent quantitative detection kit, for distinguishing EV71 type enterovirus and CA16 type enterovirus, comprising:
For the upstream primer EV71F1 of EV71 type enterovirus of increasing, for the downstream primer EV71R1 of EV71 type enterovirus of increasing, for detecting the probe EV71P1 of EV71 type enterovirus, the two ends of described probe EV71P1 are combined with the first fluorophor and the first fluorescence quencher respectively;
For the upstream primer CA16F2 of CA16 type enterovirus of increasing, for the downstream primer CA16R2 of CA16 type enterovirus of increasing, for detecting the probe CA16P2 of CA16 type enterovirus, the two ends of described probe CA16P2 are combined with the second fluorophor and the second fluorescence quencher respectively, and described first fluorophor is different from described second fluorophor; And
Real-time fluorescence quantitative PCR reaction buffer.
In one embodiment, the sequence of described upstream primer EV71F1 is the sequence shown in SEQIDNo.1, and the sequence of described downstream primer EV71R1 is the sequence shown in SEQIDNo.2, and the sequence of described probe EV71P1 is the sequence shown in SEQIDNo.3.
In one embodiment, the 5 ' end of described probe EV71P1 is combined with the first fluorophor, and the 3 ' end of described probe EV71P1 is combined with the first fluorescence quencher, and described first fluorophor is ROX, and described first fluorescence quencher is BHQ1.
In one embodiment, the sequence of described upstream primer CA16F2 is the sequence shown in SEQIDNo.4, and the sequence of described downstream primer CA16R2 is the sequence shown in SEQIDNo.5, and the sequence of described probe CA16P2 is the sequence shown in SEQIDNo.6.
In one embodiment, the 5 ' end of described probe CA16P2 is combined with the second fluorophor, and the 3 ' end of described probe CA16P2 is combined with the second fluorescence quencher, and described second fluorophor is JOE, and described second fluorescence quencher is BHQ1.
In one embodiment, also comprise for putting on trip primers F 3 in target in the noncompetitive that increases, for marking downstream primer R3 in target in the noncompetitive that increases and marking probe P3 in target for detecting in noncompetitive, the two ends of described interior mark probe P3 are combined with the 3rd fluorophor and the 3rd fluorescence quencher respectively, and described 3rd fluorophor is different from described first fluorophor and described 3rd fluorophor is different with described second fluorophor.
In one embodiment, the sequence putting on trip primers F 3 in described is the sequence shown in SEQIDNo.7, and the sequence of described interior mark downstream primer R3 is the sequence shown in SEQIDNo.8, and the sequence of described interior mark probe P3 is the sequence shown in SEQIDNo.9.
In one embodiment, the 5 ' end of described interior mark probe P3 is combined with the 3rd fluorophor, and the 3 ' end of described interior mark probe P3 is combined with the 3rd fluorescence quencher, and described 3rd fluorophor is FAM, and described 3rd fluorescence quencher is BHQ1.
In one embodiment, working standard, positive reference substance and negative controls is also comprised.
In one embodiment, described working standard is nucleic acid concentration is 1 × 10 3~ 1 × 10 6the linear gradient template of copy, described positive reference substance is the RNA extract of EV71 type enterovirus inactivation of viruses strain, and described negative controls is aseptic deionized water.
This enterovirus real time fluorescent quantitative detection kit can be used in the differentiation of EV71 type enterovirus and CA16 type enterovirus, after sample being processed by this enterovirus real time fluorescent quantitative detection kit, if the signal of the first fluorophor can be detected, and there is no the signal of the second fluorophor, then illustrate in sample containing EV71 type enterovirus and not containing CA16 type enterovirus; If the signal of the second fluorophor can be detected, and do not have the signal of the first fluorophor, then illustrate in sample containing CA16 type enterovirus and not containing EV71 type enterovirus; If the signal of the first fluorophor and the second fluorophor can be detected, then illustrate in sample simultaneously containing CA16 type enterovirus and EV71 type enterovirus; If the signal of the first fluorophor and the second fluorophor can not detected simultaneously, then illustrate in sample not containing CA16 type enterovirus and not containing EV71 type enterovirus.
Accompanying drawing explanation
Fig. 1 is that nucleic acid concentration is from 1 × 10 3~ 1 × 10 6the working standard linear gradient amplification curve of copy;
Fig. 2 is the typical curve of Fig. 1.
Embodiment
Mainly in conjunction with the drawings and the specific embodiments explanation is further explained to enterovirus real time fluorescent quantitative detection kit below.
The enterovirus real time fluorescent quantitative detection kit of one embodiment, for distinguishing EV71 type enterovirus and CA16 type enterovirus.
Enterovirus real time fluorescent quantitative detection kit comprises:
For the upstream primer EV71F1 of EV71 type enterovirus of increasing, for the downstream primer EV71R1 of EV71 type enterovirus of increasing, for detecting the probe EV71P1 of EV71 type enterovirus, the two ends of described probe EV71P1 are combined with the first fluorophor and the first fluorescence quencher respectively;
For the upstream primer CA16F2 of CA16 type enterovirus of increasing, for the downstream primer CA16R2 of CA16 type enterovirus of increasing, for detecting the probe CA16P2 of CA16 type enterovirus, the two ends of probe CA16P2 are combined with the second fluorophor and the second fluorescence quencher respectively, and the first fluorophor is different from the second fluorophor; And
Real-time fluorescence quantitative PCR reaction buffer.
In the present embodiment, the sequence of upstream primer EV71F1 is the sequence shown in SEQIDNo.1, and the sequence of downstream primer EV71R1 is the sequence shown in SEQIDNo.2, and the sequence of probe EV71P1 is the sequence shown in SEQIDNo.3.
5 ' the end of probe EV71P1 is combined with the first fluorophor, and the 3 ' end of probe EV71P1 is combined with the first fluorescence quencher, and the first fluorophor is ROX (Carboxy-X-Rhodamine), and the first fluorescence quencher is BHQ (BLACKHOLE ) serial non-fluorescence dyestuff BHQ1.
In other examples, the first fluorophor and the first fluorescence quencher also can select other materials.
In the present embodiment, the sequence of upstream primer CA16F2 is the sequence shown in SEQIDNo.4, and the sequence of downstream primer CA16R2 is the sequence shown in SEQIDNo.5, and the sequence of described probe CA16P2 is the sequence shown in SEQIDNo.6.
5 ' the end of probe CA16P2 is combined with the second fluorophor, 3 ' the end of probe CA16P2 is combined with the second fluorescence quencher, and the second fluorophor is JOE (2,7-dimethyl-4,5-bis-chloro-6-carboxyl fluorescence), the second fluorescence quencher is BHQ (BLACKHOLE ) serial non-fluorescence dyestuff BHQ1.
First fluorescence quencher can be identical with the second fluorescence quencher, also can be different, and in the present embodiment, the two is identical.
In other examples, the second fluorophor and the second fluorescence quencher also can select other materials.
Real-time fluorescence quantitative PCR reaction buffer can be 10mmol/L ~ 65mmol/L for the final concentration of Tris-HCl, the final concentration of ammonium sulfate is 10mmol/L ~ 20mmol/L, the final concentration of Repone K is 40mmol/L ~ 80mmol/L, the final concentration of magnesium sulfate is the volume ratio of 2mmol/L ~ 5mmol/L, Tween20 and reaction buffer is 0.01 ~ 0.05.
This enterovirus real time fluorescent quantitative detection kit can be used in the differentiation of EV71 type enterovirus and CA16 type enterovirus, after sample being processed by this enterovirus real time fluorescent quantitative detection kit, if the signal of the first fluorophor can be detected, and there is no the signal of the second fluorophor, then illustrate in sample containing EV71 type enterovirus and not containing CA16 type enterovirus; If the signal of the second fluorophor can be detected, and do not have the signal of the first fluorophor, then illustrate in sample containing CA16 type enterovirus and not containing EV71 type enterovirus; If the signal of the first fluorophor and the second fluorophor can be detected, then illustrate in sample simultaneously containing CA16 type enterovirus and EV71 type enterovirus; If the signal of the first fluorophor and the second fluorophor can not detected simultaneously, then illustrate in sample not containing CA16 type enterovirus and not containing EV71 type enterovirus.
In the present embodiment, above-mentioned enterovirus real time fluorescent quantitative detection kit also comprises for putting on trip primers F 3 in target in the noncompetitive that increases, for marking downstream primer R3 in target in the noncompetitive that increases and marking probe P3 in target for detecting in noncompetitive.
The two ends of interior mark probe P3 are combined with the 3rd fluorophor and the 3rd fluorescence quencher respectively, and the 3rd fluorophor is different from the first fluorophor and the 3rd fluorophor is different with the second fluorophor.
In the present embodiment, the sequence inside putting on trip primers F 3 is the sequence shown in SEQIDNo.7, and the sequence of interior mark downstream primer R3 is the sequence shown in SEQIDNo.8, and the sequence of interior mark probe P3 is the sequence shown in SEQIDNo.9.
5 ' the end of interior mark probe P3 is combined with the 3rd fluorophor, and the 3 ' end of interior mark probe P3 is combined with the 3rd fluorescence quencher, and the 3rd fluorophor is FAM, and the 3rd fluorescence quencher is BHQ1.
First fluorescence quencher, the second fluorescence quencher can be identical with the 3rd fluorescence quencher, also can be different, and in the present embodiment, three is identical.
In the present embodiment, above-mentioned enterovirus real time fluorescent quantitative detection kit also comprises working standard, positive reference substance and negative controls.
Working standard is nucleic acid concentration is 1 × 10 3~ 1 × 10 6the linear gradient template of copy, positive reference substance is the RNA extract of EV71 type enterovirus inactivation of viruses strain, and negative controls is aseptic deionized water.Working standard and positive reference substance are diluted to 1 × 10 with aseptic deionized water when using 3~ 1 × 10 6copy.
Preferably, above-mentioned enterovirus real time fluorescent quantitative detection kit also comprises reaction enzymes system.Reaction enzymes system comprises Taq DNA polymerase, dNTPs, BSA, reversed transcriptive enzyme and RNasin.
This enterovirus real time fluorescent quantitative detection kit is used for the detection of sample, should be stored in-20 DEG C of environment, and avoid multigelation as far as possible when not using.
Sample is generally the viral RNA extracted from clinical samples or Viral isolation thing.
The RNA extract of sample and the strain of EV71 type enterovirus inactivation of viruses all can adopt commercialization nucleic acid RNA extraction test kit OMEGAE.Z.N.A.TotalRNAKitI (OMEGA, R6834-01) to extract RNA and obtain.
Sample is the viral RNA extracted from clinical samples or Viral isolation thing.
Augmentation detection can carry out fluorescence quantitative PCR detection at ABI7500 instrument (or can comprise other quantitative real time PCR Instruments of above three kinds of fluorescent signals), cumulative volume 50 μ L, wherein 45 μ L real-time fluorescence quantitative PCR reaction buffers, 5 μ L detect samples (sample, make standard substance, positive reference substance and negative controls); Probe in detecting pattern is set to: ReporterDye:ROX, JOE and FAM passage, is respectively used to EV71 type, the detection of CA16 C-type virus C and interior target and detects; Reaction conditions: 50 DEG C, 15min, 1 circulation; 95 DEG C, 10min, 1 circulation; 94 DEG C, 15s → 55 DEG C, 40s, 50 circulations.
After being provided with, preserve file, working procedure.
Fluorescent quantitation result judges:
If detect amplification curve thing increased logarithmic phase or Ct value >=40 of sample, can judgement sample be negative;
If detect sample Ct value≤38, and curve has obvious increased logarithmic phase, can be judged as the positive;
If detect sample 38<Ct value <40, need to re-start nucleic acid extraction and detection to sample, if laboratory test results Ct value≤38 again, and curve has obvious increased logarithmic phase, and judgement sample is sample; If Ct is value >38, can judgement sample be negative.
Specifically belong to EV71 type or virus advocated by CA16 type, please refer to table 1 and determine.
Table 1
Sample RT-PCR detected result to be checked Qualification result
FAM(-),ROX(±),JOE(±) Detection system is abnormal, need again detect
FAM(+),ROX(-),JOE(-) Non-EV71 type or CA16 type
FAM(+),ROX(+),JOE(-) EV71 type
FAM(+),ROX(-),JOE(+) CA16 type
This enterovirus real time fluorescent quantitative detection kit carries out detection and somatotype to enterovirus EV 71 type and CA16 type by single stage method simultaneously, more independent test kit, reduces testing cost on the one hand, improves detection efficiency on the other hand; Also introduce mark in noncompetitive, interior target exists and effectively to detection reagent monitoring performance, can prevent false cloudy appearance, avoids the problem because of operation or reagent itself, occurs undetected situation simultaneously.
Be specific embodiment part below.
In following examples, if no special instructions, the experimental technique of unreceipted actual conditions, usual conveniently condition, for example, see Pehanorm Brooker, the EF not (Jin Dongyan such as Ritchie, T Manny A Disi, Li Mengfeng etc. translate) show the condition described in Molecular Cloning: A Laboratory guide [M] (Beijing: Science Press, 1992) or test kit manufacturer recommend method realize.All operations all adopts this area standard openating procedure, and the reagent adopted or carrier etc. are conventional reagent or conventional carrier.
Embodiment 1
There is provided a kind of enterovirus real time fluorescent quantitative detection kit, for distinguishing EV71 type enterovirus and CA16 type enterovirus.
This enterovirus real time fluorescent quantitative detection kit comprises: for the upstream primer EV71F1 of EV71 type enterovirus of increasing, for the downstream primer EV71R1 of EV71 type enterovirus of increasing, for detecting the probe EV71P1 of EV71 type enterovirus, the two ends of described probe EV71P1 are combined with the first fluorophor and the first fluorescence quencher respectively; For the upstream primer CA16F2 of CA16 type enterovirus of increasing, for the downstream primer CA16R2 of CA16 type enterovirus of increasing, for detecting the probe CA16P2 of CA16 type enterovirus, the two ends of probe CA16P2 are combined with the second fluorophor and the second fluorescence quencher respectively, and the first fluorophor is different from the second fluorophor; For target upstream primer F3 in the noncompetitive that increases, for marking downstream primer R3 in target in the noncompetitive that increases and marking probe P3 in target for detecting in noncompetitive; Working standard, positive reference substance, negative controls, reaction enzymes system and real-time fluorescence quantitative PCR reaction buffer.
Mentioned component above in all according to described in above, be not repeated.
Wherein, positive reference substance adopts commercialization nucleic acid RNA to extract test kit OMEGAE.Z.N.A.TotalRNAKitI (OMEGA, R6834-01) from the strain of EV71 type enterovirus inactivation of viruses, to extract viral RNA obtain.
Embodiment 2 test kit limit of identification
Adopt commercialization nucleic acid RNA to extract test kit OMEGAE.Z.N.A.TotalRNAKitI (OMEGA, R6834-01) and extract viral RNA from Viral isolation thing, obtain sample.
Adopt the nucleic acid concentration in ultraviolet-visible photometric determination sample, with aseptic deionized water, the viral nucleic acid of sample is diluted to 1.0 × 10 downwards successively 6copy, 1.0 × 10 5copy, 1.0 × 10 4copy, 1.0 × 10 3copy, 1.0 × 10 2copy, 10 copies and 1 copy.
Through detecting quantitatively, the detection limit of this enterovirus real time fluorescent quantitative detection kit is 10 copies.
The accuracy of embodiment 3 test kit and specificity
To adopt after the isolated culture mixing of EV71 type enterovirus and CA16 type enterovirus virus as experimental group, choose hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human cytomegalic inclusion disease virus (HCMV), mycoplasma pneumoniae (MP) and treponema pallidum (TP) isolated culture mix after as a control group.
Wherein, it is corresponding pathogenic micro-organism that the hepatitis B virus (HBV) chosen, hepatitis C virus (HCV), Epstein-Barr virus (EBV), human cytomegalic inclusion disease virus (HCMV), mycoplasma pneumoniae (MP) and treponema pallidum (TP) all detect through corresponding test kit the also sequence verification that is all positive.
Above-mentioned enterovirus real time fluorescent quantitative detection kit is adopted to detect experimental group and control group, detected result: the FAM passage of all detection systems is the positive, experimental group detected result is the two channels positive, and EV71 C-type virus C phosphor collection is at ROX passage, CA16 type phosphor collection is at JOE passage, and control group only has single FAM passage positive.
Detected result shows, this enterovirus real time fluorescent quantitative detection kit has the specificity of good accuracy.
Fig. 1 and Fig. 2 is obtained after adopting above-mentioned enterovirus real time fluorescent quantitative detection kit to detect the working standard after gradient dilution.In Fig. 2, coefficient R 2>0.999.
This shows, linear gradient template has higher reliability.
The above embodiment only have expressed one or more embodiments of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims (10)

1. an enterovirus real time fluorescent quantitative detection kit, for distinguishing EV71 type enterovirus and CA16 type enterovirus, is characterized in that, comprise:
For the upstream primer EV71F1 of EV71 type enterovirus of increasing, for the downstream primer EV71R1 of EV71 type enterovirus of increasing, for detecting the probe EV71P1 of EV71 type enterovirus, the two ends of described probe EV71P1 are combined with the first fluorophor and the first fluorescence quencher respectively;
For the upstream primer CA16F2 of CA16 type enterovirus of increasing, for the downstream primer CA16R2 of CA16 type enterovirus of increasing, for detecting the probe CA16P2 of CA16 type enterovirus, the two ends of described probe CA16P2 are combined with the second fluorophor and the second fluorescence quencher respectively, and described first fluorophor is different from described second fluorophor; And
Real-time fluorescence quantitative PCR reaction buffer.
2. enterovirus real time fluorescent quantitative detection kit according to claim 1, it is characterized in that, the sequence of described upstream primer EV71F1 is the sequence shown in SEQIDNo.1, the sequence of described downstream primer EV71R1 is the sequence shown in SEQIDNo.2, and the sequence of described probe EV71P1 is the sequence shown in SEQIDNo.3.
3. enterovirus real time fluorescent quantitative detection kit according to claim 1 and 2, it is characterized in that, 5 ' the end of described probe EV71P1 is combined with the first fluorophor, 3 ' the end of described probe EV71P1 is combined with the first fluorescence quencher, described first fluorophor is ROX, and described first fluorescence quencher is BHQ1.
4. enterovirus real time fluorescent quantitative detection kit according to claim 1, it is characterized in that, the sequence of described upstream primer CA16F2 is the sequence shown in SEQIDNo.4, the sequence of described downstream primer CA16R2 is the sequence shown in SEQIDNo.5, and the sequence of described probe CA16P2 is the sequence shown in SEQIDNo.6.
5. the enterovirus real time fluorescent quantitative detection kit according to claim 1 or 4, it is characterized in that, 5 ' the end of described probe CA16P2 is combined with the second fluorophor, 3 ' the end of described probe CA16P2 is combined with the second fluorescence quencher, described second fluorophor is JOE, and described second fluorescence quencher is BHQ1.
6. enterovirus real time fluorescent quantitative detection kit according to claim 1, it is characterized in that, also comprise for putting on trip primers F 3 in target in the noncompetitive that increases, for marking downstream primer R3 in target in the noncompetitive that increases and marking probe P3 in target for detecting in noncompetitive, the two ends of described interior mark probe P3 are combined with the 3rd fluorophor and the 3rd fluorescence quencher respectively, and described 3rd fluorophor is different from described first fluorophor and described 3rd fluorophor is different with described second fluorophor.
7. enterovirus real time fluorescent quantitative detection kit according to claim 6, it is characterized in that, the sequence putting on trip primers F 3 in described is the sequence shown in SEQIDNo.7, the sequence of described interior mark downstream primer R3 is the sequence shown in SEQIDNo.8, and the sequence of described interior mark probe P3 is the sequence shown in SEQIDNo.9.
8. the enterovirus real time fluorescent quantitative detection kit according to claim 6 or 7, it is characterized in that, 5 ' the end of described interior mark probe P3 is combined with the 3rd fluorophor, 3 ' the end of described interior mark probe P3 is combined with the 3rd fluorescence quencher, described 3rd fluorophor is FAM, and described 3rd fluorescence quencher is BHQ1.
9. enterovirus real time fluorescent quantitative detection kit according to claim 1, is characterized in that, also comprise working standard, positive reference substance and negative controls.
10. enterovirus real time fluorescent quantitative detection kit according to claim 9, is characterized in that, described working standard is nucleic acid concentration is 1 × 10 3~ 1 × 10 6the linear gradient template of copy, described positive reference substance is the RNA extract of EV71 type enterovirus inactivation of viruses strain, and described negative controls is aseptic deionized water.
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CN110607404A (en) * 2019-10-30 2019-12-24 广西大学 Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer for porcine enterovirus G type and kit thereof
CN112111609A (en) * 2020-10-29 2020-12-22 上海伯杰医疗科技有限公司 Universal nucleic acid detection kit for enteroviruses

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