CN110144422A - The quadruple fluorescence quantitative detection kit of four kinds of human corona virus is detected simultaneously - Google Patents
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Abstract
The present invention relates to a kind of quadruple fluorescence quantitative detection kits for detecting four kinds of human corona virus simultaneously, belong to human corona virus's detection technique field.The human corona virus is specially MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI；The detection kit specifically includes: specific primer to and four groups of probe, positive quality control product and negative quality-control product；Every group of specific primer to and probe include a pair of of specific primer to and a probe sequence；The positive quality control product is specially an artificial synthesized target sequence；Negative quality-control product is deionized water or sterile water.Kit of the present invention can single tube be realized while detecting four kinds of human corona virus, and not only detection high specificity, detection sensitivity are high, but also detection time is fast, avoid the disadvantages of time-consuming, testing cost is high, easy cross contamination brought by detection one by one.There are no similar Related product currently on the market to report, kit of the present invention disease detection, infectious disease prevention and control, in terms of there is broad prospect of application.
Description
Technical field
The present invention relates to a kind of quadruple fluorescence quantitative detection kits for detecting four kinds of human corona virus simultaneously, belong to people's hat
Shape technical field of virus detection.
Background technique
Human corona virus (Human Coronaviruses, hCoVs) infection is the important disease for causing human breathing tract disease
One of original, coronavirus recall rate is about 3%-10% in common respiratory pathogen, is to infect infant, old man and be immunized
The adult of hypofunction, to cause the important pathogen of the clinical symptoms such as cough, fever, laryngitis, bronchitis and pneumonia.Mesh
Before, it is known that human corona virus share six kinds, be respectively the sixties in 19th century discovery human corona virus 229E (Human
Coronavirus 229E, HCoV-229E) and HCoV-OC43, the serious acute respiratory occurred in China Guangdong is comprehensive within 2003
(Severe acute respiratory syndrome, SARS) coronavirus SARS-CoV is levied, 2004 in Holland's separation mirror
Fixed novel human corona virus NL63 (HCoV-NL63), the novel human corona virus HKU1 in Hong Kong separation identification in 2005
(HCoV-HKU1) and Middle East respiration syndrome (the Middle East that occurs in Middle East in June, 2012
Respiratory syndrome, MERS) coronavirus MERS-CoV.
Epidemiological study shows that these four people of HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKUI are coronal
Virus has prevalence all over the world, be in global distribution, clinical symptoms be mainly shown as fever, cough, rhinitis, laryngitis and
Bronchitis, pneumonia etc. cause certain burden to human life and health.Both coronavirus of SARS-CoV and MERS-CoV
But acute respiratory symptom can be caused after infection people, serious person causes respiratory system and renal function system failure and causes death.
SARS-CoV caused global about 9000 people infection from 2003 altogether after China Guangdong first appears, and about 800 is dead, and the death rate is close
10%.Have benefited from global effective control and prevention of disease measure, SARS-CoV, which has disappeared, at present has no popular report.MERS-CoV makees
To occur the 6th kind of human corona virus recently, existing 26 countries in the whole world and area report confirmed cases 2428 at present, extremely
839 are died, the death rate is up to 34.6%.MERS-CoV is mainly popular in Middle East Saudi Arabia area at present, and South Korea is 2015
Year occurred once concentrating eruption and prevalence, and China also has an example from South Korea's introduced cases.MERS-CoV is not only domestic disease control
The infectious disease of emphasis monitoring, while being also that current frontier port prevents incoming one of Important Infectious Diseases.
Early stage prevention and control are the premises for preventing infectious disease input from sending out, ensureing public health security, safeguarding human life and health
Condition, and developing quick, sensitive, special molecular detection technology method is the key that early stage prevention and control, while being also guidance clinic,
It reasonably selects antiviral drugs and important evidence is provided.Currently, the detection technique method for human corona virus mainly includes virus
It is separately cultured, serological detection method, regular-PCR method, isothermal duplication method, solubility curve method and fluorescence quantitative RT-RCR method.Disease
Poison is separately cultured, serological detection method and regular-PCR method are the conventional detection methods of comparison, these methods are during the experiment
Face that detection sensitivity is lower, detection time is too long, high, higher to operator's technical requirements etc. all to laboratory hardware requirement
More disadvantages not can guarantee the timeliness of detection and the accuracy of result.Isothermal duplication method and fluorescence quantitative RT-RCR method are current
Most of commonly used molecular detection technology methods in laboratory.There are detection device, detection reagent valuableness, poles for isothermal duplication method
Cross contamination, which easily occurs, leads to the outstanding problems such as testing result uncertainty, and is only able to achieve single pathogen detection, can not
The problems such as realizing single tube Multiple detection.The position that solubility curve method mainly utilizes the difference of amplified production Tm value to pass through melting curve
Difference is distinguished, and this requires design of primers must have Tm to be worth difference, and the difference of single base will change Tm value, cause
The unreliability of testing result.Fluorescence quantitative RT-RCR method equally faces that be only able to achieve single tube substance (at most double or three at present
Again) the problem of pathogen detection, the number of pathogen screening is not only increased, so that the time of testing result greatly prolongs, and
And operating process is increased, testing cost is greatly increased, the quick diagnosis and early stage prevention and control of disease are unfavorable for.HCoVs quickly,
Accurate detection is not only of great significance to the prevalence of monitoring and prevention and control hCoVs, is also clinic early diagnosis, reasonably selects anti-
The reliable laboratory foundation of the offers such as viral drug treatment.
Summary of the invention
Present invention aim to address taken a long time in hCoVs detection process, detection efficiency is lower, testing cost is higher,
The defects of timeliness is not strong is detected, quadruple fluorescence quantitative detection kit that is a kind of while detecting four kinds of human corona virus is provided；
It can single tube detect tetra- kinds of human corona virus of MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-HKUI simultaneously.
Technical solution of the present invention, while the quadruple fluorescence quantitative detection kit of four kinds of human corona virus is detected, it is described
Human corona virus is specially MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI；The detection kit is specifically wrapped
Include: specific primer to and four groups of probe, positive quality control product and negative quality-control product；
Every group of specific primer to and probe include a pair of of specific primer to and a probe sequence；The positive
Quality-control product is specially an artificial synthesized target sequence；Negative quality-control product is deionized water or sterile water.
According to tetra- kinds of human corona virus's genome sequences of MERS-CoV, HCoV-NL63, HCoV-OC43 and HCoV-HKUI,
The conserved sequence of each coronavirus is found respectively, preferably, having separately designed the specific primer group suitable for qRT-PCR
With target probe, primer information is as follows:
For human corona virus MERS-CoV, specific primer is to including primer 1 and primer 2, the sequence of primer 1 such as SEQ
ID No.1, the sequence of primer 2 such as SEQ ID No.2；1 sequence of probe such as SEQ ID No.3；
For human corona virus HCoV-NL63, specific primer is to including primer 3 and primer 4, the sequence of primer 3 such as SEQ
ID No.4, the sequence of primer 4 such as SEQ ID No.5；2 sequence of probe such as SEQ ID No.6；
For human corona virus HCoV-OC43, specific primer is to including primer 5 and primer 6, the sequence of primer 5 such as SEQ
ID No.7, the sequence of primer 6 such as SEQ ID No.8；3 sequence of probe such as SEQ ID No.9；
For human corona virus HCoV-HKUI, specific primer is to including primer 7 and primer 8, the sequence of primer 7 such as SEQ
ID No.10, the sequence of primer 8 such as SEQ ID No.11；4 sequence of probe such as SEQ ID No.12.
Further, fluorescent marker is carried out to four primed probes using different iridescent.
Further, the fluorophor the 5 ' of primed probe ends being marked be specially FAM, VIC, ROX and
Cy5, the quenching group that 3 ' ends are marked is MGB.
Further, the end the 5' flag F AM fluorophor of 1 middle probe 1 is organized, the end 3' marks MGB fluorescent quenching group；Group 2
The end 5' of middle probe 2 marks VIC fluorophor, and the end 3' marks MGB fluorescent quenching group；The end 5' of 3 middle probes 3 of group marks ROX
Fluorophor, the end 3' mark MGB fluorescent quenching group；The end 5' of 4 middle probes 4 of group marks Cy5 fluorophor, and the end 3' marks MGB
Fluorescent quenching group.
Shown in table 1 specific as follows:
Table 1
Further, one section of covering MERS-CoV specificity is contained in the positive quality control product quality-control product sequence to draw
Object, probe 102bp target fragment, covering HCoV-NL63 specific primer, probe 109bp target fragment, covering
HCoV-OC43 specific primer, probe 122bp target fragment, covering HCoV-HKU1 primer, probe 143bp purpose
Segment；The quality-control product sequence by be directly used as after artificial synthesized positive quality control product or it is artificial synthesized after be building up on plasmid vector,
Cloned plasmids are extracted as positive quality control product by shaking bacterium.
Further, the positive quality control product target sequence is specifically such as SEQ ID No.13.
Specifically:
The application of the quadruple fluorescence quantitative detection kit, be applied to and meanwhile to MERS-CoV, HCoV-NL63,
Tetra- kinds of human corona virus of HCoV-OC43, HCoV-HKUI detect.
The method detected using quadruple fluorescence quantitative detection kit, steps are as follows:
(1) extraction of sample nucleic acid: taking sample to be tested, carries out nucleic acid extraction to it, obtains sample nucleic acid；
(2) qRT-PCR is detected: using MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI specific primer and
The sample nucleic acid that probe sequence, 2 Х reaction solutions, archaeal dna polymerase and RT reverse transcriptase and step 1 are extracted configures reaction system；It takes
Positive quality control product and negative quality-control product are respectively configured and the consistent reaction system of sample nucleic acid；Above three reaction system is carried out
QRT-PCR detection；
(3) analyze and determine: the fluorescence signal highest point as obtained by step (2) negative quality-control product adjusts threshold values, and control is positive
Quality-control product has S type amplification curve in four sense channels；Then by amplification curve obtained by sample nucleic acid and positive quality control product into
Row control, interpretation result.
Further, the instrument and equipment that can be applied to includes but is not limited to ABI7000/7300/7500/7500Fast/
7900HT/StepOnedTMEtc., the instrument of Roche company, the instrument of Bio-Rad company, the instrument etc. of QIAGEN company.
Preferably, amplification program is provided that 42 DEG C of reaction 10min using Bio-Rad CFX96 instrument as embodiment；
95 DEG C of reaction 5min；It then carries out circular response 40 times, cyclic program are as follows: 94 DEG C of 10~15s of reaction, 58~60 DEG C of reactions 30~
60s collects fluorescence.
Detection setting: by taking the setting of Bio-Rad CFX96 instrument as an example, " plate " window is clicked, into " create New "
Sample information editing interface selects then FAM, VIC, ROX and Cy5 fluorescence channel in " select Folurophore " respectively, point
The detection of MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-HKUI are not corresponded to.
Step (3) analyzes and determines that process is specific as follows:
A, adjust threshold value: threshold value setting principle is using the fluorescence signal highest point just above negative quality-control product as threshold value
Line, or be adjusted according to instrument noise situation；
B, quality controls: negative quality-control product is without amplification curve, and positive quality control product is examined at FAM, VIC, ROX and Cy5 tetra-
Surveying channel has S type amplification curve, and experiment is set up；Otherwise experimental result is invalid；
C, result judgement:
If c1, test sample have the amplification of S type and value≤38 Ct or Cq, carried out according to fluorescence channel corresponding to detection target
Interpretation；
If c2, having the amplification of S type, and 38 < Ct or value < 40 Cq, it is determined as uncertain sample, is carried out after nucleic acid need to be extracted again
Detection；
If c3, reinspection sample are consistent with the result of c2, it can determine that sample is weakly positive；
If c4, without obvious S type amplification curve, but report has Ct or Cq value, is non-specific amplification, is determined as feminine gender.
Beneficial effects of the present invention: the present invention can quickly screen four kinds of human corona virus MERS-CoV/HCoV- simultaneously
NL63/HCoV-OC43/HCoV-HKUI；Its with detection sensitivity height, high specificity, multiple quick detection, operation can be achieved
It simply, is that the pathogen in the fields such as clinical diagnosis, disease surveillance detection, inspection and quarantine is quick using the prominent advantage such as facilitating
Detection provides practicable technical method.
Detailed description of the invention
Fig. 1 is 1 sample amplification curve schematic diagram of embodiment.
Fig. 2 is 2 sample amplification curve schematic diagram of embodiment.
Fig. 3 is 3 sample amplification curve schematic diagram of embodiment.
Fig. 4 is 4 sample amplification curve schematic diagram of embodiment.
Fig. 5 is kit of the present invention and company A kit to HCoV-HKU1 sample amplification curve schematic diagram.
Specific embodiment
In the embodiment of the present invention, MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-HKUI tetra- is detected while offer
Kind human corona virus qRT-PCR kit contains amplification system, detection method following steps:
(1) sample preparation: sample nucleic acid can be tried from Nasopharyngeal swabs, bronchoalveolar lavage fluid, sputum equal samples with nucleic acid extraction
Agent box directly extracts, or can manual extraction, it is also possible to which Full automatic instrument for extracting nucleic acid extracts, and the nucleic acid of extraction is directly used in next step
Amplified reaction.
(2) qRT-PCR is detected:
Select One-step qRT-PCR Kit (TOYOBO) concrete configuration by taking 25 μ L reaction systems as an example as follows: 2 Х are anti-
Answer 12.5 μ L of liquid；1 μ L of archaeal dna polymerase；1 μ L of RT reverse transcriptase；Four group-specific primers are to totally 8, every 0.5 μ L of primer, eventually
0.5 μM of concentration；Four probe sequences each 0.375 μ L, 0.2 μM of final concentration；The three above-mentioned reaction system of pipe of configuration, is respectively added wherein
Sample rna, positive quality control product and the negative quality-control product that 5 μ L steps (1) are extracted, are then settled to 25 μ L using ultrapure water；
Using Bio-Rad CFX96 instrument as embodiment, amplification program setting is as shown in table 2.
Table 2
Detection setting: by taking the setting of Bio-Rad CFX96 instrument as an example, " plate " window is clicked, into " create New "
Sample information editing interface selects then FAM, VIC, ROX and Cy5 fluorescence channel in " select Folurophore " respectively, point
The detection of MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-HKUI are not corresponded to.
(3) interpretation of result and judgement:
A, adjust threshold value: threshold value setting principle is using the fluorescence signal highest point just above negative quality-control product as threshold value
Line, or be adjusted according to instrument noise situation；
B, quality controls: negative quality-control product is without amplification curve, and positive quality control product is examined at FAM, VIC, ROX and Cy5 tetra-
Surveying channel has S type amplification curve, and experiment is set up；Otherwise experimental result is invalid；
C, result judgement:
If c1, test sample have S type expand and value≤38 Ct or Cq, according to detection target corresponding to fluorescence channel according to
Table 3 carries out interpretation；
If c2, having the amplification of S type, and 38 < Ct or value < 40 Cq, it is determined as uncertain sample, is carried out after nucleic acid need to be extracted again
Detection；
If c3, reinspection sample are consistent with the result of c2, it can determine that sample is weakly positive；
If c4, without obvious S type amplification curve, but report has Ct or Cq value, is non-specific amplification, is determined as feminine gender.
Table 3
| Fluorescence channel | FAM | VIC | ROX | Cy5 |
| MERS-CoV | Positive (+) | Negative (-) | Negative (-) | Negative (-) |
| HCoV-NL63 | Negative (-) | Positive (+) | Negative (-) | Negative (-) |
| HCoV-OC43 | Negative (-) | Negative (-) | Positive (+) | Negative (-) |
| HCoV-HKU1 | Negative (-) | Negative (-) | Negative (-) | Positive (+) |
Embodiment 1
Sample to be tested are as follows: Nasopharyngeal swabs.
Corresponding table 3 carries out result interpretation, and specific amplification curve is as shown in Figure 1.
Such as Fig. 1, negative quality-control product or blank control are without amplification curve, and positive quality control product is in FAM, VIC, ROX and Cy5 tetra-
A sense channel has S type amplification curve, and measuring samples only S type amplification curve, and Ct (or Cq) value occur in FAM fluorescence channel
≤ 38, interpretation sample to be examined MERS-CoV is positive.
Embodiment 2
Sample to be tested are as follows: Nasopharyngeal swabs.
Corresponding table 3 carries out result interpretation, and specific amplification curve is as shown in Figure 2.
Such as Fig. 2, negative quality-control product or blank control are without amplification curve, and positive quality control product is in FAM, VIC, ROX and Cy5 tetra-
A sense channel has S type amplification curve, and measuring samples only S type amplification curve, and Ct (or Cq) value occur in VIC fluorescence channel
≤ 38, interpretation sample to be examined HCoV-NL63 is positive.
Embodiment 3
Sample to be tested are as follows: Nasopharyngeal swabs.
Corresponding table 3 carries out result interpretation, and specific amplification curve is as shown in Figure 3.
Such as Fig. 3, negative quality-control product or blank control are without amplification curve, and positive quality control product is in FAM, VIC, ROX and Cy5 tetra-
A sense channel has S type amplification curve, and measuring samples only S type amplification curve, and Ct (or Cq) value occur in ROX fluorescence channel
≤ 38, interpretation sample to be examined HCoV-OC43 is positive.
Embodiment 4
Sample to be tested are as follows: Nasopharyngeal swabs.
Corresponding table 3 carries out result interpretation, and specific amplification curve is as shown in Figure 4.
Such as Fig. 4, negative quality-control product or blank control are without amplification curve, and positive quality control product is in FAM, VIC, ROX and Cy5 tetra-
A sense channel has S type amplification curve, and measuring samples only S type amplification curve, and Ct (or Cq) value occur in Cy5 fluorescence channel
≤ 38, interpretation sample to be examined HCoV-HKU1 is positive.
Embodiment 5
Selection has homology with human corona virus's nucleic acid sequence, easily causes the same or similar clinical symptoms, sampling unit
The normal parasitic or easily concurrent other pathogens in position, such as influenza A virus, influenza B virus, influenza virus C, rhinopathy
Poison, Respiratory Syncytial Virus(RSV), adenovirus, measles virus, rubella virus, mumps virus are sample to be examined, are sent out with this
Bright kit is detected, and operation is carried out in strict accordance with kit specification, enterprising in CFX96 real-time fluorescence quantitative PCR instrument
Row detection, testing result is feminine gender, shows that kit of the present invention has preferably detection specificity.
Embodiment 6
Kit of the present invention is selected to detect with company A substance detection kit to HCoV-HKU1, as a result as schemed
Shown in 5, using quadruple qRT-PCR kit of the present invention and company A substance detection kit detection HCoV-HKU1 virus, this hair
The bright kit positive quality control product Ct value is far below company A, and can detect the HCoV-HKU1 positive.Company A fails to detect
HCoV-HKU1, detection sensitivity are lower than kit of the present invention.
Embodiment 7
Respectively using the kit of the present invention of 3 batches to positive quality control product, negative quality-control product, in, low three it is dense
The standard items of degree carry out 10 repetitions and detect, the CV range of imprecision Ct value in batch are as follows: and MERS-CoV is 0.68~1.42%,
It is 0.77~1.47%, HCoV-HKU1 is 0.89~1.65% that HCoV-NL63, which is 0.72~1.55%, HCoV-OC43,；Between batch
The CV range of imprecision Ct value are as follows: MERS-CoV is that 1.12~1.19%, HCoV-NL63 is 1.04~1.40%, HCoV-
OC43 is that 1.03~1.52%, HCoV-HKU1 is 0.95~1.40%；It is respectively less than in batch with the CV value of betweenrun precision Ct value
5%, negative control is feminine gender, shows that kit precision of the present invention is good, testing result is as shown in table 4 below.
Table 4
Reagent of the present invention can single tube realize and meanwhile detect MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-HKUI tetra-
It is high not only to detect high specificity, detection sensitivity by kind human corona virus, but also detection time is fast, avoids detection institute's band one by one
Carry out the disadvantages of time-consuming, testing cost is high, easy cross contamination.It there are no similar Related product currently on the market to report, this
Invention kit disease detection, infectious disease prevention and control, in terms of have broad prospect of application.
The advantages of basic principles and main features and invention of invention have been shown and described above.The technical staff of the industry
It should be appreciated that invention is not restricted to the described embodiments, what is described in the above embodiment and the description is only the principle of invention,
Inventing under the premise of not departing from spirit and range will also have various changes and improvements, these changes and improvements both fall within requirement
In the range of the invention of protection.The protection scope that invention requires is defined by appended claims and its equivalent.
Sequence table
<110>People's Republic of China (PRC) Wuxi customs
China Sickness Prevention Control Center Virus Disease Prevention Control Institute
<120>the quadruple fluorescence quantitative detection kit of four kinds of human corona virus is detected simultaneously
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tcttgtccct attggtaagg gtaataaaga tgagcagatt ggttattgga atgttcaaga 180
gcgttggcgt atgcgcaggg ggcaacgtgt tgatttgcct cctaacagga ccgcatgcta 240
aagaccagta cggcaccgat attgacggag tctactgggt cgctagcaac caggctgatg 300
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atgccaatgc atcctatggt gaatccctcg aaggggtctt ctgggttgct aatcaccaag 420
ctgacacttc tactccctcc gatgtttcgt caagggatcc tactactcaa gaagctatcc 480
ctactaggtt tccgcctggt 500
Claims (10)
1. the quadruple fluorescence quantitative detection kit of four kinds of human corona virus is detected simultaneously, it is characterized in that: the human corona virus
Specially MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI；The detection kit specifically includes: specificity is drawn
Object to and four groups of probe, positive quality control product and negative quality-control product；
Every group of specific primer to and probe include a pair of of specific primer to and a probe sequence；The positive quality control
Product are specially an artificial synthesized target sequence；Negative quality-control product is deionized water or sterile water.
2. detect the quadruple fluorescence quantitative detection kit of four kinds of human corona virus simultaneously as described in claim 1, it is characterized in that:
For human corona virus MERS-CoV, specific primer is to including primer 1 and primer 2, the sequence of primer 1 such as SEQ ID
No.1, the sequence of primer 2 such as SEQ ID No.2；1 sequence of probe such as SEQ ID No.3；
For human corona virus HCoV-NL63, specific primer is to including primer 3 and primer 4, the sequence of primer 3 such as SEQ ID
No.4, the sequence of primer 4 such as SEQ ID No.5；2 sequence of probe such as SEQ ID No.6；
For human corona virus HCoV-OC43, specific primer is to including primer 5 and primer 6, the sequence of primer 5 such as SEQ ID
No.7, the sequence of primer 6 such as SEQ ID No.8；3 sequence of probe such as SEQ ID No.9；
For human corona virus HCoV-HKUI, specific primer is to including primer 7 and primer 8, the sequence of primer 7 such as SEQ ID
No.10, the sequence of primer 8 such as SEQ ID No.11；4 sequence of probe such as SEQ ID No.12.
3. detect the quadruple fluorescence quantitative detection kit of four kinds of human corona virus simultaneously as described in claim 1, it is characterized in that:
Contained in the positive quality control product quality-control product sequence one section covering MERS-CoV specific primer, probe 102bp mesh
Segment, covering HCoV-NL63 specific primer, probe 109bp target fragment, covering HCoV-OC43 specific primer,
The target fragment of the 122bp of probe, covering HCoV-HKU1 primer, probe 143bp target fragment；The quality-control product sequence by
Be directly used as after artificial synthesized positive quality control product or it is artificial synthesized after be building up on plasmid vector, by shake bacterium extract clone matter
Grain is used as positive quality control product.
4. detect the quadruple fluorescence quantitative detection kit of four kinds of human corona virus simultaneously as claimed in claim 3, it is characterized in that:
The positive quality control product target sequence is specifically such as SEQ ID No.13.
5. detect the quadruple fluorescence quantitative detection kit of four kinds of human corona virus simultaneously as claimed in claim 2, it is characterized in that:
Fluorescent marker is carried out to four probe sequences using different iridescent.
6. detect the quadruple fluorescence quantitative detection kit of four kinds of human corona virus simultaneously as claimed in claim 5, it is characterized in that:
The fluorophor that 5 ' ends of the probe sequence are marked is specially FAM, VIC, ROX and Cy5, and 3 ' ends are marked
Quenching group is MGB.
7. using the application of quadruple fluorescence quantitative detection kit described in one of claim 1-6, it is characterized in that: being applied to
Tetra- kinds of human corona virus of MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-HKUI are detected simultaneously.
8. the method detected using quadruple fluorescence quantitative detection kit, it is characterized in that steps are as follows:
(1) extraction of sample nucleic acid: taking sample to be tested, carries out nucleic acid extraction to it, obtains sample nucleic acid；
(2) qRT-PCR is detected: using MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI specific primer and probe
The sample nucleic acid that sequence, 2 Х reaction solutions, archaeal dna polymerase and RT reverse transcriptase and step 1 are extracted configures reaction system；Take the positive
Quality-control product and negative quality-control product are respectively configured and the consistent reaction system of sample nucleic acid；QRT- is carried out to above three reaction system
PCR detection；
(3) analyze and determine: the fluorescence signal highest point as obtained by step (2) negative quality-control product adjusts threshold values, controls positive quality control
Product have S type amplification curve in four sense channels；Then amplification curve obtained by sample nucleic acid and positive quality control product are carried out pair
According to interpretation result.
9. the method detected as claimed in claim 8 using quadruple fluorescence quantitative detection kit, it is characterized in that step (2)
The qRT-PCR amplification program is as follows: 42 DEG C of 10 min of reaction；95 DEG C of reaction 5min；It then carries out circular response 40 times, circulation
Program are as follows: 94 DEG C of reactions 10 ~ 30s, 58 ~ 60 DEG C of 30 ~ 60s of reaction collect fluorescence.
10. the method detected as claimed in claim 8 using quadruple fluorescence quantitative detection kit, it is characterized in that step
(3) analyze and determine that process is specific as follows:
A, adjust threshold value: threshold value setting principle be using the fluorescence signal highest point just above negative quality-control product as threshold line, or
It is adjusted according to instrument noise situation；
B, quality controls: negative quality-control product is without amplification curve, and positive quality control product is logical in FAM, VIC, ROX and Cy5 tetra- detections
There is S type amplification curve in road, and experiment is set up；Otherwise experimental result is invalid；
C, result judgement:
If c1, test sample have the amplification of S type and value≤38 Ct or Cq, sentenced according to fluorescence channel corresponding to detection target
It reads；
If c2, having the amplification of S type, and 38 < Ct or value < 40 Cq, it is determined as uncertain sample, is examined after nucleic acid need to being extracted again
It surveys；
If c3, reinspection sample are consistent with the result of c2, it can determine that sample is weakly positive；
If c4, without obvious S type amplification curve, but report has Ct or Cq value, is non-specific amplification, is determined as feminine gender.
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| CN111100954A (en) * | 2020-02-08 | 2020-05-05 | 中华人民共和国无锡海关 | Quadruple fluorescent quantitative detection kit for simultaneously detecting four human coronaviruses including 2019-nCoV |
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