CN108034762A - Multiplex PCR detects six kinds of diarrhea virus primed probe groups - Google Patents
Multiplex PCR detects six kinds of diarrhea virus primed probe groups Download PDFInfo
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Abstract
This disclosure relates to a kind of multiplex PCR detect six kinds of diarrhea viruses with primed probe group and and detection method, six kinds of diarrhea viruses are rotavirus, norovirus, astrovirus, letter such as virus, adenovirus and bocavirus.The disclosure additionally provides the kit that a kind of multiple fluorescence quantitative PCR detects six kinds of diarrhea viruses, and the kit includes the primed probe group described in the disclosure.The Pass through above-mentioned technical proposal disclosure considerably improves sensitiveness, specificity and the simplicity of diarrhea virus detection.
Description
Technical field
The present invention relates to biological technical field, and in particular, to multiplex PCR detects six kinds of diarrhea virus primed probes
Group.
Background technology
Baby diarrhea is global common disease, seriously endangers the health and life security of children.Cause children's abdomen
The common virus rushed down has rotavirus, norovirus, astrovirus, letter such as virus, adenovirus and bocavirus.It is above-mentioned various
Cause the serotype of diarrhea virus numerous, clinically diarrheic syndromes are often caused jointly by one or more kinds of viruses, this is
The universal phenomenon of viral infectious diarrhea.
Real-Time Fluorescent Quantitative PCR Technique has totally-enclosed Single tube amplification, simple and efficient, reproducible, real-time quantitative and dirt
The defects of contaminating the advantage such as few, overcoming conventional clinical diagnosis, has the specificity and sensitiveness of height, is provided for clinical diagnosis
Accurately and reliably foundation, can be as a kind of effective, quick detection means of clinical etiological diagnosis.In the prior art, had more
Kind detects the list of rotavirus, astrovirus, intestinal adenovirus, human bocavirus and human calicivirus using Taqman sonde methods
Heavy, double or triple fluorescent quantitative PCR method.But it is limited because of the viral species that one-time detection can be identified, still compare
Compared with labor intensive, reagent and time, and need to carry out quality control to detecting each time, ensure the homogeneity of testing result.
Six kinds of viral detection methods of the above are not detected at the same time in the prior art.
The content of the invention
A kind of the problem of being detected at the same time with identification present invention aim to address a variety of diarrhea viruses, there is provided six kinds of diarrhoeal diseases
The fluorescence quantification PCR primer probe groups and kit of poison, while six kinds of diarrhea viruses of precise Identification, are established one kind and are determined based on fluorescence
Measure the multi-PCR detection method of PCR platforms.
To achieve the above objectives, the present invention provides a kind of multiplex PCR and detects six kinds of diarrhea virus primed probe groups, including
Component A and component B,
Wherein, the component A includes：
The rotavirus sense primer of nucleotide sequence shown in SEQ ID NO.1,
The rotavirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.2,
Rotavirus probe with nucleotide sequence shown in SEQ ID NO.3,5 ' end marks of the rotavirus probe
Note has FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The norovirus sense primer of nucleotide sequence shown in SEQ ID NO.4,
The norovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.5,
Norovirus probe with nucleotide sequence shown in SEQ ID NO.6,5 ' end marks of the norovirus probe
Note has VIC luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The astrovirus sense primer of nucleotide sequence shown in SEQ ID NO.7,
The astrovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.8,
Astrovirus probe with nucleotide sequence shown in SEQ ID NO.9,5 ' end marks of the astrovirus probe
Note has CY5 luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ3；
Wherein, the component B includes：
Such as viral sense primer of the letter of nucleotide sequence shown in SEQ ID NO.10,
Such as viral anti-sense primer of the letter of nucleotide sequence shown in SEQ ID NO.11,
Letter such as Viral Probe with nucleotide sequence shown in SEQ ID NO.12,5 ' end marks of the letter such as Viral Probe
Note has FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2；
The adenovirus sense primer of nucleotide sequence shown in SEQ ID NO.13,
The adenovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.14,
Adenovirus probe with nucleotide sequence shown in SEQ ID NO.15,5 ' ends of the adenovirus probe are marked with
VIC luminophores, 3 ' ends are marked with fluorescent quenching group BHQ1；
The bocavirus sense primer of nucleotide sequence shown in SEQ ID NO.16,
The bocavirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.17,
Bocavirus probe with nucleotide sequence shown in SEQ ID NO.18,5 ' end marks of the bocavirus probe
Note has CY5 luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2.
Second aspect of the disclosure provides the detection method that a kind of multiple fluorescence quantitative PCR detects six kinds of diarrhea viruses,
Wherein, the detection method includes the following steps：
(1) total nucleic acid of sample to be tested is extracted；
(2) using the total nucleic acid as pcr template, using the primed probe group described in disclosure the first aspect to described
Pcr template carries out multiple fluorescence quantitative PCR amplification；
Wherein, the multiple fluorescence quantitative PCR carries out in two systems at the same time,
In first system, the first amplification is carried out to the pcr template using the component A,
In second system, the second amplification is carried out to the pcr template using the component B；
(3) FAM, VIC and CY5 fluorescence channel signal in two systems are collected respectively.
3rd aspect of the disclosure provides the kit that a kind of multiple fluorescence quantitative PCR detects six kinds of diarrhea viruses, institute
Stating kit includes the primed probe group described in disclosure the first aspect.
Rotavirus that the present invention establishes, norovirus, astrovirus, letter such as virus, adenovirus and bocavirus sixfold
Real-time fluorescence RT-PCR detection technique, can quickly realize the examination and discriminating of important diarrhea virus in clinical sample, avoid blood
The troublesome operation of the methods of clear, cause of disease culture, reaches following detection result：
(1) Multiple detection
The detection method that the present invention is established can examination colyliform be sick at the same time in 2 systems of a RT-PCR reaction
Poison, norovirus, astrovirus, letter such as virus, adenovirus and bocavirus, fast and convenient acquisition are caused a disease relevant cause of disease kind
Class, saves time, manpower and reagent cost.
(2) high sensitivity
The detection method that the present invention is established detects while can realizing 6 genes, each target base in reaction system
The detection sensitivity of cause can reach 102Copy/ml, can reach substance real-time fluorescence RT-PCR detection sensitivity.
(3) specificity is high
The detection method specificity that the present invention is established is mainly reflected in the specificity of a whole set of specific primer probe, institute
There is primer all to compare analysis by BLAST, the conservative and specificity with height, not only detecting target in the present invention can
Mutually distinguish, and can also virus close with other kinds, that living environment is identical differentiate, this knows viral, double angstroms including love
Can virus, enterovirns type 71, Coxsackie virus A type, Coxsackie B virus, poliovirus, hepatitis A virus
Deng, it was demonstrated that detection method has the specificity of height, can accurately distinguish non-detection target.
The present invention is rotavirus in clinical sample, norovirus, astrovirus, letter such as virus, adenovirus and Bo Ka diseases
The rapid screening of poison provides complete solution, can realize normal after the daily monitoring viral to this 6 kinds and epidemic outbreak
See and cause diarrhoeal viral examination, reliable laboratory is provided to disposal rationally appropriate after Prevalence of diseases and Epidemic outbreak of disease
Foundation.
Embodiment
It is that the embodiment of the disclosure is described in detail below.It should be appreciated that tool described herein
Body embodiment is only used for describing and explaining the disclosure, is not limited to the disclosure.
One embodiment of the present invention provides multiplex PCR and detects six kinds of diarrhea virus primed probe groups.
Six kinds of diarrhea viruses are rotavirus, norovirus, astrovirus, letter such as virus, adenovirus and Bo Ka diseases
Poison.
The specific detection gene of primed probe group selection or conserved sequence of the present invention, wherein, detection rotavirus, promise
As virus, astrovirus and letter such as virus primed probe group by detect respectively NSP3 genes, ORF2 genes, ORF1A genes,
6 sets of ORF1 genes, Hexon genes and NS1 genes primed probe compositions.
The primed probe of 6 detection targets is designed for target sequence.Different target gene is taken into full account in design process
Primed probe in a reaction system the problem of coamplification, therefore, what primed probe to be integrated when designing considers Tm values
Uniformity, G/C content homogenization, while situations such as avoid the occurrence of hairpin structure, primer dimer as far as possible, to ensure the later stage not
The probability expanded at the same time with primed probe.Finally obtain a set of special primer probe sequence provided by the invention.The primer
Probe groups include component A and component B,
Wherein, the component A includes：
The rotavirus sense primer of nucleotide sequence shown in SEQ ID NO.1,
The rotavirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.2,
Rotavirus probe with nucleotide sequence shown in SEQ ID NO.3,5 ' end marks of the rotavirus probe
Note has FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The norovirus sense primer of nucleotide sequence shown in SEQ ID NO.4,
The norovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.5,
Norovirus probe with nucleotide sequence shown in SEQ ID NO.6,5 ' end marks of the norovirus probe
Note has VIC luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The astrovirus sense primer of nucleotide sequence shown in SEQ ID NO.7,
The astrovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.8,
Astrovirus probe with nucleotide sequence shown in SEQ ID NO.9,5 ' end marks of the astrovirus probe
Note has CY5 luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ3；
Wherein, the component B includes：
Such as viral sense primer of the letter of nucleotide sequence shown in SEQ ID NO.10,
Such as viral anti-sense primer of the letter of nucleotide sequence shown in SEQ ID NO.11,
Letter such as Viral Probe with nucleotide sequence shown in SEQ ID NO.12,5 ' end marks of the letter such as Viral Probe
Note has FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2；
The adenovirus sense primer of nucleotide sequence shown in SEQ ID NO.13,
The adenovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.14,
Adenovirus probe with nucleotide sequence shown in SEQ ID NO.15,5 ' ends of the adenovirus probe are marked with
VIC luminophores, 3 ' ends are marked with fluorescent quenching group BHQ1；
The bocavirus sense primer of nucleotide sequence shown in SEQ ID NO.16,
The bocavirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.17,
Bocavirus probe with nucleotide sequence shown in SEQ ID NO.18,5 ' end marks of the bocavirus probe
Note has CY5 luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2.
In a kind of preferred embodiment of the disclosure, also respectively containing Quality Control in the positive in the component A and component B
Primed probe,
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.19 in the positive,
The nucleotide sequence of Quality Control anti-sense primer is as shown in SEQ ID NO.20 in the positive,
The nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.21 in the positive, 5 ' ends of Quality Control probe in the positive
ROX luminophores are marked with, 3 ' ends are marked with fluorescent quenching group BHQ2.
Quality Control primed probe is used to expand plasmid template pET28a in the positive.
The detection method of six kinds of diarrhea viruses, the detection method are detected in a kind of multiple fluorescence quantitative PCR of the present invention
Include the following steps：
(1) total nucleic acid of sample to be tested is extracted；
(2) using the total nucleic acid as pcr template, using the primed probe group described in disclosure the first aspect to described
Pcr template carries out multiple fluorescence quantitative PCR amplification；
Wherein, the multiple fluorescence quantitative PCR carries out in two systems at the same time,
In first system, the first amplification is carried out to the pcr template using the component A,
In second system, the second amplification is carried out to the pcr template using the component B；
(3) FAM, VIC and CY5 fluorescence channel signal in two systems are collected respectively.
Optionally, the detection method further includes adds Quality Control pET28a plasmid moulds in the positive in two systems respectively
Plate.
In first system, FAM fluorescence channels are used to detect rotavirus, VIC fluorescence channels for detecting promise such as disease
Poison, CY5 fluorescence channels are used to detect astrovirus, ROX fluorescence channels for detecting Quality Control in the positive.
In second system, FAM fluorescence channels be used for detect letter as virus, VIC fluorescence channels be used for detect adenovirus,
CY5 fluorescence channels are used to detect bocavirus, ROX fluorescence channels for detecting Quality Control in the positive.
In a kind of embodiment of the disclosure, judge by the following method in each fluorescence channel detected material whether be
It is positive：In each fluorescence detection channel：
If a) there is the amplification of S types, and CTValue is less than 35, then is determined as that corresponding detected material is positive；
If b) there is the amplification of S types, and CTValue is then determined as uncertain sample, need to extract again less than 40 and more than or equal to 35
Rechecked after nucleic acid；If reinspection still has the amplification of S types, and CTValue be less than 40, then judge corresponding detected material for the positive, otherwise for
It is negative；
If c) without obvious S types amplification curve, but report has CTValue, then be determined as non-specific amplification.
In a kind of specific embodiment of the disclosure, such as following table can be passed through in the case of Quality Control establishment in the positive
1 method is detected the judgement of result.
Table 1
Optionally, the reaction system of two multiple fluorescence quantitative PCRs of the disclosure includes each component of following content：
Hotstar archaeal dna polymerases containing 1-3U in 25 μ L reaction solutions, the RNase inhibitor of 20-60U, 100-300U
MMLV reverse transcriptases, the Tris-HCl buffer solutions that concentration is 20mM and pH is 8.3, the pET28a of 0.00003-0.0003ng/ μ L,
0.2-0.4mM dNTP, 2-6mM MgCl2。
In a kind of specific embodiment of the disclosure, multiple fluorescence quantitative PCR primed probe concentration and reaction system
Configuration is as follows：
Hotstar archaeal dna polymerases 1-2U, 5 × PCR buffer solution (Tris-HCl 100mM (pH8.3), KCl 100mM,
Tween-20 0.2%, pET28a 0.0003-0.01ng) 5 μ L, 10mM dNTP 0.5-1.25 μ L, 25mM MgCl2 2-4μ
L, 10 × primed probe mixture, 2.5 μ L, 2 μ L of DNA profiling, ultra-pure water are mended to 25 μ L.Wherein, every primer in reaction system
Final concentration is respectively 0.3-0.6 μM, and the final concentration of every probe is respectively 0.1-0.3 μM.
To improve the sensitivity of PCR reactions, present disclose provides specific reaction temperature, this is different from generally acknowledged setting, and
And there is good effect.
The reaction condition of multiple fluorescence quantitative PCR includes the following steps：
a：45℃9-11min；
b：95℃4-6min；
c：95 DEG C of 15-60s,
d：50-60 DEG C of 15-60s, c-d circulate 40 reactions.
The direct purpose of the detection method of the disclosure is not to obtain diagnostic result and health status, and simply to having been detached from
The sample of human body or animal body is detected, and what is obtained is merely possible to the information of intermediate result.
Present invention also offers a kind of kit containing foregoing multiple fluorescence quantitative PCR detection primer probe groups.
Preferably, also containing Quality Control plasmid template pET28a in the positive in the kit.
Preferably, the kit further includes reaction system buffer solution, Hotstar archaeal dna polymerases, primed probe mixing
Thing, positive control and ultra-pure water.
Preferably, the final concentration of every primer is respectively 0.3-0.6 μM in the kit, and every probe is most
Whole concentration is respectively 0.1-0.3 μM.
Hereinafter, the disclosure is further described by embodiment, in following embodiments, rotavirus is purchased from Chinese science
Wuhan institute of viruses of institute, numbering GDV023；Norovirus is purchased from Wuhan Virology Institute,Chinan academy of Sciences, and numbering is
GDV120；Astrovirus is purchased from Wuhan Virology Institute,Chinan academy of Sciences, numbering GDV039；Letter is purchased from Chinese science such as virus
Wuhan institute of viruses of institute, numbering GDV201；Adenovirus is purchased from Wuhan Virology Institute,Chinan academy of Sciences, and numbering is
GDV080；Bocavirus is purchased from Wuhan Virology Institute,Chinan academy of Sciences, numbering GDV174.
Embodiment 1
The establishment of first system：
Primed probe includes：
The rotavirus sense primer of nucleotide sequence shown in SEQ ID NO.1,
The rotavirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.2,
Rotavirus probe with nucleotide sequence shown in SEQ ID NO.3,5 ' end marks of the rotavirus probe
Note has FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The norovirus sense primer of nucleotide sequence shown in SEQ ID NO.4,
The norovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.5,
Norovirus probe with nucleotide sequence shown in SEQ ID NO.6,5 ' end marks of the norovirus probe
Note has VIC luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The astrovirus sense primer of nucleotide sequence shown in SEQ ID NO.7,
The astrovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.8,
Astrovirus probe with nucleotide sequence shown in SEQ ID NO.9,5 ' end marks of the astrovirus probe
Note has CY5 luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ3；
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.19 in the positive,
The nucleotide sequence of Quality Control anti-sense primer is as shown in SEQ ID NO.20 in the positive,
The nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.21 in the positive, 5 ' ends of Quality Control probe in the positive
ROX luminophores are marked with, 3 ' ends are marked with fluorescent quenching group BHQ2.
Enzyme mixation pipe includes Hotstar archaeal dna polymerases, RNase inhibitor, MMLV reverse transcriptases, and 5 × RT-PCR delays
Fliud flushing (Tris-HCl 100Mm (pH 8.3), KCl 100mM, Tween-20 0.2%, pET28a 0.0003ng, 5mM
DNTP, 20mM MgCl2, 10 × primed probe mixture, draws comprising every including Quality Control in the positive (pET28a plasmid templates)
The concentration of thing is 0.3 μM, and the concentration of every probe is 0.2 μM；Positive control (rotavirus, norovirus, astrovirus it is mixed
Shuttering, every kind of is 106Copy/ml).
The reaction system of kit detection is 25 μ L, its configuration is as follows：5 × RT-PCR buffer solutions, 5 μ L；10 × enzymatic mixture
2.5μL；10 × primer mixture, 2.5 μ L；5 μ L of template, 10 μ L of ultra-pure water.
The establishment of second system：
Primed probe includes：
Such as viral sense primer of the letter of nucleotide sequence shown in SEQ ID NO.10,
Such as viral anti-sense primer of the letter of nucleotide sequence shown in SEQ ID NO.11,
Letter such as Viral Probe with nucleotide sequence shown in SEQ ID NO.12,5 ' end marks of the letter such as Viral Probe
Note has FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2；
The adenovirus sense primer of nucleotide sequence shown in SEQ ID NO.13,
The adenovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.14,
Adenovirus probe with nucleotide sequence shown in SEQ ID NO.15,5 ' ends of the adenovirus probe are marked with
VIC luminophores, 3 ' ends are marked with fluorescent quenching group BHQ1；
The bocavirus sense primer of nucleotide sequence shown in SEQ ID NO.16,
The bocavirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.17,
Bocavirus probe with nucleotide sequence shown in SEQ ID NO.18,5 ' end marks of the bocavirus probe
Note has CY5 luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2.
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.19 in the positive,
The nucleotide sequence of Quality Control anti-sense primer is as shown in SEQ ID NO.20 in the positive,
The nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.21 in the positive, 5 ' ends of Quality Control probe in the positive
ROX luminophores are marked with, 3 ' ends are marked with fluorescent quenching group BHQ2.
Enzyme mixation pipe includes Hotstar archaeal dna polymerases, RNase inhibitor, MMLV reverse transcriptases, and 5 × RT-PCR delays
Fliud flushing (Tris-HCl 100mM (PH 8.3), KCl 100mM, Tween-20 0.2%, pET28a 0.0003ng, 5mM
DNTP, 20mM MgCl2, 10 × primed probe mixture, draws comprising every including Quality Control in the positive (pET28a plasmid templates)
The concentration of thing is 3 μM, and the concentration of every probe is 2 μM；Positive control (letter such as the hybrid guided mode of virus, adenovirus and bocavirus
Plate, every kind of is 106Copy/ml).
The reaction system of kit detection is 25 μ L, its configuration is as follows：5 × RT-PCR buffer solutions, 5 μ L；10 × enzymatic mixture
2.5μL；10 × primer mixture, 2.5 μ L；5 μ L of template, 10 μ L of ultra-pure water.
The operation of 2 kit of embodiment and result judge
1st, the extraction of genome
Take 0.2g fecal samples to be added in EP pipes, add 1.5mL physiological saline, shake mixing 3 times, each 10s, then
10min is stored at room temperature, 5min is centrifuged with 8000r/min.Draw 200 μ L of supernatant to be added in EP pipes, using Geneaid companies
The QIAmp DNA Stool Mini Kit of Viral N μ cleic Acid extraction Kit II or QIAGEN companies,
Extraction is carried out according to kit specification, takes the sample to be tested nucleic acid that 5 μ L have been extracted as template.
2nd, the preparation of reaction system
The PCR pipe of 200 μ L is taken to configure the reaction system of 25 μ L, manner of formulation is the same as embodiment 1.
3rd, RT-PCR reacts
The PCR pipe of two systems is put into fluorescence quantitative PCR instrument, RT-PCR reactions are carried out according to following program：45℃
10min；95℃5min；95 DEG C of 15s, 60 DEG C of 40s, circulate 40 reactions.
4th, result judges
1) threshold value is adjusted：Instrument is according to preceding 15 circular response background values, adjust automatically decision threshold.
2) quality control：Quality Control is set up in blank control, positive control and the positive, and it is invalid otherwise to regard experiment.
3) judgement and explanation of each fluorescence detection channel：If a) there is the amplification of S types, and CTValue is less than 35, then is determined as phase
Answer detected material positive；If b) there is the amplification of S types, and CTValue is then determined as uncertain sample, needs weight less than 40 and more than or equal to 35
Rechecked after new extraction nucleic acid；If reinspection still has the amplification of S types, and CTValue is less than 40, then judges corresponding detected material for the positive,
Otherwise it is feminine gender；If c) without obvious S types amplification curve, but report has CTValue, then be determined as non-specific amplification.It may be probe
The discharged non-specific fluorescence signal of degraded.Specific decision procedure is with reference to table 1.
Testing result is as shown in table 2：
Table 2
| Sample number into spectrum | A-FAM | A-VIC | A-CY5 | B-FAM | B-VIC | B-CY5 | Result judgement |
| 1 | + | - | - | - | - | - | Rotavirus |
| 2 | - | + | - | - | - | - | Norovirus |
| 3 | - | - | + | - | - | - | Astrovirus |
| 4 | - | - | - | + | - | - | Letter such as virus |
| 5 | - | - | - | - | + | - | Adenovirus |
| 6 | - | - | - | - | - | + | Bocavirus |
| 7 | - | - | - | - | - | - | It is negative |
| 8 | - | - | - | - | - | - | It is negative |
The storage life experiment of 3 kit of embodiment
With 105Copy/mL rotavirus, norovirus, astrovirus, letter such as virus, adenovirus and bocavirus mix
Shuttering detects sample for assessment, at the 0th day, is distributed into 9 parts and freezes in -70 DEG C of refrigerators.The reagent finished will be set up
Box is positioned over -20 DEG C of preservations, takes the kit of 0,10,15,30,60,90,120,150,180 and 360 day to carry out storage life respectively
Experiment.Storage life testing result is as shown in table 3：
3 storage life result of the test of table
| Storage life | Detect the validity of six kinds of target virals |
| 0th day | 6 effective |
| 10th day | 6 effective |
| 15th day | 6 effective |
| 30th day | 6 effective |
| 60th day | 6 effective |
| 90th day | 6 effective |
| 120th day | 6 effective |
| 150th day | 6 effective |
| 180th day | 6 effective |
| 360th day | 6 effective |
As shown in Table 3, kit is stored in -20 DEG C of refrigerators, effective in different storage lives six target virals of detection, real
Test the result shows that the storage life of the kit is at least 6 months.
The specific test of 4 kit of embodiment
AIDS virus, double echovirus, the Enterovirus 71 preserved using the separation of infectious disease diagnosis and treatment National Key Laboratory
The poison such as type, Coxsackie virus A type, Coxsackie B virus, Respiratory Syncytial Virus(RSV), poliovirus, hepatitis A virus
Strain, is detected after extracting nucleic acid with this kit.Quality Control is to set up in negative control, positive control and the positive, it was demonstrated that detection system
System is set up；Do not occur nonspecific fluorescence signal in virus to be checked above, show that kit of the present invention can be distinguished effectively
Non-targeted virus, has preferable specificity.
The minimum detectability experiment of 5 kit of embodiment
Assessment detection sample：By rotavirus, norovirus, astrovirus, letter such as virus, adenovirus and bocavirus
Viral suspension be separately adjusted to angularly 108Copy/mL, extracts the nucleic acid of six kinds of target virals respectively.It is dilute that 6 templates are distinguished into gradient
It is interpreted into equivalent to 107Copy/mL, 106Copy/mL, 105Copy/mL, 104Copy/mL, 103Copy/mL, 102Copy/mL, 10
The detection sample of copy/mL.
Using kit of the present invention detect respectively rotavirus, norovirus, astrovirus, letter as virus, adenovirus and
The template of bocavirus difference dilution factor.Operation is carried out using the method for embodiment 2 and result judges, kit detects 6 mesh
The minimum detectability result of the test of virus is marked as shown in table 4-5：
4 kit of table detection rotavirus, norovirus and astrovirus minimum detectability result of the test
5 kit of table detection letter such as virus, adenovirus and bocavirus minimum detectability result of the test
Find out from table 4 and table 5, the minimum detectability that kit detects six kinds of target virals reaches 102Copy/mL, examination
The minimum detectability of agent box totality is the target molecule of each reaction detection 1 copy.
Comparative example 1
Contrast agent box is assembled in the same manner as shown in Example 1.Differ only in, adjust the inspection of kit A, B pipe
Target is surveyed, the primed probe shown in SEQ ID NO.1-3 is adjusted to B and is managed, by the primed probe shown in SEQ ID NO.10-12
Adjust to A and manage, obtain contrast agent box 1.
Wherein, the component A includes：
Such as viral sense primer of the letter of nucleotide sequence shown in SEQ ID NO.10,
Such as viral anti-sense primer of the letter of nucleotide sequence shown in SEQ ID NO.11,
Letter such as Viral Probe with nucleotide sequence shown in SEQ ID NO.12,5 ' end marks of the letter such as Viral Probe
Note has FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2；
The norovirus sense primer of nucleotide sequence shown in SEQ ID NO.4,
The norovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.5,
Norovirus probe with nucleotide sequence shown in SEQ ID NO.6,5 ' end marks of the norovirus probe
Note has VIC luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The astrovirus sense primer of nucleotide sequence shown in SEQ ID NO.7,
The astrovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.8,
Astrovirus probe with nucleotide sequence shown in SEQ ID NO.9,5 ' end marks of the astrovirus probe
Note has CY5 luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ3；
Wherein, the component B includes：
The rotavirus sense primer of nucleotide sequence shown in SEQ ID NO.1,
The rotavirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.2,
Rotavirus probe with nucleotide sequence shown in SEQ ID NO.3,5 ' end marks of the rotavirus probe
Note has FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The adenovirus sense primer of nucleotide sequence shown in SEQ ID NO.13,
The adenovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.14,
Adenovirus probe with nucleotide sequence shown in SEQ ID NO.15,5 ' ends of the adenovirus probe are marked with
VIC luminophores, 3 ' ends are marked with fluorescent quenching group BHQ1；
The bocavirus sense primer of nucleotide sequence shown in SEQ ID NO.16,
The bocavirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.17,
Bocavirus probe with nucleotide sequence shown in SEQ ID NO.18,5 ' end marks of the bocavirus probe
Note has CY5 luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2.
Also contain Quality Control primed probe in the positive in the component A and component B respectively,
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.19 in the positive,
The nucleotide sequence of Quality Control anti-sense primer is as shown in SEQ ID NO.20 in the positive,
The nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.21 in the positive, 5 ' ends of Quality Control probe in the positive
ROX luminophores are marked with, 3 ' ends are marked with fluorescent quenching group BHQ2.
Quality Control primed probe is used to expand plasmid template pET28a in the positive.
Specificity and minimum detectability experiment are carried out according to the method identical with embodiment 4 and 5, the results showed that kit 1
Non-targeted virus can be effectively distinguished, there is preferable specificity.
Kit 1 detects rotavirus, norovirus, astrovirus, letter such as virus, adenovirus and bocavirus not respectively
With the template of dilution factor.Operation is carried out using the method for embodiment 2 and result judges, contrast agent box 1 detects 6 target virals
Minimum detectability result of the test as shown in table 6-7：
6 kit of table detection letter such as virus, norovirus and astrovirus minimum detectability result of the test
7 kit of table detection rotavirus, adenovirus and bocavirus minimum detectability result of the test
Find out from table 6 and table 7, contrast agent box 1 detects six kinds of target viral stellate viruses, letters such as the minimum inspection of virus
Rising limit 103Copy/mL, is worse than the kit set up in embodiment 1.
It is further to note that each particular technique feature described in above-mentioned embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should equally be considered as content disclosed in this invention.
The preferred embodiment of the present invention described in detail above, still, during present invention is not limited to the embodiments described above
Detail, in the range of the technology design of the present invention, a variety of simple variants can be carried out to technical scheme, this
A little simple variants belong to protection scope of the present invention.
Sequence table
<110>Beijing Zhuo Cheng Hui Sheng biotech inc
<120>Multiplex PCR detects six kinds of diarrhea virus primed probe groups
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<400> 19
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<210> 20
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Claims (8)
1. multiplex PCR detects six kinds of diarrhea virus primed probe groups, it is characterised in that including component A and component B,
Wherein, the component A includes：
The rotavirus sense primer of nucleotide sequence shown in SEQ ID NO.1,
The rotavirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.2,
Rotavirus probe with nucleotide sequence shown in SEQ ID NO.3,5 ' ends of the rotavirus probe are marked with
FAM luminophores, 3 ' ends are marked with fluorescent quenching group BHQ1；
The norovirus sense primer of nucleotide sequence shown in SEQ ID NO.4,
The norovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.5,
Norovirus probe with nucleotide sequence shown in SEQ ID NO.6,5 ' ends of the norovirus probe are marked with
VIC luminophores, 3 ' ends are marked with fluorescent quenching group BHQ1；
The astrovirus sense primer of nucleotide sequence shown in SEQ ID NO.7,
The astrovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.8,
Astrovirus probe with nucleotide sequence shown in SEQ ID NO.9,5 ' ends of the astrovirus probe are marked with
CY5 luminophores, 3 ' ends are marked with fluorescent quenching group BHQ3；
Wherein, the component B includes：
Such as viral sense primer of the letter of nucleotide sequence shown in SEQ ID NO.10,
Such as viral anti-sense primer of the letter of nucleotide sequence shown in SEQ ID NO.11,
Letter such as Viral Probe with nucleotide sequence shown in SEQ ID NO.12,5 ' ends of the letter such as Viral Probe are marked with
FAM luminophores, 3 ' ends are marked with fluorescent quenching group BHQ2；
The adenovirus sense primer of nucleotide sequence shown in SEQ ID NO.13,
The adenovirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.14,
Adenovirus probe with nucleotide sequence shown in SEQ ID NO.15,5 ' ends of the adenovirus probe are marked with VIC
Luminophore, 3 ' ends are marked with fluorescent quenching group BHQ1；
The bocavirus sense primer of nucleotide sequence shown in SEQ ID NO.16,
The bocavirus anti-sense primer of nucleotide sequence shown in SEQ ID NO.17,
Bocavirus probe with nucleotide sequence shown in SEQ ID NO.18,5 ' ends of the bocavirus probe are marked with
CY5 luminophores, 3 ' ends are marked with fluorescent quenching group BHQ2.
2. primed probe group according to claim 1, wherein, also respectively containing Quality Control in the positive in the component A and component B
Primed probe,
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.19 in the positive,
The nucleotide sequence of Quality Control anti-sense primer is as shown in SEQ ID NO.20 in the positive,
The nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.21 in the positive, 5 ' end marks of Quality Control probe in the positive
There are ROX luminophores, 3 ' ends are marked with fluorescent quenching group BHQ2.
3. a kind of multiple fluorescence quantitative PCR detects the detection method of six kinds of diarrhea viruses, it is characterised in that the detection method bag
Include following steps：
(1) total nucleic acid of sample to be tested is extracted；
(2) using the total nucleic acid as pcr template, using the primed probe group described in claim 1 or 2 to the pcr template into
Row multiple fluorescence quantitative PCR expands；
Wherein, the multiple fluorescence quantitative PCR carries out in two systems at the same time,
In first system, the first amplification is carried out to the pcr template using the component A,
In second system, the second amplification is carried out to the pcr template using the component B；
(3) FAM, VIC and CY5 fluorescence channel signal in two systems are collected respectively.
4. detection method according to claim 3, wherein, the detection method further includes adds in two systems respectively
Quality Control pET28a plasmid templates in the positive.
5. the detection method according to claim 3 or 4, wherein, in the primed probe group, the final use of every primer
Concentration is respectively 0.3-0.6 μM, and the final concentration of every probe is respectively 0.1-0.3 μM.
6. detection method according to claim 5, wherein, the reaction system of two multiple fluorescence quantitative PCRs is including following
The each component of content：
Hotstar archaeal dna polymerases containing 1-3U, the RNase inhibitor of 20-60U, the MMLV of 100-300U in 25 μ L reaction solutions
Reverse transcriptase, the Tris-HCl buffer solutions that concentration is 20mM and pH is 8.3, the pET28a, 0.2- of 0.00003-0.0003ng/ μ L
0.4mM dNTP, 2-6mM MgCl2。
7. detection method according to claim 6, wherein, the reaction condition of multiple fluorescence quantitative PCR includes the following steps：
a：45 DEG C of 9-11min,
b：95 DEG C of 4-6min,
c：95 DEG C of 15-60s,
d：50-60 DEG C of 15-60s, c-d circulate 40 reactions.
8. multiple fluorescence quantitative PCR detects the kit of six kinds of diarrhea viruses, it is characterised in that the kit will including right
Seek the primed probe group described in 1 or 2.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593890A (en) * | 2018-12-29 | 2019-04-09 | 深圳市刚竹医疗科技有限公司 | Detect the nucleic acid compositions of diarrhea virus, the application method of kit and kit |
| CN110055354A (en) * | 2019-04-23 | 2019-07-26 | 深圳市亚辉龙生物科技股份有限公司 | Nucleic acid compositions, detection unit, micro-fluidic chip and detection device |
| CN111518950A (en) * | 2020-04-30 | 2020-08-11 | 中国农业大学 | Complete set of reagent and kit for detecting four viruses causing bovine diarrhea |
| CN111826464A (en) * | 2020-07-16 | 2020-10-27 | 亚能生物技术（深圳）有限公司 | Primer probe for detecting multiple gastrointestinal viruses in one tube, screening method and kit |
| CN112501358A (en) * | 2021-02-04 | 2021-03-16 | 爱科睿特生物医疗科技(南京)有限公司 | Primer probe combination and kit for detecting 9 children digestive tract pathogens |
| CN112526130A (en) * | 2020-12-15 | 2021-03-19 | 武汉大学 | Reagent kit for detecting rotavirus/norovirus and application thereof |
| CN114292956A (en) * | 2021-12-17 | 2022-04-08 | 江苏汇先医药技术有限公司 | Kit, primer probe composition and method for combined detection of multiple enteroviruses |
| CN114836581A (en) * | 2022-06-02 | 2022-08-02 | 昆明理工大学 | Primer combination for detecting pathogens of infectious diseases of digestive tract |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671746A (en) * | 2009-10-21 | 2010-03-17 | 中华人民共和国北京出入境检验检疫局 | Kit and oligonucleotide sequences for detecting rotavirus A |
| CN103074449A (en) * | 2013-01-25 | 2013-05-01 | 海尔施生物医药股份有限公司 | Kit for synchronously detecting thirteen diarrhea viruses and detection method of kit |
| CN103215379A (en) * | 2013-04-10 | 2013-07-24 | 深圳市疾病预防控制中心 | Diarrhea virus detection kit and method |
| WO2016085267A1 (en) * | 2014-11-28 | 2016-06-02 | 가톨릭대학교 산학협력단 | Probe for detecting virus |
| CN106191316A (en) * | 2016-07-27 | 2016-12-07 | 国家食品安全风险评估中心 | Multiplex PCR detects seven kinds of diarrhea virus primer sets and test kit and detection method thereof |
-
2017
- 2017-12-21 CN CN201711395100.9A patent/CN108034762A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671746A (en) * | 2009-10-21 | 2010-03-17 | 中华人民共和国北京出入境检验检疫局 | Kit and oligonucleotide sequences for detecting rotavirus A |
| CN103074449A (en) * | 2013-01-25 | 2013-05-01 | 海尔施生物医药股份有限公司 | Kit for synchronously detecting thirteen diarrhea viruses and detection method of kit |
| CN103215379A (en) * | 2013-04-10 | 2013-07-24 | 深圳市疾病预防控制中心 | Diarrhea virus detection kit and method |
| WO2016085267A1 (en) * | 2014-11-28 | 2016-06-02 | 가톨릭대학교 산학협력단 | Probe for detecting virus |
| CN106191316A (en) * | 2016-07-27 | 2016-12-07 | 国家食品安全风险评估中心 | Multiplex PCR detects seven kinds of diarrhea virus primer sets and test kit and detection method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 李自刚等: "《生物检测技术 第二版》", 31 August 2016, 中国轻工业出版社 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593890A (en) * | 2018-12-29 | 2019-04-09 | 深圳市刚竹医疗科技有限公司 | Detect the nucleic acid compositions of diarrhea virus, the application method of kit and kit |
| CN110055354A (en) * | 2019-04-23 | 2019-07-26 | 深圳市亚辉龙生物科技股份有限公司 | Nucleic acid compositions, detection unit, micro-fluidic chip and detection device |
| CN110055354B (en) * | 2019-04-23 | 2023-11-03 | 深圳市亚辉龙生物科技股份有限公司 | Nucleic acid composition, detection unit, microfluidic chip and detection device |
| CN111518950A (en) * | 2020-04-30 | 2020-08-11 | 中国农业大学 | Complete set of reagent and kit for detecting four viruses causing bovine diarrhea |
| CN111826464A (en) * | 2020-07-16 | 2020-10-27 | 亚能生物技术（深圳）有限公司 | Primer probe for detecting multiple gastrointestinal viruses in one tube, screening method and kit |
| CN111826464B (en) * | 2020-07-16 | 2023-08-08 | 亚能生物技术（深圳）有限公司 | Primer probe for detecting various gastrointestinal viruses in one tube, screening method and kit |
| CN112526130A (en) * | 2020-12-15 | 2021-03-19 | 武汉大学 | Reagent kit for detecting rotavirus/norovirus and application thereof |
| CN112501358A (en) * | 2021-02-04 | 2021-03-16 | 爱科睿特生物医疗科技(南京)有限公司 | Primer probe combination and kit for detecting 9 children digestive tract pathogens |
| CN112501358B (en) * | 2021-02-04 | 2021-11-09 | 爱科睿特生物医疗科技(南京)有限公司 | Primer probe combination and kit for detecting 9 children digestive tract pathogens |
| CN114292956A (en) * | 2021-12-17 | 2022-04-08 | 江苏汇先医药技术有限公司 | Kit, primer probe composition and method for combined detection of multiple enteroviruses |
| CN114836581A (en) * | 2022-06-02 | 2022-08-02 | 昆明理工大学 | Primer combination for detecting pathogens of infectious diseases of digestive tract |
| CN114836581B (en) * | 2022-06-02 | 2024-03-12 | 昆明理工大学 | Primer combination for detecting pathogens of digestive tract infectious diseases |
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