CN109593890A - Detect the nucleic acid compositions of diarrhea virus, the application method of kit and kit - Google Patents
Detect the nucleic acid compositions of diarrhea virus, the application method of kit and kit Download PDFInfo
- Publication number
- CN109593890A CN109593890A CN201811643012.0A CN201811643012A CN109593890A CN 109593890 A CN109593890 A CN 109593890A CN 201811643012 A CN201811643012 A CN 201811643012A CN 109593890 A CN109593890 A CN 109593890A
- Authority
- CN
- China
- Prior art keywords
- amplimer
- seq
- sequence
- group
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the application methods of a kind of nucleic acid compositions for detecting diarrhea virus, kit and kit.The nucleic acid compositions of the detection diarrhea virus include amplimer pair and detection probe corresponding with amplimer.The nucleic acid compositions of above-mentioned detection diarrhea virus can at least detect three kinds in A group rotavirus, B group rotavirus, C group rotavirus, norovirus, norovirus I type, norovirus II type, letter such as virus, astrovirus and intestinal adenovirus simultaneously, and detection sensitivity is high, specificity is good.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of nucleic acid compositions, kit for detecting diarrhea virus
And the application method of kit.
Background technique
Diarrhoeal diseases is global important public hygiene problem, is one of the principal disease for influencing global human health, is sending out
Country and developed country, which have, in exhibition widely distributes prevalence and breaks out.According to statistics, developing country 5 years old or less children year morbidity
About 7.5~1,000,000,000 person-times of number, the died about 5,000,000 due to diarrhea.In a variety of pathogen for causing diarrhea, virus has become
Most important one kind diarrhoeal diseases substance, is the Etiological of acute diarrhea.Causing the common virus of diarrhea has rotavirus
(rotavirus), norovirus (norovirus), letter such as viral (Sapovirus), astrovirus (astrovirus) and intestines
Road adenovirus (adenovirus) etc., wherein rotavirus is again based on A group, B group and C group rotavirus, and norovirus is with I type
Based on II type.The above-mentioned various serotypes for causing diarrhea virus are numerous, and clinically diarrheic syndromes are often by a kind of or more
Kind virus causes jointly.
Traditional diarrhea virus detection method mainly has cell biology method (virus purification), PCR and immunology diagnosis.
But time-consuming for cell culture processes isolated viral, complicated for operation；PCR and immunological method can only detect one or two kinds of cause of diseases
Body cannot fully meet the needs of the diarrhea virus of detection wide variety.In recent years also occur using Taqman sonde method detection wheel
The dual or triple fluorescent quantitative PCR method of shape virus, norovirus, astrovirus, letter such as virus, intestinal adenovirus,
When a variety of primers of diarrhea virus being mixed while being detected in this method, it is easy to produce between a variety of primers of diarrhea virus dry
It disturbs, thus poor specificity, sensitivity is not also high.
Summary of the invention
Based on this, it is necessary to provide that a species specificity is good and the nucleic acid compositions of the detection diarrhea virus of high sensitivity.
In addition, also providing that a species specificity is good and the kit of the detection diarrhea virus of high sensitivity and a kind of simple and direct inspection
Survey the application method of the kit of diarrhea virus.
A kind of nucleic acid compositions detecting diarrhea virus, including amplimer pair and detection corresponding with the amplimer
Probe, the amplimer is at least three kinds including following amplimer centering: sequence such as SEQ ID No.1 and SEQ ID
A group rotavirus amplimer shown in No.2 is to, sequence B group colyliform as shown in SEQ ID No.3 and SEQ ID No.4 disease
Malicious amplimer to, sequence C group rotavirus amplimer as shown in SEQ ID No.5 and SEQ ID No.6 to, sequence such as
Norovirus amplimer shown in SEQ ID No.7 and SEQ ID No.8 is to, sequence such as SEQ ID No.9 and SEQ ID
Norovirus I type amplimer shown in No.10 is to, sequence promise as shown in SEQ ID No.11 and SEQ ID No.12 such as disease
Malicious II type amplimer is to, sequence letter such as virus amplification primer pair, sequence as shown in SEQ ID No.13 and SEQ ID No.14
Arrange the astrovirus amplimer as shown in SEQ ID No.15 and SEQ ID No.16 to and sequence such as SEQ ID No.17 and
Intestinal adenovirus amplimer pair shown in SEQ ID No.18；With the amplimer to the sequence of corresponding detection probe or
Its complementary series as shown in SEQ ID No.19~SEQ ID No.27, is connected with fluorophor respectively in detection probe.
The nucleic acid compositions of above-mentioned detection diarrhea virus by amplimer pair and correspond to the well-designed of detection probe,
A group rotavirus, B group rotavirus, C group rotavirus, norovirus, norovirus I type, promise can be at least detected simultaneously such as
Three kinds of viruses in virus Type II, letter such as virus, astrovirus and intestinal adenovirus.Use the nucleic acid of above-mentioned detection diarrhea virus
The progress real-time fluorescence detection of composition the results show that the specificity of the nucleic acid compositions of above-mentioned detection diarrhea virus is high, respectively
The high sensitivity for influencing each other small, and detecting between a corresponding amplimer of virus and corresponding detection probe, Neng Gouda
To 102copies/mL。
A kind of kit detecting diarrhea virus, the nucleic acid compositions including above-mentioned detection diarrhea virus.
A kind of application method for the kit detecting diarrhea virus, comprising the following steps: the nucleic acid for extracting sample to be tested obtains
To sample of nucleic acid；Using the sample of nucleic acid as template, be added amplimer in the kit of above-mentioned detection diarrhea virus to
Corresponding detection probe carries out real-time fluorescent PCR amplification reaction；And in real-time fluorescent PCR amplification reaction process detection fluorescence letter
Number, obtain testing result.
Detailed description of the invention
The amplification curve diagram of first group of reaction solution of Fig. 1 embodiment 1；Fig. 2 is the amplification of first group of reaction solution of embodiment 1
Curve graph；Fig. 3 is the amplification curve diagram of first group of reaction solution of embodiment 1.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing
Give section Example of the invention.But the invention can be realized in many different forms, however it is not limited to this paper institute
The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the disclosure of invention more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
An embodiment of the present invention provides a kind of nucleic acid compositions of detection diarrhea virus, can at least examine simultaneously
Survey A group rotavirus, B group rotavirus, C group rotavirus, norovirus, norovirus I type, norovirus II type, letter such as
Three kinds in virus, astrovirus and intestinal adenovirus.Carry out using the nucleic acid compositions of above-mentioned detection diarrhea virus is real-time
The results show that the specificity of the nucleic acid compositions of above-mentioned detection diarrhea virus is high, corresponding expand of each virus is drawn for fluorescence detection
The high sensitivity for influencing each other small, and detecting between object and corresponding detection probe, can reach 102copies/mL。
The nucleic acid compositions of the detection diarrhea virus include amplimer pair and detection probe corresponding with amplimer.Expand
Increasing primer pair includes at least three kinds: sequence A as shown in SEQ ID No.1 and SEQ ID No.2 of following amplimer centering
Group rotavirus amplimer is to, sequence B group rotavirus amplimer as shown in SEQ ID No.3 and SEQ ID No.4
To, sequence C group rotavirus amplimer as shown in SEQ ID No.5 and SEQ ID No.6 to, sequence such as SEQ ID
Norovirus amplimer shown in No.7 and SEQ ID No.8 is to, sequence as shown in SEQ ID No.9 and SEQ ID No.10
Norovirus I type amplimer, sequence norovirus II type as shown in SEQ ID No.11 and SEQ ID No.12 is expanded
Increase primer pair, sequence letter such as virus amplification primer pair, sequence such as SEQ as shown in SEQ ID No.13 and SEQ ID No.14
Astrovirus amplimer shown in ID No.15 and SEQ ID No.16 to and sequence such as SEQ ID No.17 and SEQ ID
Intestinal adenovirus amplimer pair shown in No.18.Wherein, the sequence of SEQ ID No.1 are as follows: 5 '-
CTGTTGAAAGAAAATTGGTGAAGT-3'；The sequence of SEQ ID No.2 are as follows: 5 '-
TGTTCTCATAATCCAATTCATTCACT-3'；The sequence of SEQ ID No.3 are as follows: 5 '-TTGAAAGGATGGCAAGAGTCT-
3'；The sequence of SEQ ID No.4 are as follows: 5 '-CGGAAGTGCGGTATACGATT-3 '；The sequence of SEQ ID No.5 are as follows: 5 '-
AAGAGAATAACACAATGCCACTATTAC-3'；The sequence of SEQ ID No.6 are as follows: 5 '-CACAGTCGATGAGGATCCTTT-
3'；The sequence of SEQ ID No.7 are as follows: 5 '-ATGTTCAGGTGGATGAGATTC-3 '；The sequence of SEQ ID No.8 are as follows: 5 '-
TCGACGCCATCTTCATTCAC-3'；The sequence of SEQ ID No.9 are as follows: 5 '-GGGCTCACGACGGTGTCT-3 '；SEQ ID
The sequence of No.10 are as follows: 5 '-TGTCGTGCTCCTTTGGTCAA-3 '；The sequence of SEQ ID No.11 are as follows: 5 '-
GGACCTCTCGTTATTTCTGAAGA-3'；The sequence of SEQ ID No.12 are as follows: 5 '-GGGTCGCGGGTCACA-3 '；SEQ ID
The sequence of No.13 are as follows: 5 '-TTCTGGCCCCATGAATTATT-3 '；The sequence of SEQ ID No.14 are as follows: 5 '-
ATGGGCACCATCACACTCAA-3'；The sequence of SEQ ID No.15 are as follows: 5 '-CAAGGCGCGCATTCC-3 '；SEQ ID
The sequence of No.16 are as follows: 5 '-CGAGACAAGCAGGTCCATGT-3 '；The sequence of SEQ ID No.17 are as follows: 5 '-
CGAGGACAGGGCCTCTCA-3'；The sequence of SEQ ID No.18 are as follows: 5 '-TGGCTACGTGAATGGTCTTCAG-3 '.
A group rotavirus amplimer is to, B group rotavirus amplimer to, C group rotavirus amplimer to, promise
Amplification such as virus amplification primer pair, norovirus I type amplimer to, norovirus II type amplimer to, letter such as virus
Primer pair, astrovirus amplimer to and intestinal adenovirus amplimer to be respectively used to detection A group rotavirus, B group wheel
Shape virus, C group rotavirus, norovirus, norovirus I type, norovirus II type, letter such as virus, astrovirus and enteron aisle
Adenovirus.
Specifically, A group rotavirus amplimer to for sequence upstream primer as shown in SEQ ID No.1 and sequence such as
Downstream primer shown in SEQ ID No.2.B group rotavirus amplimer is to for sequence upstream as shown in SEQ ID No.3
Primer and the sequence downstream primer as shown in SEQ ID No.4.C group rotavirus amplimer is to for sequence such as SEQ ID
The downstream primer as shown in SEQ ID No.6 of upstream primer and sequence shown in No.5.Norovirus amplimer is to for sequence
The upstream primer as shown in SEQ ID No.7 and the sequence downstream primer as shown in SEQ ID No.8.The amplification of norovirus I type
Primer pair is sequence upstream primer as shown in SEQ ID No.9 and the sequence downstream primer as shown in SEQ ID No.10.Promise
If virus Type II amplimer is to for sequence upstream primer as shown in SEQ ID No.11 and sequence such as SEQ ID No.12 institute
The downstream primer shown.The amplimer of letter such as virus is to for upstream primer shown in SEQ ID No.13 and sequence such as SEQ ID
Downstream primer shown in No.14.Astrovirus amplimer is to for sequence upstream primer and sequence as shown in SEQ ID No.15
Arrange the downstream primer as shown in SEQ ID No.16.Intestinal adenovirus amplimer draws to for upstream shown in SEQ ID No.17
Object and the sequence downstream primer as shown in SEQ ID No.18.
And complementation of the amplimer to the sequence of corresponding detection probe or with amplimer to corresponding detection probe
Sequence as shown in SEQ ID No.19~SEQ ID No.27, is connected with fluorophor respectively in detection probe.Specifically, with A
Group rotavirus amplimer is to the sequence of corresponding probe or its complementary series as shown in SEQ ID No.19.With B group colyliform
The sequence of the corresponding probe of virus amplification primer pair or its complementary series are as shown in SEQ ID No.20.Expand with C group rotavirus
The sequence for increasing the corresponding probe of primer pair or its complementary series are as shown in SEQ ID No.21.With norovirus amplimer to right
The sequence for the probe answered or its complementary series are as shown in SEQ ID No.22.With norovirus I type amplimer to corresponding spy
The sequence of needle or its complementary series are as shown in SEQ ID No.23.With norovirus II type amplimer to the sequence of corresponding probe
Column or its complementary series are as shown in SEQ ID No.24.The sequence of probe corresponding with letter such as virus amplification primer pair or its complementation
Sequence is as shown in SEQ ID No.25.With astrovirus amplimer to the sequence of corresponding probe or its complementary series such as SEQ
Shown in ID No.26.With intestinal adenovirus amplimer to the sequence of corresponding probe or its complementary series such as SEQ ID No.27
It is shown.Wherein, the sequence of SEQ ID No.19 are as follows: 5 '-CGAAAGGCAAACCATTGGAGGCAGAC-3 '；SEQ ID No.20
Sequence are as follows: 5 '-TGGTTTCCATATTCAGCCGACTTCGC-3 '；The sequence of SEQ ID No.21 are as follows: 5 '-
AGCTATTTCGGTCTGAGAACACACGACTATG-3'；The sequence of SEQ ID No.22 are as follows: 5 '-
AGCACGTGGGAGGGCGATCG-3'；The sequence of SEQ ID No.23 are as follows: 5 '-CCTGAGTGGAATATGTGCCCGCTACC-
3'；The sequence of SEQ ID No.24 are as follows: 5 '-TTAGATGGTTTGACTTTCCTGCGGAGA-3 '；The sequence of SEQ ID No.25
Are as follows: 5 '-ATGAGGCACCGACCCCACTAGTGG-3 '；The sequence of SEQ ID No.26 are as follows: 5 '-
CGCTTTCCGATAGGATCAAGCGTG-3'；The sequence of SEQ ID No.26 are as follows: 5 '-ACCTGCTCGGACGGCAACTGTCA-
3’。
The both ends of detection probe are connected separately with fluorophor and quenching group.Preferably, fluorophor is located at detection and visits
5 ' ends of needle, quenching group are located at 3 ' ends of detection probe.
In one of the embodiments, the fluorophor in detection probe in FAM, HEX, VIC, CY5 and ROX one
Kind.
In one of the embodiments, amplimer to include sequence as shown in SEQ ID No.1 and SEQ ID No.2
A group rotavirus amplimer is to, sequence B group rotavirus amplimer as shown in SEQ ID No.3 and SEQ ID No.4
To, sequence C group rotavirus amplimer as shown in SEQ ID No.5 and SEQ ID No.6 to, sequence such as SEQ ID
Norovirus amplimer shown in No.7 and SEQ ID No.8 is to, sequence as shown in SEQ ID No.9 and SEQ ID No.10
Norovirus I type amplimer, sequence norovirus II type as shown in SEQ ID No.11 and SEQ ID No.12 is expanded
Increase primer pair, sequence letter such as virus amplification primer pair, sequence such as SEQ as shown in SEQ ID No.13 and SEQ ID No.14
Astrovirus amplimer shown in ID No.15 and SEQ ID No.16 to and sequence such as SEQ ID No.17 and SEQ ID
Intestinal adenovirus amplimer pair shown in No.18；Amplimer to being divided into multiple groups, the different amplimers pair in same group
The fluorophor marked in detection probe is different.
Further, amplimer is to being three groups；Wherein, first group to include A group rotavirus amplimer take turns, B group
Shape virus amplification primer pair and C group rotavirus amplimer pair；Second group includes norovirus amplimer to, norovirus
I type amplimer to and norovirus II type amplimer pair；Third group includes that letter such as virus amplification primer pair, astrovirus expand
Increase primer pair and intestinal adenovirus amplimer pair.
The fluorophor in each detection probe in each group is selected from FAM, HEX, VIC, CY5 in one of the embodiments,
And one of ROX.
Specifically, the fluorophor in the corresponding detection probe in described first group is respectively FAM, HEX and ROX；Institute
The fluorophor stated in the corresponding detection probe in second group is respectively FAM, HEX and ROX；It is corresponding in the third group
Fluorophor in detection probe is respectively FAM, HEX and ROX.
Nucleic acid compositions further include sequence such as SEQ ID No.28 and SEQ ID No.29 institute in one of the embodiments,
Show interior label primer to and interior label primer to corresponding detection probe, with interior label primer to the sequence of corresponding detection probe or with it is interior
The complementary series of the corresponding detection probe of primer pair is marked as shown in SEQ ID No.30, with interior label primer to corresponding detection probe
On be connected with the fluorophor different to corresponding detection probe from amplimer.Wherein, the sequence of SEQ ID No.28 are as follows:
5'-TGGTTGCAGTGAGCCAAGATC-3'；The sequence of SEQ ID No.29 are as follows: 5 '-
TTTATTTTGAGATGGAGTCTCTCTCTGT-3'；The sequence of SEQ ID No.30 are as follows: 5 '-
CACCACTGGCCTGTAGCCTGGGC-3’。
It in one of the embodiments, include the interior index as shown in SEQ ID No.28 and SEQ ID No.29 in each group
Object to as shown in SEQ ID No.30 with interior label primer to corresponding detection probe.Specifically, corresponding in first group
Fluorophor in detection probe is respectively FAM, HEX and ROX；The fluorophor in corresponding detection probe point in second group
It Wei not FAM, HEX and ROX；The fluorophor in corresponding detection probe in third group is respectively FAM, HEX and ROX；Each group
Interior interior label primer is CY5 to the fluorogene in corresponding detection probe.
The nucleic acid compositions of above-mentioned detection diarrhea virus are avoided by amplimer pair and the ingehious design of detection probe
Interfering with each other between multiple amplimers and corresponding detection probe can once detect nine kinds of diarrhea virus simultaneously.And
And it is verified, using above-mentioned detection diarrhea virus Nucleic acid combinations analyte detection when high sensitivity, specificity it is good.
An embodiment of the present invention provides a kind of kit for detecting diarrhea virus, the kit of the detection diarrhea virus
Nucleic acid compositions including above-mentioned detection diarrhea virus.
The kit of above-mentioned detection diarrhea virus further includes that PCR reaction buffer, RNA are mentioned in one of the embodiments,
Take at least one of reagent, PCR reinforcing agent and PCR stabilizer.Specifically, PCR reaction buffer includes 10 × HS
Buffer, thermal starting HS Taq enzyme, MMLV reverse transcriptase and dNTPs.
The kit of above-mentioned detection diarrhea virus further includes positive control and negative control in one of the embodiments,.
Further, positive control is the Plasmid DNA of primer corresponding sequence, and negative control is the H of free nucleic acid2O。
An embodiment of the present invention provides a kind of application method of kit for detecting diarrhea virus, including following step
It is rapid:
S110, the nucleic acid for extracting sample to be tested, obtain sample of nucleic acid.
Specifically, sample of nucleic acid is the RNA extracted from clinical sample.Clinical sample be nose swab, throat swab or
The types such as secretion.
S130, using sample of nucleic acid as template, the amplimer pair in the nucleic acid compositions of above-mentioned detection diarrhea virus is added
With corresponding detection probe, real-time fluorescent PCR amplification reaction is carried out.
Specifically, the expansion in the nucleic acid compositions of sample of nucleic acid and PCR reaction buffer and above-mentioned detection diarrhea virus
Increase primer pair and the mixing of corresponding detection probe, obtain reaction solution, reaction solution is then subjected to real-time fluorescent PCR amplification reaction.
Further, by the amplimer in the nucleic acid compositions of PCR reaction buffer and above-mentioned detection diarrhea virus to and it is corresponding
Detection probe mixing, obtain pre-reaction liquid.Then after pre-reaction liquid being mixed with sample of nucleic acid, reaction solution is obtained, then
Reaction solution is subjected to real-time fluorescent PCR amplification reaction.Wherein, the concentration of the RNA of reaction solution center acid sample be 10ng~
100ng；In reaction solution the upstream primer of each amplimer pair and the final concentration of downstream primer be respectively 0.1 μM~0.5 μM and
0.1 μM~0.5 μM；Final concentration of 0.1 μM~0.5 μM to corresponding detection probe of each amplimer in reaction solution.Reaction
Final concentration of 0.1U~5U of thermal starting HS Taq enzyme in liquid；Final concentration of 0.4U~5U of MMLV reverse transcriptase in reaction solution；
Final concentration of 0.1 μM~0.5 μM of dNTPs in reaction solution.
Sample of nucleic acid is divided into multiple groups in one of the embodiments, by the Nucleic acid combinations of above-mentioned detection diarrhea virus
Amplimer in object is mixed with the sample of nucleic acid of each group respectively to corresponding detection probe and PCR reaction buffer, is obtained
Multiple groups reaction solution.Wherein, the concentration of the RNA of every kind of viral sample of nucleic acid is 10ng~100ng in every group of reaction solution；Every group anti-
Answer in liquid the upstream primer of each amplimer pair and the final concentration of downstream primer be respectively 0.1 μM~0.5 μM and 0.1 μM~
0.5μM；Final concentration of 0.1 μM~0.5 μM to corresponding detection probe of each amplimer in every group of reaction solution.In reaction solution
Final concentration of 1U~5U of thermal starting HS Taq enzyme；The final concentration of 1U-5U of MMLV reverse transcriptase in reaction solution；In reaction solution
Final concentration of 0.1 μM~0.5 μM of dNTPs.
Specifically, the amplification condition of real-time fluorescent PCR amplification reaction are as follows: 90 DEG C~98 DEG C, 1min~10min；And 90 DEG C
~98 DEG C, 10s~30s, 55 DEG C~65 DEG C, 10s~30s, 30~40 circulations.
The amplification condition of real-time fluorescent PCR amplification reaction in one of the embodiments, are as follows: 50 DEG C, 30min；95℃,
5min；95 DEG C, 15s, 60 DEG C, 40s recycle 40.
S130, fluorescence signal is detected in real-time fluorescent PCR amplification reaction process, obtains testing result.
The application method of the kit of above-mentioned detection diarrhea virus is easy, quickly.
Specific embodiment
It is described in detail below in conjunction with specific embodiment.In embodiment if not otherwise indicated using drug and instrument,
For this field conventional selection.Test method without specific conditions in embodiment, according to normal conditions, such as document, books
In condition or manufacturer recommend method realize.
Embodiment 1
(1) RNA extracts kit (the bacteria RNA extracts kit of Tiangeng) is used, contains different virus to 9 respectively
Positive sample carries out RNA extraction, obtains 9 positive samples to be tested for containing only a kind of RNA of virus.9 positive sample difference
Corresponding to containing only A group rotavirus, B group rotavirus, C group rotavirus, norovirus, norovirus I type, norovirus
The positive sample of II type, letter such as virus, astrovirus or intestinal adenovirus.Every kind of the specific of positive sample to be tested is walked
Suddenly it is carried out according to product description.
(2) the positive sample to be tested mixing of 9 kinds for taking step (1) to obtain, obtains sample to be examined.Three groups of sample to be examined are taken respectively
It is mixed with nucleic acid compositions and PCR reaction buffer, obtains three groups of reaction solutions.Wherein, PCR reaction buffer is 10 × HS
Buffer, thermal starting HS Taq enzyme, MMLV reverse transcriptase and dNTPs mixture.Nucleic acid compositions include amplimer pair and
Detection probe corresponding with amplimer.Amplimer is to being three groups, and every group of amplimer is to by three amplimers pair and right
The detection probe answered and an interior label primer and corresponding detection probe composition.The particular sequence of each group reaction solution is as shown in table 1,
Wherein the fluorophor at the 5 ' ends of SEQ ID No.19, SEQ ID No.22 and SEQ ID No.25 is FAM, the quenching base at 3 ' ends
Group is BHQ1；The fluorophor at the 5 ' ends of SEQ ID No.20, SEQ ID No.23 and SEQ ID No.26 is HEX, 3 ' ends
Quencher is BHQ1；SEQ ID No.21, SEQ ID No.24 and SEQID No.27 5 ' end fluorophors be ROX, 3 '
The quencher at end is BHQ2；The fluorophor at the 5 ' ends of SEQ ID No.30 is CY5, and the quencher at 3 ' ends is BHQ3.Often
The final concentration of 10ng/ μ L of the RNA of every kind of positive sample in group reaction solution.
Table 1
Specifically, the concrete composition of every group of reaction solution is as shown in table 2.Synchronization process positive control and negative control, it is positive
Control is 9 groups, and 9 groups of positive controls respectively correspond the RNA for containing only the virus of one of 9 kinds of virus, and the concentration of RNA is
10ng/μL.Negative control is free nucleic acid H2O。
Table 2
(3) the resulting each group reaction solution of step (2), positive control and negative control are subjected to real-time fluorescence quantitative PCR.
RT-PCR condition are as follows: 50 DEG C of 30min；95℃5min；95 DEG C of 15s, 60 DEG C of 40s recycle 40 reactions.Expand in real-time fluorescence PCR
Increase reaction process and detect fluorescence signal, show that testing result is as shown in Figures 1 to 3.Abscissa is recurring number in Fig. 1~3
(Cycle), ordinate is fluorescent value (△ Rn).
(4) result judgement: 1) adjust threshold value: instrument is according to preceding 15 circular response background values, adjust automatically decision threshold.
2) quality controls: negative control: without amplification curve, Ct value is shown as Undet or No for the channel FAM, the channel HEX and the channel ROX
Ct；Positive control: there is amplification curve in the channel FAM, the channel HEX and the channel ROX, and Ct value is ≤32；Internal standard: the channel CY5 will have
Amplification curve；Three above requires to meet simultaneously in same primary experiment, and otherwise, this experiment is invalid, needs to re-start
Experiment.3) judgement and explanation of each fluorescence detection channel:, can when and CT value≤35 when there is an amplification curve in sample detection channel
Interpretation is that corresponding detection project is positive；When sample detection channel is without amplification curve, and when Ct value shows no numerical value or Ct value=40,
This time result judges corresponding detection project for feminine gender；As 35 < sample Ct value < 40, it is considered as gray area, then repeats primary
Experiment is considered as the positive when rechecking result < 37, is otherwise considered as feminine gender.
9 kinds of viral positive samples in sample to be examined it can be seen from Fig. 1~3 are detected, and specificity is good, and 9
It is noiseless between kind virus.Detection sensitivity can achieve 102copies/mL。
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Shenzhen Gang Zhu medical science and technology Co., Ltd
<120>nucleic acid compositions of diarrhea virus, the application method of kit and kit are detected
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ctgttgaaag aaaattggtg aagt 24
<210> 2
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tgttctcata atccaattca ttcact 26
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ttgaaaggat ggcaagagtc t 21
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cggaagtgcg gtatacgatt 20
<210> 5
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
aagagaataa cacaatgcca ctattac 27
<210> 6
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cacagtcgat gaggatcctt t 21
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
atgttcaggt ggatgagatt c 21
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tcgacgccat cttcattcac 20
<210> 9
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gggctcacga cggtgtct 18
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
tgtcgtgctc ctttggtcaa 20
<210> 11
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ggacctctcg ttatttctga aga 23
<210> 12
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gggtcgcggg tcaca 15
<210> 13
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
ttctggcccc atgaattatt 20
<210> 14
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
atgggcacca tcacactcaa 20
<210> 15
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
caaggcgcgc attcc 15
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
cgagacaagc aggtccatgt 20
<210> 17
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
cgaggacagg gcctctca 18
<210> 18
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
tggctacgtg aatggtcttc ag 22
<210> 19
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
cgaaaggcaa accattggag gcagac 26
<210> 20
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
tggtttccat attcagccga cttcgc 26
<210> 21
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
agctatttcg gtctgagaac acacgactat g 31
<210> 22
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
agcacgtggg agggcgatcg 20
<210> 23
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
cctgagtgga atatgtgccc gctacc 26
<210> 24
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
ttagatggtt tgactttcct gcggaga 27
<210> 25
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
atgaggcacc gaccccacta gtgg 24
<210> 26
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
cgctttccga taggatcaag cgtg 24
<210> 27
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
acctgctcgg acggcaactg tca 23
<210> 28
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
tggttgcagt gagccaagat c 21
<210> 29
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
tttattttga gatggagtct ctctctgt 28
<210> 30
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
caccactggc ctgtagcctg ggc 23
Claims (10)
1. it is a kind of detect diarrhea virus nucleic acid compositions, which is characterized in that including amplimer pair and with the amplimer
Corresponding detection probe；
The amplimer is at least three kinds including following amplimer centering: sequence such as SEQ ID No.1 and SEQ ID
A group rotavirus amplimer shown in No.2 is to, sequence B group colyliform as shown in SEQ ID No.3 and SEQ ID No.4 disease
Malicious amplimer to, sequence C group rotavirus amplimer as shown in SEQ ID No.5 and SEQ ID No.6 to, sequence such as
Norovirus amplimer shown in SEQ ID No.7 and SEQ ID No.8 is to, sequence such as SEQ ID No.9 and SEQ ID
Norovirus I type amplimer shown in No.10 is to, sequence promise as shown in SEQ ID No.11 and SEQ ID No.12 such as disease
Malicious II type amplimer is to, sequence letter such as virus amplification primer pair, sequence as shown in SEQ ID No.13 and SEQ ID No.14
Arrange the astrovirus amplimer as shown in SEQ ID No.15 and SEQ ID No.16 to and sequence such as SEQ ID No.17 and
Intestinal adenovirus amplimer pair shown in SEQ ID No.18；
With the amplimer to the sequence of corresponding detection probe or its complementary series respectively such as SEQ ID No.19~SEQ
Shown in ID No.27, fluorophor is connected in the detection probe.
2. the nucleic acid compositions of detection diarrhea virus according to claim 1, which is characterized in that the amplimer is to packet
The A group rotavirus amplimer is included to, the B group rotavirus amplimer to, the C group rotavirus amplimer
To, the norovirus amplimer to, the norovirus I type amplimer to, the norovirus II type amplimer
To, the letter such as virus amplification primer pair, the astrovirus amplimer to and the intestinal adenovirus amplimer pair；
For the amplimer to for multiple groups, the fluorophor in the detection probe of the different amplimers pair in same group is different.
3. it is according to claim 2 detection diarrhea virus nucleic acid compositions, which is characterized in that the amplimer to for
Three groups；Wherein, first group include the A group rotavirus amplimer to, the B group rotavirus amplimer to it is described
C group rotavirus amplimer pair；Second group to include the norovirus amplimer draw, norovirus I type amplification
Object to the norovirus II type amplimer pair；Third group includes the letter such as virus amplification primer pair, the starlike disease
Malicious amplimer to the intestinal adenovirus amplimer pair.
4. the nucleic acid compositions of detection diarrhea virus according to claim 3, which is characterized in that each inspection in each group
Fluorophor on probing needle is selected from one of FAM, HEX, VIC, CY5 and ROX.
5. the nucleic acid compositions of detection diarrhea virus according to claim 4, which is characterized in that pair in described first group
The fluorophor in detection probe answered is respectively FAM, HEX and ROX；It is glimmering in corresponding detection probe in described second group
Light group is respectively FAM, HEX and ROX；The fluorophor in corresponding detection probe in the third group be respectively FAM,
HEX and ROX.
6. the nucleic acid compositions of any detection diarrhea virus according to claim 1~5, which is characterized in that the nucleic acid
Composition further include sequence interior label primer as shown in SEQ ID No.28 and SEQ ID No.29 to with the interior label primer
To corresponding detection probe, it is described with the interior label primer to the sequence of corresponding detection probe or its complementary series such as SEQ ID
Shown in No.30, described and interior label primer is visited corresponding detection with the amplimer to being connected in corresponding detection probe
The different fluorophor of needle.
7. a kind of kit for detecting diarrhea virus, which is characterized in that including the described in any item detection abdomens of claim 1~6
The nucleic acid compositions of diarrhea virus.
8. it is according to claim 7 detection diarrhea virus kit, which is characterized in that further include PCR reaction buffer,
RNA extracts at least one of reagent, PCR stabilizer and PCR reinforcing agent.
9. a kind of application method for the kit for detecting diarrhea virus, which comprises the following steps:
The nucleic acid for extracting sample to be tested, obtains sample of nucleic acid；
Using the sample of nucleic acid as template, it is added in the kit of detection diarrhea virus described in any one of claim 7~8
Amplimer to corresponding detection probe, carry out real-time fluorescent PCR amplification reaction；And
Fluorescence signal is detected in real-time fluorescent PCR amplification reaction process, obtains testing result.
10. the application method of the kit of detection diarrhea virus according to claim 9, which is characterized in that described real-time
The amplification condition of fluorescent PCR amplified reaction are as follows: 90 DEG C~98 DEG C, 1min~10min；And 90 DEG C~98 DEG C, 10s~30s, 55 DEG C
~65 DEG C, 10s~30s, 30~40 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811643012.0A CN109593890A (en) | 2018-12-29 | 2018-12-29 | Detect the nucleic acid compositions of diarrhea virus, the application method of kit and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811643012.0A CN109593890A (en) | 2018-12-29 | 2018-12-29 | Detect the nucleic acid compositions of diarrhea virus, the application method of kit and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109593890A true CN109593890A (en) | 2019-04-09 |
Family
ID=65965675
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811643012.0A Pending CN109593890A (en) | 2018-12-29 | 2018-12-29 | Detect the nucleic acid compositions of diarrhea virus, the application method of kit and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109593890A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988869A (en) * | 2019-04-23 | 2019-07-09 | 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 |
| CN111411175A (en) * | 2020-04-29 | 2020-07-14 | 深圳市儿童医院 | PCR fluorescence detection kit for digestive tract adenovirus and application thereof |
| CN111748552A (en) * | 2019-12-24 | 2020-10-09 | 深圳市人民医院 | Kit for detecting five infant diarrhea disease series viruses and application thereof |
| 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 |
| CN112575118A (en) * | 2020-07-15 | 2021-03-30 | 江苏硕世生物科技股份有限公司 | Method for simultaneously detecting various diarrhea viruses by using melting curve |
| 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 |
| CN114736991A (en) * | 2022-05-18 | 2022-07-12 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting diarrheagenic virus by one-step method and application of composition, kit and method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206713A (en) * | 2011-04-06 | 2011-10-05 | 浙江大学 | Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit and use thereof |
| CN103131798A (en) * | 2013-02-25 | 2013-06-05 | 湖北朗德医疗科技有限公司 | Norovirus real-time fluorescent RT-PCR detection kit and application thereof |
| CN103146846A (en) * | 2013-03-21 | 2013-06-12 | 广州维伯鑫生物科技有限公司 | Single standard product-based four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) method and kit |
| CN103224998A (en) * | 2013-04-24 | 2013-07-31 | 深圳市生科源技术有限公司 | Rotavirus PCR detection kit and detection method thereof |
| CN107058632A (en) * | 2017-05-23 | 2017-08-18 | 安徽安龙基因医学检验所有限公司 | A kind of multiplex PCR detects three kinds of diarrhea virus kits and its detection method |
| CN108034762A (en) * | 2017-12-21 | 2018-05-15 | 北京卓诚惠生生物科技股份有限公司 | Multiplex PCR detects six kinds of diarrhea virus primed probe groups |
| CN108330211A (en) * | 2017-01-18 | 2018-07-27 | 南京美宁康诚生物科技有限公司 | A group rotavirus/astrovirus/intestinal adenovirus nucleic acid multiple fluorescence PCR detection reagent box and application thereof |
-
2018
- 2018-12-29 CN CN201811643012.0A patent/CN109593890A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206713A (en) * | 2011-04-06 | 2011-10-05 | 浙江大学 | Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit and use thereof |
| CN103131798A (en) * | 2013-02-25 | 2013-06-05 | 湖北朗德医疗科技有限公司 | Norovirus real-time fluorescent RT-PCR detection kit and application thereof |
| CN103146846A (en) * | 2013-03-21 | 2013-06-12 | 广州维伯鑫生物科技有限公司 | Single standard product-based four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) method and kit |
| CN103224998A (en) * | 2013-04-24 | 2013-07-31 | 深圳市生科源技术有限公司 | Rotavirus PCR detection kit and detection method thereof |
| CN108330211A (en) * | 2017-01-18 | 2018-07-27 | 南京美宁康诚生物科技有限公司 | A group rotavirus/astrovirus/intestinal adenovirus nucleic acid multiple fluorescence PCR detection reagent box and application thereof |
| CN107058632A (en) * | 2017-05-23 | 2017-08-18 | 安徽安龙基因医学检验所有限公司 | A kind of multiplex PCR detects three kinds of diarrhea virus kits and its detection method |
| CN108034762A (en) * | 2017-12-21 | 2018-05-15 | 北京卓诚惠生生物科技股份有限公司 | Multiplex PCR detects six kinds of diarrhea virus primed probe groups |
Non-Patent Citations (6)
| Title |
|---|
| CANNON JL等: "Genetic and Epidemiologic Trends of Norovirus Outbreaks in the United States from 2013 to 2016 Demonstrated Emergence of Novel GII.4 Recombinant Viruses", 《J CLIN MICROBIOL》 * |
| LEE SG等: "Nationwide groundwater surveillance of noroviruses in South Korea", 《APPL ENVIRON MICROBIOL》 * |
| LOISY F等: "Real-time RT-PCR for norovirus screening in shellfish", 《J VIROL METHODS》 * |
| MATHIJS E等: "Novel norovirus recombinants and of GII.4 sub-lineages associated with outbreaks between 2006 and 2010 in Belgium", 《VIROL J》 * |
| PARK Y等: "Immunomagnetic separation combined with real-time reverse transcriptase PCR assays for detection of norovirus in contaminated food", 《APPL ENVIRON MICROBIOL》 * |
| 环境保护部主编: "《国家污染物环境健康风险名录》", 30 December 2013 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988869A (en) * | 2019-04-23 | 2019-07-09 | 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 |
| CN109988869B (en) * | 2019-04-23 | 2023-08-11 | 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 |
| CN111748552A (en) * | 2019-12-24 | 2020-10-09 | 深圳市人民医院 | Kit for detecting five infant diarrhea disease series viruses and application thereof |
| CN111411175A (en) * | 2020-04-29 | 2020-07-14 | 深圳市儿童医院 | PCR fluorescence detection kit for digestive tract adenovirus and application thereof |
| CN112575118A (en) * | 2020-07-15 | 2021-03-30 | 江苏硕世生物科技股份有限公司 | Method for simultaneously detecting various diarrhea viruses by using melting curve |
| CN112575118B (en) * | 2020-07-15 | 2023-12-01 | 江苏硕世生物科技股份有限公司 | Method for simultaneously detecting various diarrhea viruses by using melting curve |
| 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 |
| 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 |
| CN114736991A (en) * | 2022-05-18 | 2022-07-12 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting diarrheagenic virus by one-step method and application of composition, kit and method |
| CN114736991B (en) * | 2022-05-18 | 2023-11-17 | 圣湘生物科技股份有限公司 | Composition, kit, method and application for detecting diarrheal virus by one-step method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111020064B (en) | Novel coronavirus ORF1ab gene nucleic acid detection kit | |
| CN109593890A (en) | Detect the nucleic acid compositions of diarrhea virus, the application method of kit and kit | |
| CN111235316B (en) | Primer probe for identifying novel coronavirus and application of primer probe in triple fluorescence RPA | |
| US11649511B2 (en) | Multiplex PCR method for the detection of SARS-CoV-2 | |
| CN110184390A (en) | For identifying the double FQ-PCR detection kit of African swine fever and wild strains of classical swine fever virus | |
| CN107245531B (en) | Diarrhea pathogen multiple gene detection system and kit and application thereof | |
| CN110438265B (en) | Rapid differential diagnosis method for African swine fever virus gene type I and type II | |
| JPWO2016167365A1 (en) | Mental illness biomarker | |
| CN113005226A (en) | Oligonucleotide and kit for detecting SARS-CoV-2 | |
| CN110804669A (en) | CRISPR (clustered regularly interspaced short palindromic repeats) detection primer group for mycoplasma pneumoniae and application thereof | |
| CN107058632A (en) | A kind of multiplex PCR detects three kinds of diarrhea virus kits and its detection method | |
| CN110106285A (en) | A kind of dual isothermal nucleic acid amplification method containing internal reference of 3 type adenovirus hominis of quick detection | |
| CN105112558B (en) | The triple real time fluorescent quantitative RT PCR detection kits of the type of foot and mouth disease virus O, A and Asia I | |
| CN111521781B (en) | Detection kit for SARS-CoV-2 nucleic acid of new coronary pneumonia virus and detection method thereof | |
| CN111826464A (en) | Primer probe for detecting multiple gastrointestinal viruses in one tube, screening method and kit | |
| CN109762932A (en) | Identify the detection primer and probe, kit and application of HP-PRRSV and NADC30-like strain | |
| CN110484625A (en) | For detecting primer combination of probe object, kit and the detection method of PRKY gene methylation | |
| KR101857684B1 (en) | Primers and probe for detection of middle east respiratory syndrome coronavirus and detecting method for middle east respiratory syndrome coronavirus using the same | |
| CN106119421B (en) | QYYZ plants of pig blue-ear disease of fluorogenic quantitative detection primer, probe and kit | |
| CN108411014A (en) | Differentiate the primer and probe and detection method of A types and the dual real-time fluorescence quantitative PCR of Type B ox pasteurella multocida | |
| CN113046452A (en) | Composition for detecting Boeck hollandia farci and application thereof | |
| CN101812538A (en) | Enterovirus 71-detecting fluorescent quantitative RT-PCR kit | |
| CN110468223A (en) | Primer, probe, kit and the method for mycoplasma pneumoniae and Bao Te Pseudomonas detection of nucleic acids based on dUTP/UNG method | |
| CN111500768B (en) | Primer probe for identifying novel coronavirus and application of primer probe in dual-digital PCR | |
| US20220403476A1 (en) | Transcription Mediated Amplification Methods for RNA Detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190409 |
|
| RJ01 | Rejection of invention patent application after publication |