CN108977580A - A kind of kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation - Google Patents
A kind of kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation Download PDFInfo
- Publication number
- CN108977580A CN108977580A CN201810912949.7A CN201810912949A CN108977580A CN 108977580 A CN108977580 A CN 108977580A CN 201810912949 A CN201810912949 A CN 201810912949A CN 108977580 A CN108977580 A CN 108977580A
- Authority
- CN
- China
- Prior art keywords
- hepatitis
- nucleic acid
- virus nucleic
- kit
- qrt
- 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
- C12Q1/706—Specific hybridization probes for hepatitis
- C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
-
- 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
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Communicable Diseases (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The kit of the quick detection hepatitis C virus nucleic acid saved the invention discloses a kind of 2-8 DEG C, including qRT-PCR reaction solution, qRT-PCR start agent, RNA internal reference, strong positive quality-control product, weakly positive quality-control product, negative quality-control product and qualitative reference product；Wherein in qRT-PCR reaction solution include qRT-PCR reinforcing agent, reaction buffer, 2 detection hepatitis C virus nucleic acid special primer, 1 detection hepatitis C virus nucleic acid specific probe, 2 detection RNA internal reference specific primer, 1 detection RNA internal reference specific probe, dual-function dna polymerase, UNG enzyme, dATP, dGTP, dCTP, dUTP, 0.1% sodium azide.Kit high sensitivity (minimum detectable HCV virus 10IU/ml) of the present invention, specificity is good, precision is high, the quantitative accurate, range of linearity is wide, detection method is simple and required time is short, the clinic of hepatitis C virus nucleic acid, which is quickly detected, has very high reference value, is suitable for popularization and application.
Description
Technical field
The present invention relates to vitro diagnostic techniques field, the quick detection hepatitis C saved more particularly, to a kind of 2-8 DEG C
The kit of viral nucleic acid.
Background technique
Hepatitis C Virus (Hepatitis C Virus, HCV) belongs to flaviviridae, is single strand plus RNA virus, at present
6 genotype and multiple hypotypes can be divided into, viral hepatitis type C caused by HCV infection is in global prevalence.Chronic hepatitis's disease
Virus hepatitis can lead to liver chronic inflammation necrosis and fibrosis, some patientss can develop for cirrhosis even hepatocellular carcinoma, it is right
The health and lives of patient are very harmful, it has also become serious society and public health problem, the nucleic acid inspection of Hepatitis C Virus
It surveys and provides effective method for the clinical diagnosis and treatment of hepatitis C.The nucleic acid detection method of HCV is based primarily upon real-time fluorescence at present
Reverse transcriptase polymerase even reaction (qRT-polymerase chain reaction, qRT-PCR), it is poly- using reverse transcriptase and DNA
Synthase carries out.Since reverse transcriptase thermal stability is poor, detection reagent is usually stored under -20 DEG C of freezing conditions, in solid
Body state, when use, again thaw to reagent, inconvenient, extend detection used time, and detection of the thawing to reagent
Performance has certain influence；And the reagent of freezen protective requires height to traffic condition, further increases testing cost.City at present
The hepatitis C virus nucleic acid detection reagent for only having Roche Holding Ag on face is 2-8 DEG C of liquid condition preservation, though operation is more convenient,
But nucleic acid extraction adds nucleic acid amplification detection to need could complete for 4.5 hours, time-consuming longer.Up to now there has been no 2-8 DEG C save and
Hepatitis C virus nucleic acid detection reagent with quick detectability comes out.
Summary of the invention
The kit of the quick detection hepatitis C virus nucleic acid saved the purpose of the present invention is to provide a kind of 2-8 DEG C,
Not only facilitate storage and transport, while detection time can also be greatly shortened.
To achieve the above object, the present invention can take following technical proposals:
The kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation of the present invention, including qRT-PCR reaction
Liquid, qRT-PCR start agent, RNA internal reference, strong positive quality-control product, weakly positive quality-control product, negative quality-control product and qualitative reference product；
The special of hepatitis C virus nucleic acid is detected including qRT-PCR reinforcing agent, reaction buffer, 2 in the qRT-PCR reaction solution
Primer, 1 detection hepatitis C virus nucleic acid specific probe, 2 detection RNA internal reference specific primer, 1 detection
Specific probe, dual-function dna polymerase, UNG enzyme, dATP, dGTP, dCTP, dUTP, 0.1% sodium azide of RNA internal reference.
Wherein the qRT-PCR reinforcing agent is by 1%-10%DMSO, 200-500mM glycine betaine, 0.01%-0.05%
The mixture of Tween20 and 1%-10% glycerol.
The reaction buffer is made of 50mM tri- (methylol) methylglycine (pH8.3) and 100mM potassium acetate.
The concentration of the special primer of 2 detections hepatitis C virus nucleic acid is 0.3 ~ 0.9uM, a detection third
The concentration of the specific probe of Hepatitis virus nucleic acid is 0.1 ~ 0.3uM, and base sequence is respectively as follows:
Target primer 1(SEQ ID No.1): 5'-GTCTAGCCATGGCGTTAGTA-3',
Target primer 2 (SEQ ID No.2): 5'-AAGCACCCTATCAGGCAGTA-3'；
Target probe (SEQ ID No.5): FAM-CGGAACCGGTGAGTACACCGGAAT-BHQ1.
The concentration of the specific primers of 2 detections RNA internal reference is 0.3 ~ 0.9uM, right in 1 detection RNA
According to specific probe concentration be 0.1 ~ 0.3uM；Its base sequence is respectively as follows:
Internal reference primer 1(SEQ ID No.3): 5'-GGCTGCTCGCGGATACCC-3',
Internal reference primer 2 (SEQ ID No.4): 5'-CTGAAGAACTTGCGTTCTCG-3'；
Internal reference probe (SEQ ID No.6): ROX-ACCTCGGGTTTCCGTCTTGCTCGT-BHQ2.
The dual-function dna polymerase is rTth archaeal dna polymerase, content 5-10U.
The qRT-PCR starting agent is 1-3mM Mn (OAc)2With 0.1% sodium azide.
The RNA internal reference is RNA pseudovirus solution prepared by one section of nucleotide sequence of engineer；Its nucleotides sequence
It is classified as (SEQ ID No.7):
5'-ggctgctcgcggatacccgtacctcgggtttccgtcttgctcgtatcgctcgagaacgcaagttcttcag
cgaaaagcacgacagtggtcgctacatagcgtggttccatactggaggtgaaatcaccgacagcatgaattccgccg
gcgtgcgcgttatacgcacttcggagtggctaacgccggttcccacattccctca-3'。
The feminine gender quality-control product is the serum or blood plasma without hepatitis C virus nucleic acid or without Hepatitis C Virus；Institute
Stating strong positive quality-control product, weakly positive quality-control product and qualitative reference product is the nucleic acid sequence system according to the 5 ' area UTR of Hepatitis C Virus
Standby pseudovirion, through without hepatitis C virus nucleic acid or serum without Hepatitis C Virus or diluted plasma to difference
Concentration gradient obtains, and nucleic acid sequence is (SEQ ID No.8):
5'-gtctagccatggcgttagtatgagtgtcgtgcagcctccaggaccccccctcccgggagagccatagtg
gtctgcggaaccggtgagtacaccggaattgccaggacgaccgggtcctttcttggataaacccgctcaatgcctgg
agatttgggcgtgcccccgcaagactgctagccgagtagtgttgggtcgcgaaaggccttgtggtactgcctgatag
ggtgctt-3'。
The method that one kind of the invention quickly detects hepatitis C virus nucleic acid, using kit prepared by the present invention, tool
Body step includes:
The first step, the extraction of template ribonucleic acid
It extracts and purifies the hepatitis C virus nucleic acid in clinical serum or plasma sample, and add RNA internal reference wherein, it is dense
Degree is 1.0E+03-1.0E+04copies/ml；
Second step, negative quality-control product, strong positive quality-control product, weakly positive quality-control product and qualitative reference product A, B, C, D and sample synchronization
Carry out the extraction and purifying of template ribonucleic acid
Negative quality-control product, for serum or blood plasma without hepatitis C virus nucleic acid, appearance clarification；
Strong positive quality-control product carries out hepatitis C pseudovirion using the serum or blood plasma that are free of hepatitis C virus nucleic acid
Dilution, concentration 1.0E+04-1.0E+06IU/ml；(being quantified using WHO plasmid standards for quantitation),
Weakly positive quality-control product carries out hepatitis C pseudovirion using the serum or blood plasma that are free of hepatitis C virus nucleic acid
Dilution, concentration are that 5.0E+01-1.0E+03IU/ml(is quantified using WHO plasmid standards for quantitation)；
Qualitative reference product A, B, C, D, using the serum or blood plasma for being free of hepatitis C virus nucleic acid to hepatitis C pseudovirus
Grain carries out 10 times of gradient dilutions, determines its concentration (using WHO plasmid standards for quantitation between 1.0E+02-1.0E+08IU/ml
Amount), concentration differs about 100 times between each adjacent qualitative reference product；
Third step, qRT-PCR reaction
Prepare reaction system in 200 μ l PCR pipes: qRT-PCR reaction solution 20ul, qRT-PCR start agent 10ul, template
RNA40-60ul；Above-mentioned reaction tube is subjected to quantitative fluorescent PCR reaction in the following conditions:
4th step, as a result interpretation
The channel FAM No Ct is answered in strong feminine gender Quality Control, and value≤35.0 ROX channel C t, strong positive Quality Control detectable concentration should be in 1.0E+04-
Within the scope of 1.0E+06IU/mL, weakly positive Quality Control detectable concentration should within the scope of 5.0E+01-1.0E+03IU/mL, meet with
Under the conditions of upper 3, fluorescence quantitative PCR instrument can be automatically generated according to the calibration curve that 4 concentration gradient qualitative reference product are drawn
The detectable concentration result of sample to be tested.
The advantage of the invention is that be added in reaction solution qRT-PCR reinforcing agent (by DMSO, glycine betaine, Tween20 and
The mixture of glycerol composition), which can prevent the denaturation of dual-function dna polymerase as a kind of permeation protective agent, increase
The stability of strong qRT-PCR reaction solution is, it can be achieved that detection kit is at least stable under the conditions of 2-8 DEG C saves 1 year, in reagent
The use of box, saves the limitation for getting rid of freezing conditions in the process at transport, greatly reduces time, economic cost, improves behaviour
The convenience of work；The reinforcing agent is as synergist simultaneously, and reduces the mispairing in amplification procedure, improves the effect of PCR amplification
Rate and specificity improve the rate that primer and template annealing extend, so shorten in amplification cycles and anneal, extend when
Between, the detection of sample can be completed in 1 hour, and there is apparent advantage on detection efficiency.
Kit high sensitivity (minimum detectable HCV virus 10IU/ml) of the present invention, specificity is good, precision is high, quantitative
Accurately, the range of linearity is wide, and detection method is simple and required time is short, the clinic quickly detection for hepatitis C virus nucleic acid
With very high reference value, it is suitable for popularization and application.
Detailed description of the invention
Fig. 1 is the Linear Fit Chart of the embodiment of the present invention 1.
Specific embodiment
The present invention is described further combined with specific embodiments below, to facilitate those skilled in the art more preferable
The understanding present invention and be practiced, but illustrated embodiment can not be used as limitation of the invention.
The composition of the kit of the present invention of embodiment 1
1,20ulqRT-PCR reaction solution: 5%DMSO, 300mM glycine betaine, 0.05%Tween20,5% glycerol, 50mM tri- (methylol)
Methylglycine (pH8.3), 100mM potassium acetate, 0.6uM primer 1,0.6uM primer 2,0.2uM primer 3,0.2uM primer 4,
0.3uM probe 5,0.1uM probe 6,10U rTth archaeal dna polymerase, 0.5U UNG enzyme, 0.2mMdATP, 0.2mMdGTP,
0.2mMdCTP, 0.4mMdUTP, 0.1% sodium azide；4 specific primers and 2 specific probes are respectively as follows:
Primer 1(SEQ ID No.1): 5'-GTCTAGCCATGGCGTTAGTA-3'；
Primer 2 (SEQ ID No.2): 5'-AAGCACCCTATCAGGCAGTA-3'；
Primer 3(SEQ ID No.3): 5'-GGCTGCTCGCGGATACCC-3'；
Primer 4(SEQ ID No.4): 5'-CTGAAGAACTTGCGTTCTCG-3'；
Probe 1(SEQ ID No.5): FAM-CGGAACCGGTGAGTACACCGGAAT-BHQ1；
Probe 2(SEQ ID No.6): ROX-ACCTCGGGTTTCCGTCTTGCTCGT-BHQ2.
2,10ulqRT-PCR starts agent: by 1.5mM Mn (OAc)2It is formed with 0.1% sodium azide.
3, RNA internal reference: for the RNA pseudovirus of one section of nucleotide sequence (SEQ ID No.7) preparation of engineer
Grain solution, concentration 1.0E+03-1.0E+04copies/ml；SEQ ID No.7 are as follows:
5'-ggctgctcgcggatacccgtacctcgggtttccgtcttgctcgtatcgctcgagaacgcaagttcttcag
cgaaaagcacgacagtggtcgctacatagcgtggttccatactggaggtgaaatcaccgacagcatgaattccgccg
gcgtgcgcgttatacgcacttcggagtggctaacgccggttcccacattccctca-3'。
4, negative quality-control product: serum or blood plasma without hepatitis C virus nucleic acid, appearance clarification.
5, strong positive quality-control product: using the serum or blood plasma for being free of hepatitis C virus nucleic acid to hepatitis C pseudovirus
Grain is diluted, and concentration is that 1.0E+04-1.0E+06IU/ml(is quantified using WHO plasmid standards for quantitation).
6, weakly positive quality-control product: using the serum or blood plasma for being free of hepatitis C virus nucleic acid to hepatitis C pseudovirus
Grain is diluted, and concentration is that 5.0E+01-1.0E+03IU/ml(is quantified using WHO plasmid standards for quantitation).
7, qualitative reference product A, B, C, D: using the serum or blood plasma for being free of hepatitis C virus nucleic acid to hepatitis C vacation
Virion carries out 10 times of gradient dilutions, makes its concentration between 1.0E+02-1.0E+08IU/ml (using WHO plasmid standards for quantitation
Quantified), between each adjacent qualitative reference product, concentration differs about 100 times.
The extraction of 2 hepatitis C virus nucleic acid of embodiment
The present embodiment extract and purifying sample in hepatitis C virus nucleic acid when, using clinical serum or plasma sample and
The nucleic acid extraction purified reagent (paramagnetic particle method) of Zhengzhou Autobio Engineering Co., Ltd., concrete operation method is to specifications
It carries out.
The minimum detectability of the kit of the present invention of embodiment 3
By WHO plasmid standards for quantitation (WHO International Standard 5th WHO International Standard
ForHCV NAT NIBSC code:14/150) with negative plasma 15,10,7.5,5,2.5IU/ml are diluted to, pacified using Zhengzhou
The extraction that the nucleic acid extraction purified reagent (paramagnetic particle method) of figure bioengineering limited liability company carries out hepatitis C virus nucleic acid is pure
Change, and detected using this kit, each concentration samples repeat 60 times, the recall rate of each concentration samples are calculated, thus really
Determine minimum detectability (minimum concentration of Jian Chu Shuai≤95% is set to minimum detectability), the results are shown in Table 1.As can be seen from the results,
The minimum detectability of this kit is 10IU/ml.
Table 1
The precision of the kit of the present invention of embodiment 4
The hepatitis C pseudovirion by WHO plasmid standards for quantitation quantitatively calibrating is taken, it is dilute to carry out gradient to it with negative plasma
It releases, is diluted to 5.0E+06IU/ml, 5.0E+02IU/ml.Using the nucleic acid extraction of Zhengzhou Autobio Engineering Co., Ltd.
Purified reagent (paramagnetic particle method) carries out the extraction purification of hepatitis C virus nucleic acid, and is detected using this kit, Mei Genong
It spends sample and makees 20 repetitions, calculate the coefficient of variation (CV, %) of each concentration of specimens logarithm, the results are shown in Table 2.By result
It is found that the coefficient of variation of the sample of this kit detection two concentration of height is respectively less than 5%, it is good to illustrate that this kit has
Detect precision.
Table 2
The range of linearity of the kit of the present invention of embodiment 5
The hepatitis C pseudovirion by WHO plasmid standards for quantitation quantitatively calibrating is taken, it is dilute to carry out gradient to it with negative plasma
Release, be diluted to 1.0E+08IU/ml, 1.0E+07IU/ml, 1.0E+06IU/ml, 1.0E+05IU/ml, 1.0E+04IU/ml,
1.0E+03IU/ml,1.0E+02IU/ml,50IU/ml,20IU/ml,10IU/ml.Had using Zhengzhou Antu bioengineering share
The nucleic acid extraction purified reagent (paramagnetic particle method) of limit company carries out the extraction purification of hepatitis C virus nucleic acid, and uses this kit
It is detected, each concentration samples make 4 repetitions, calculate absolute deviation (measured concentration and the mark of each concentration of specimens logarithm
Show the deviation of log concentration value), it is to meet the requirements to be no more than ± 0.5log value range, is then to the logarithm of mark concentration
X-axis carries out linear fit by Y-axis of the logarithm of measured concentration, calculates its linearly dependent coefficient, if | r | it is accorded with if≤0.9800
Zygonema area requirement, testing result are as shown in table 3.
Table 3
As can be known from the results, concentration of specimens absolute deviation of testing result in the range of 10-1.0E+08IU/ml is all satisfied requirement,
Data are subjected to linear fit, as a result as shown in Figure 1, the results show that R2=0.9997, r=0.9998.Therefore, this kit
The range of linearity is 10IU/ml-1.0E+08IU/ml.
The stability that the kit of the present invention of embodiment 6 is saved at 2-8 DEG C
This kit is placed in 2-8 DEG C of environment, is detected respectively at 1 month, 3 months, 6 months, 12 months, is examined respectively
The minimum detectability of test agent box, precision, linear, the kit with -20 DEG C compares, and the results are shown in Table 4.Show to try
Agent box is after 2-8 DEG C saves 1 year, minimum detection limit, precision, the linear kit test result indifference with -20 DEG C.
Table 4
。
Sequence table
<110>Zhengzhou Autobio Engineering Co., Ltd.
<120>a kind of kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation
<141> 2018-08-08
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (ordo artificialis)
<400> 1
gtctagccat ggcgttagta 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (ordo artificialis)
<400> 2
aagcacccta tcaggcagta 20
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (ordo artificialis)
<400> 3
ggctgctcgc ggataccc 18
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (ordo artificialis)
<400> 4
ctgaagaact tgcgttctcg 20
<210> 5
<211> 24
<212> DNA
<213>artificial sequence (ordo artificialis)
<400> 5
cggaaccggt gagtacaccg gaat 24
<210> 6
<211> 24
<212> DNA
<213>artificial sequence (ordo artificialis)
<400> 6
acctcgggtt tccgtcttgc tcgt 24
<210> 7
<211> 202
<212> DNA
<213>artificial sequence (ordo artificialis)
<400> 7
ggctgctcgc ggatacccgt acctcgggtt tccgtcttgc tcgtatcgct cgagaacgca 60
agttcttcag cgaaaagcac gacagtggtc gctacatagc gtggttccat actggaggtg 120
aaatcaccga cagcatgaat tccgccggcg tgcgcgttat acgcacttcg gagtggctaa 180
cgccggttcc cacattccct ca 202
<210> 8
<211> 230
<212> DNA
<213>artificial sequence (ordo artificialis)
<400> 8
gtctagccat ggcgttagta tgagtgtcgt gcagcctcca ggaccccccc tcccgggaga 60
gccatagtgg tctgcggaac cggtgagtac accggaattg ccaggacgac cgggtccttt 120
cttggataaa cccgctcaat gcctggagat ttgggcgtgc ccccgcaaga ctgctagccg 180
agtagtgttg ggtcgcgaaa ggccttgtgg tactgcctga tagggtgctt 230
Claims (10)
1. a kind of kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation, it is characterised in that: including qRT-PCR
Reaction solution, qRT-PCR start agent, RNA internal reference, strong positive quality-control product, weakly positive quality-control product, negative quality-control product and qualitative reference
Product；It include qRT-PCR reinforcing agent, reaction buffer, 2 detection hepatitis C virus nucleic acids in the qRT-PCR reaction solution
Special primer, 1 detection hepatitis C virus nucleic acid specific probe, 2 detection RNA internal reference specific primer, 1
Detect specific probe, dual-function dna polymerase, UNG enzyme, dATP, dGTP, dCTP, dUTP, 0.1% nitrine of RNA internal reference
Change sodium.
2. the kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation according to claim 1, feature exist
In: the qRT-PCR reinforcing agent is by 1%-10%DMSO, 200-500mM glycine betaine, 0.01%-0.05%Tween20 and 1%-10%
The mixture of glycerol.
3. the kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation according to claim 1, feature exist
In: the reaction buffer is made of (methylol) methylglycine of 50mM tri- and 100mM potassium acetate.
4. the kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation according to claim 1, feature exist
In: the concentration of the special primer of 2 detections hepatitis C virus nucleic acid is 0.3 ~ 0.9uM, the third type liver of a detection
The concentration of the specific probe of scorching viral nucleic acid is 0.1 ~ 0.3uM, and base sequence is respectively as follows:
Target primer 1:5'-GTCTAGCCATGGCGTTAGTA-3',
Target primer 2: 5'-AAGCACCCTATCAGGCAGTA-3'；
Target probe: FAM-CGGAACCGGTGAGTACACCGGAAT-BHQ1.
5. the kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation according to claim 1, feature exist
In: the concentration of the specific primer of 2 detections RNA internal reference is 0.3 ~ 0.9uM, the spy of 1 detection RNA internal reference
The concentration of specific probes is 0.1 ~ 0.3uM；Its base sequence is respectively as follows:
Internal reference primer 1:5'-GGCTGCTCGCGGATACCC-3',
Internal reference primer 2: 5'-CTGAAGAACTTGCGTTCTCG-3'；
Internal reference probe: ROX-ACCTCGGGTTTCCGTCTTGCTCGT-BHQ2.
6. the kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation according to claim 1, feature exist
In: the dual-function dna polymerase is rTth archaeal dna polymerase, content 5-10U.
7. the kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation according to claim 1, feature exist
In: the qRT-PCR starting agent is 1-3mM Mn (OAc)2With 0.1% sodium azide.
8. the kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation according to claim 1, feature exist
In: the RNA internal reference is RNA pseudovirus solution prepared by one section of nucleotide sequence of engineer；Its nucleotide sequence are as follows:
5'-ggctgctcgcggatacccgtacctcgggtttccgtcttgctcgtatcgctcgagaacgcaagttcttcag
cgaaaagcacgacagtggtcgctacatagcgtggttccatactggaggtgaaatcaccgacagcatgaattccgccg
gcgtgcgcgttatacgcacttcggagtggctaacgccggttcccacattccctca-3'。
9. the kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation according to claim 1, feature exist
In: the feminine gender quality-control product is the serum or blood plasma without hepatitis C virus nucleic acid or without Hepatitis C Virus；It is described strong
Positive quality control product, weakly positive quality-control product and qualitative reference product are to be prepared according to the nucleic acid sequence in the 5 ' area UTR of Hepatitis C Virus
Pseudovirion, through without hepatitis C virus nucleic acid or serum without Hepatitis C Virus or diluted plasma to various concentration
Gradient obtains, nucleic acid sequence are as follows:
5'-gtctagccatggcgttagtatgagtgtcgtgcagcctccaggaccccccctcccgggagagccatagtgg
tctgcggaaccggtgagtacaccggaattgccaggacgaccgggtcctttcttggataaacccgctcaatgcctgga
gatttgggcgtgcccccgcaagactgctagccgagtagtgttgggtcgcgaaaggccttgtggtactgcctgatagg
gtgctt-3'。
10. a kind of method of quickly detection hepatitis C virus nucleic acid, it is characterised in that: kit prepared by the present invention is used,
Specific steps include:
The first step, the extraction of template ribonucleic acid
It extracts and purifies the hepatitis C virus nucleic acid in clinical serum or plasma sample, and add RNA internal reference wherein, it is dense
Degree is 1.0E+03-1.0E+04copies/ml；
Second step, negative quality-control product, strong positive quality-control product, weakly positive quality-control product and qualitative reference product A, B, C, D and sample synchronization
Carry out the extraction and purifying of template ribonucleic acid；
Negative quality-control product, for serum or blood plasma without hepatitis C virus nucleic acid, appearance clarification；
Strong positive quality-control product carries out hepatitis C pseudovirion using the serum or blood plasma that are free of hepatitis C virus nucleic acid
Dilution, concentration 1.0E+04-1.0E+06IU/ml；
Weakly positive quality-control product carries out hepatitis C pseudovirion using the serum or blood plasma that are free of hepatitis C virus nucleic acid
Dilution, concentration 5.0E+01-1.0E+03IU/ml；
Qualitative reference product A, B, C, D, using the serum or blood plasma for being free of hepatitis C virus nucleic acid to hepatitis C pseudovirus
Grain carry out 10 times of gradient dilutions, make its concentration between 1.0E+02-1.0E+08IU/ml, each adjacent qualitative reference product it
Between concentration differ about 100 times；
Third step, qRT-PCR reaction
Prepare reaction system in 200 μ l PCR pipes: qRT-PCR reaction solution 20ul, qRT-PCR start agent 10ul, template
RNA40-60ul；By above-mentioned reaction tube in the following conditions progress quantitative fluorescent PCR reaction: 50 DEG C of 2min, 55 DEG C of 30min, 95 DEG C
2min, 95 DEG C of 5s, 60 DEG C of 15s are recycled 40 times, 40 DEG C of 10s；
4th step, as a result interpretation
The channel FAM No Ct is answered in strong feminine gender Quality Control, and value≤35.0 ROX channel C t, strong positive Quality Control detectable concentration should be in 1.0E+04-
Within the scope of 1.0E+06IU/mL, weakly positive Quality Control detectable concentration should within the scope of 5.0E+01-1.0E+03IU/mL, meet with
Under the conditions of upper 3, fluorescence quantitative PCR instrument can be automatically generated according to the calibration curve that 4 concentration gradient qualitative reference product are drawn
The detectable concentration result of sample to be tested.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810912949.7A CN108977580A (en) | 2018-08-13 | 2018-08-13 | A kind of kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810912949.7A CN108977580A (en) | 2018-08-13 | 2018-08-13 | A kind of kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108977580A true CN108977580A (en) | 2018-12-11 |
Family
ID=64552962
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810912949.7A Pending CN108977580A (en) | 2018-08-13 | 2018-08-13 | A kind of kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108977580A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109852726A (en) * | 2019-01-21 | 2019-06-07 | 上海科华生物工程股份有限公司 | A kind of reagent using noncompetitive internal standard single-point quantitative detection HCV |
| CN110656189A (en) * | 2019-10-30 | 2020-01-07 | 郑州安图生物工程股份有限公司 | Fluorescent PCR kit for single-tube multiple rapid detection of chlamydia trachomatis, gonococcus and ureaplasma urealyticum |
| CN110982945A (en) * | 2020-03-04 | 2020-04-10 | 珠海丽珠试剂股份有限公司 | Nucleic acid composition, kit and method for detecting 2019 novel coronavirus |
| CN110982885A (en) * | 2019-12-20 | 2020-04-10 | 郑州安图生物工程股份有限公司 | Gene polymorphism detection primer, probe and kit |
| CN111172323A (en) * | 2020-01-17 | 2020-05-19 | 郑州安图生物工程股份有限公司 | Herpes simplex virus 1 type and 2 type typing nucleic acid detection kit |
| CN112034186A (en) * | 2020-09-07 | 2020-12-04 | 南京立顶医疗科技有限公司 | Glycosylated hemoglobin kit based on biotin-streptavidin amplification and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827831A (en) * | 2012-08-31 | 2012-12-19 | 武汉百泰基因工程有限公司 | Nucleic acid releasing reagent and nucleic acid releasing method |
| CN105177154A (en) * | 2015-10-10 | 2015-12-23 | 山东出入境检验检疫局检验检疫技术中心 | Test and quality control kit of Norovirus in shellfish and non-diagnostic test method |
| CN105555972A (en) * | 2013-07-25 | 2016-05-04 | 伯乐生命医学产品有限公司 | Genetic assays |
| CN105861743A (en) * | 2016-03-21 | 2016-08-17 | 珠海丽珠试剂股份有限公司 | Internal standard-containing kit and detection method for detecting hepatitis C virus nucleic acid |
| CN107022651A (en) * | 2017-06-02 | 2017-08-08 | 辽宁润基生物科技有限公司 | The kit and its detection method of a kind of quick detection hepatitis C virus nucleic acid |
-
2018
- 2018-08-13 CN CN201810912949.7A patent/CN108977580A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827831A (en) * | 2012-08-31 | 2012-12-19 | 武汉百泰基因工程有限公司 | Nucleic acid releasing reagent and nucleic acid releasing method |
| CN105555972A (en) * | 2013-07-25 | 2016-05-04 | 伯乐生命医学产品有限公司 | Genetic assays |
| CN105177154A (en) * | 2015-10-10 | 2015-12-23 | 山东出入境检验检疫局检验检疫技术中心 | Test and quality control kit of Norovirus in shellfish and non-diagnostic test method |
| CN105861743A (en) * | 2016-03-21 | 2016-08-17 | 珠海丽珠试剂股份有限公司 | Internal standard-containing kit and detection method for detecting hepatitis C virus nucleic acid |
| CN107022651A (en) * | 2017-06-02 | 2017-08-08 | 辽宁润基生物科技有限公司 | The kit and its detection method of a kind of quick detection hepatitis C virus nucleic acid |
Non-Patent Citations (5)
| Title |
|---|
| DOMINGO CALAP,P. 等: "JF719743.1 Enterobacteria phage MS2 isolate MS2 3 FR, partial genome", 《GENBANK》 * |
| FAHIMEH RANJBAR KERMANI 等: "Evaluation of the Performance Characteristics of an In-House One Step TaqMan Real Time-Polymerase Chain Reaction Assay for Detection and Quantification of Hepatitis C Virus", 《JUNDISHAPUR J MICROBIOL》 * |
| GHAFARI,S. 等: "MG436881.1 Hepatitis C virus isolate 26 5 UTR", 《GENBANK》 * |
| JI HOON PARK 等: "Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics", 《PLOS ONE》 * |
| 王香玲 等: "Tth DNA 聚合酶在丙型肝炎病毒RNA检测中的应用", 《中华医学检验杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109852726A (en) * | 2019-01-21 | 2019-06-07 | 上海科华生物工程股份有限公司 | A kind of reagent using noncompetitive internal standard single-point quantitative detection HCV |
| CN110656189A (en) * | 2019-10-30 | 2020-01-07 | 郑州安图生物工程股份有限公司 | Fluorescent PCR kit for single-tube multiple rapid detection of chlamydia trachomatis, gonococcus and ureaplasma urealyticum |
| CN110656189B (en) * | 2019-10-30 | 2023-08-11 | 郑州安图生物工程股份有限公司 | Fluorescent PCR (polymerase chain reaction) kit for single-tube multiplex rapid detection of chlamydia trachomatis, gonococcus and ureaplasma urealyticum |
| CN110982885A (en) * | 2019-12-20 | 2020-04-10 | 郑州安图生物工程股份有限公司 | Gene polymorphism detection primer, probe and kit |
| CN111172323A (en) * | 2020-01-17 | 2020-05-19 | 郑州安图生物工程股份有限公司 | Herpes simplex virus 1 type and 2 type typing nucleic acid detection kit |
| CN110982945A (en) * | 2020-03-04 | 2020-04-10 | 珠海丽珠试剂股份有限公司 | Nucleic acid composition, kit and method for detecting 2019 novel coronavirus |
| CN110982945B (en) * | 2020-03-04 | 2020-06-09 | 珠海丽珠试剂股份有限公司 | Nucleic acid composition, kit and method for detecting 2019 novel coronavirus |
| CN112034186A (en) * | 2020-09-07 | 2020-12-04 | 南京立顶医疗科技有限公司 | Glycosylated hemoglobin kit based on biotin-streptavidin amplification and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108977580A (en) | A kind of kit of the quick detection hepatitis C virus nucleic acid of 2-8 DEG C of preservation | |
| WO2022057060A1 (en) | Method and kit for multiple detection of respiratory virus nucleic acids | |
| CN107058623B (en) | High-sensitivity and specific blood HBV pgRNA fluorescent quantitative PCR detection system and detection method | |
| CN111363858B (en) | 2019-nCoV S gene detection nucleic acid composition, kit and production method | |
| US20090226888A1 (en) | Diagnostic Primers And Method For Detecting Avian Influenza Virus Subtype H5 And H5N1 | |
| CN107022651B (en) | Kit for rapidly detecting hepatitis C virus nucleic acid and detection method thereof | |
| CN108676913A (en) | A kind of human parainfluenza viruses' nucleic acid is hands-free to take gene parting detecting reagent | |
| WO2020125246A1 (en) | Primers, probe, kit and detection method for detecting hepatitis b virus nucleic acid | |
| CN109182600B (en) | Fluorescent quantitative PCR kit for synchronously detecting hepatitis B virus, hepatitis C virus and human immunodeficiency virus type 1 | |
| CN109196124A (en) | The kit and method of the multiple Taqman probe qPCR of detection and four kinds of haematogenous virus of quantitative analysis simultaneously | |
| JP2012055311A (en) | Generic buffer for amplification | |
| CN110923361B (en) | Primer, probe and kit for blood source screening based on digital PCR | |
| CN105861743A (en) | Internal standard-containing kit and detection method for detecting hepatitis C virus nucleic acid | |
| CN117265186B (en) | TaqMan fluorescent quantitative PCR primer group, kit and method for detecting pangolin east yang virus | |
| CN111363859B (en) | Nucleic acid composition for 2019-nCoV detection, kit and production method thereof | |
| WO2006132601A1 (en) | Diagnostic primers and method for detecting avian influenza virus subtype h5 and h5n1 | |
| CN105525036B (en) | A kind of hepatitis b virus hbv real-time fluorescence nucleic acid isothermal amplification detection kit | |
| WO2023116064A1 (en) | Composition, kit and method for detection of sars-cov-2 and use thereof | |
| CN106011308B (en) | Hepatitis C virus genotyping detection kit, oligonucleotide and application thereof | |
| CN109593887B (en) | Kit for quantitative detection of hepatitis C virus nucleic acid | |
| CN110684862B (en) | Microdroplet digital PCR kit for quantitatively detecting hepatitis B virus and detection method | |
| WO2020125294A1 (en) | Primers for hepatitis c virus nucleic acid detection, probe, kit, and detection method | |
| CN107034312B (en) | Nucleotide composition, kit and application thereof | |
| CN111206117A (en) | Kit for detecting human immunodeficiency virus | |
| CN106754900B (en) | Nucleic acid packaging reagent and application thereof |
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: 20181211 |
|
| RJ01 | Rejection of invention patent application after publication |