CN105063235A - Kit for quickly and quantitatively detecting hepatitis b virus nucleic acid DNA (deoxyribonucleic acid) and using method of kit - Google Patents
Kit for quickly and quantitatively detecting hepatitis b virus nucleic acid DNA (deoxyribonucleic acid) and using method of kit Download PDFInfo
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- CN105063235A CN105063235A CN201510452653.8A CN201510452653A CN105063235A CN 105063235 A CN105063235 A CN 105063235A CN 201510452653 A CN201510452653 A CN 201510452653A CN 105063235 A CN105063235 A CN 105063235A
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- nucleic acid
- kit
- hbv
- pcr
- releasing agent
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- 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
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- 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
Abstract
The invention discloses a kit for extracting HBV (hepatitis b virus) DNA (deoxyribonucleic acid) by using micro-nucleic acid releasing reagent and a using method, and belongs to the technical field of molecular biology. According to the kit for extracting and amplifying HBV virus-based nucleic acid (DNA) by using the micro-nucleic acid releasing reagent and the using method of the kit, real-time fluorescence quantification PCR (polymerase chain reaction) principle is adopted. A kit extracting method comprises the following steps of splitting, releasing promoting, and amplifying. The kit has the advantages that extracting and amplifying are carried out by the micro-nucleic acid releasing reagent simply, conveniently, sensitively, quickly and accurately, and minimum detectability is 30 IU (international unit)/ml; while nucleic acid is released completely by the micro-nucleic acid releasing reagent, functions of sealing various substances such as protein, medicines and hemolysis which have interference effects on PCR amplification in samples are fulfilled, and false negative in a detection process is avoided; and internal standard quality-control products are added in an amplification process, and extracting and amplifying efficiency of the samples can be monitored effectively.
Description
Technical field
The invention belongs to technical field of molecular biology, utilize real-time fluorescence quantitative PCR reaction principle, the test kit of the extraction of employing micro-nucleic acid releasing agent, amplification HBV virus base nucleic acid (DNA) and using method thereof.
Background technology
Real-time fluorescence quantitative PCR (Real-timePCR) is the amount immediately being measured specific product during PCR exponential amplification by the change of monitoring fluorescent signal power continuously, and infers the original bulk of goal gene accordingly, does not need to take out PCR primer and is separated; Real-time quantitative PCR, as an extremely effective experimental technique, has been widely used in the every field of molecular biology research.Real-timePCR can be quick, sensitive detection viral RNA and DNA and DNA of bacteria; The method is widely used in the diagnosis of human infectious disease and cause of disease is quantitative, in the detection of animal pathogen gene, and the inspection and quarantine of animal products, the fields such as the qualification of biological products.
Real-Time Fluorescent Quantitative PCR Technique adds fluorophor in PCR reaction system, utilizes fluorescent signal to accumulate the whole PCR process of Real-Time Monitoring, finally by typical curve, unknown template is carried out to the method for quantitative analysis; Amplified fluorescence curve can divide three phases: the fluorescence background stage, fluorescent signal exponential amplification stage and plateau; In the fluorescence background stage, the fluorescent signal of amplification cover by fluorescence background, the change of product amount cannot be judged; And in plateau, the increase of amplified production no longer exponentially level, does not have linear relationship, can not calculate initiate dna copy number according to final PCR primer amount between the end product amount of PCR and starting template amount; Only in the fluorescent signal exponential amplification stage, between the logarithmic value of PCR primer amount and starting template amount, there is linear relationship, can select to carry out quantitative analysis in this stage.
Summary of the invention
The present invention is the quick release adopting a kind of micro-nucleic acid releasing agent single stage method to operate HBV-DNA in serum, blood plasma, the nucleic acid obtained is applicable to the detected downstream such as quantitative fluorescent PCR, there is easy and simple to handle, use safety, advantage with low cost, be adapted at applying during clinical gene detects.
According to a preferred embodiment of the invention, reagent constituents is as follows: nucleic acid releasing agent, short releasing agent, enzyme mixation, HBV-PCR reaction solution, interior mark, negative quality control product, positive quality control product, qualitative reference product A ~ D; HBV micro-nucleic acid releasing agent contains KCl, the 0.5 ~ 20%TritonX-100 of 5 ~ 500mM, the Proteinase K of 1 ~ 100mg/ml, the NaOH of 1 ~ 20mM; Short releasing agent contains the DMSO of pH value 5 ~ 8, the BSA of final concentration 0.5 ~ 10%; Enzyme mixation contains Taq DNA polymerase, uridylic-N glycosylase; HBV-PCR reaction solution contains HBV conservative region primed probe, interior mark probe, MgCl
2, deoxyribonucleotide.
HBV upstream primer sequence: 5 '-AGAGTCTAGACTCGTGGTGGAC-3 '.
HBV downstream primer sequence: 5 '-GAAAAAGTTRCATGGTGCTGGTG-3 '.
HBV fluorescent probe thing sequence and fluorescein-labelled thing:
FAM--5′-ACCAATTTATGCCTACAGCCTARTACAAAG-3′BHQ-1。
Inside put on trip primer sequence: 5 '-AATTGGTCAACATGTGAAAGC-3 '.
Interior mark downstream primer sequence: 5 '-GAATGTGGCCAAGGTTCCGTCATTTGG-3 '.
Interior mark fluorescent probe thing sequence and fluorescein-labelled thing:
CY5--5′-AATCTTCTAATTACTGTATATGGAAG-3′BHQ-2。
In the preferred case, micro-nucleic acid releasing agent compound method is: in sterilizing deionized water, add KCl final concentration is 5 ~ 500mM, the Proteinase K of final concentration 0.5 ~ 20%TritonX-100, final concentration 1 ~ 100mg/ml, the NaOH of final concentration 1 ~ 20mM; Short releasing agent: add final concentration 10% in sterilizing deionized water, the DMSO of pH value 5 ~ 8, the BSA of final concentration 0.5 ~ 10%; Quality or volume percent are to extract reagent volume for Calculation Basis; It is frozen that need-20 DEG C preserved for a long time by the reagent prepared.
Concrete, in a preferred embodiment, the method for the invention comprises the steps:
1) get micro-nucleic acid releasing agent 5 μ l and add isopyknic serum or plasma sample, with pipettor piping and druming mixing 10 times;
2) often pipe adds 30 μ l encapsulants, and build pipe lid and be placed in regular-PCR instrument and react, design parameter is as follows:
The first step: 95 DEG C, 10 minutes; Second step: 4 DEG C, 2 minutes;
3) taken out from regular-PCR instrument by the PCR reaction tubes through cracking process, carefully open pipe lid, often pipe adds 2.5 μ l and urgees releasing agent and repeatedly blow and beat mixing 10 times with pipettor;
4) often pipe adds the PCR-mix of 35 μ l, brief centrifugation, is placed in quantitative real time PCR Instrument amplification.
According to a preferred embodiment of the invention, the minimum detectability of this test kit can reach 30IU/ml.
Micro-nucleic acid releasing agent of the present invention adopts protein denaturant, HBVDNA can be discharged by rapid damage HBV virus particle shell protein structure, combine with traditional thermo-cracking mode and synchronously carry out, more effective guarantee sample amplifying nucleic acid lysis efficiency, micro-nucleic acid releasing agent is while discharging nucleic acid completely, also have in closed sample, to pcr amplification, there is the protein of interference effect, the function of the various factors material such as medicine and haemolysis, avoid the false negative of testing process; And containing the component suppressing nuclease in micro-nucleic acid releasing agent, can not have an impact to PCR subsequent experimental.
Accompanying drawing explanation
Fig. 1 is qualitative reference product A ~ D amplification curve.
Fig. 2 is national ginseng dish sensitivity reference material L0 ~ L5 amplification curve.
Fig. 3 is national ginseng dish sensitivity reference material L6 amplification curve.
Embodiment
Reference once embodiment can be described in further detail the present invention; But following examples are only illustrations, and the present invention is not limited to these embodiments.
Embodiment 1: country ginseng dish sensitivity reference material L0 ~ L6 is extracted by the inventive method.
Each for test kit component is recovered room temperature, concussion, brief centrifugation; According to the quantity of sample to be tested, (HBV-PCR reaction solution 33 μ l/ person-portion+interior mark 1 μ l/ person-portion+enzyme mixation 1 μ l/ person-portion) gets the HBV-PCR reaction solution of respective amount, interior mark and enzyme mixation in proportion, is fully mixed into PCR-mix, for subsequent use after brief centrifugation.
Get national ginseng dish sensitivity reference material L0 ~ L6 for subsequent use.
Prepare respective numbers 200 μ l nuclease free PCR reaction tubes, often pipe adds 5 μ l micro-nucleic acid releasing agents, adds each 5 μ l of national ginseng dish sensitivity reference material L0 ~ L6 different concns sample, repeatedly blows and beats 10 times with pipettor.
Add 30 μ l encapsulants, build pipe lid and be placed in the cracking of regular-PCR instrument, design parameter is as follows:
The first step: 95 DEG C, 10 minutes; Second step: 4 DEG C, 2 minutes.
Taken out from regular-PCR instrument by PCR reaction tubes through cracking process, carefully open pipe lid, often pipe adds 2.5 μ l and urgees releasing agent and repeatedly blow and beat mixing 10 times with pipettor.
Often pipe adds PCR-mix(33 μ l reaction solution, 1 μ l enzyme mixation, the interior mark of 1 μ l of 35 μ l), brief centrifugation, is placed in quantitative real time PCR Instrument amplification;
The first step: UNG enzyme reaction, 50 DEG C 2 minutes, 1 circulation;
Second step: denaturation, 95 DEG C 5 minutes, 1 circulation;
3rd step: comprise sex change, annealing, extension and fluorescent collecting; Sex change, 95 DEG C 15 seconds, 45 circulations;
Annealing, extend and fluorescent collecting, 60 DEG C 45 seconds, 45 circulations;
4th step: instrument cool, 25 DEG C 10 seconds, 1 circulation.
Interpretation of result condition sets
After reaction terminates, adjust baseline and threshold value manually or automatically according to PCR instrument specification sheets and fluorescence curve; During manual setting, the starting point (Start) of baseline (Baseline) is set between 3 ~ 8, terminating point (Stop) is set between 12 ~ 15, threshold line (Threshold) be usually set in 1000 ~ 5000 between (being determined on a case-by-case basis).
The explanation of assay
1) if target gene FAM passage does not occur standard amplification curve, mark HEX(VIC simultaneously) there is standard amplification curve, and between 16≤Ct≤35, be judged to HBVDNA and detect negative in passage;
2) for measured value 300 ~ 2.5 × 10
9sample between IU/ml, there is standard amplification curve in target gene FAM passage, mark HEX(VIC simultaneously) there is standard amplification curve, and between 16≤Ct≤35, then report corresponding measurement result in passage;
3) for measured value > 2.5 × 10
9the sample of IU/ml, there is standard amplification curve in target gene FAM passage, simultaneously, standard amplification curve appears in mark HEX passage, and between 16≤Ct≤35, report indicates > 2.5 × 10
9iU/ml; If need accurate quantification, can according to result by Sample Dilution to 2.5 × 10
9below IU/ml is repetition measurement again, and according to following formulae discovery concentration of specimens, result indicates can report numerical value;
4) for the sample of measured value at 30 ~ 300IU/ml, there is standard amplification curve in target gene FAM passage, mark HEX(VIC simultaneously) there is standard amplification curve in passage, and 16≤Ct≤35, show that virus load is low, measured value is only for reference;
5) for the sample of measured value < 30IU/ml, interior mark HEX(VIC) there is standard amplification curve in passage, and between 16≤Ct≤35, then report that HBV-DNA content is lower than test kit Monitoring lower-cut, should judge in conjunction with other clinical diagnosis indexs;
6) if interior mark HEX(VIC) channel C t value > 35 or without amplification curve occur, then this pattern detection result is invalid, should search and get rid of reason, and carries out duplicate detection to this sample.
Result judges
1) as can be seen from accompanying drawing 1, qualitative reference product A ~ D all has standard amplification curve; And typical curve relation conefficient | r| >=0.980, inside indicates standard amplification curve, and 16≤Ct≤35;
2) can find out that country dish sensitivity reference material L0-L5 respectively extracts 3 times from accompanying drawing 2, all have standard amplification curve, and in indicate standard amplification curve, and 16≤Ct≤35;
3) can find out that minimum detectability L68 time extracts amplification and all has amplification curve from accompanying drawing 3, and in indicate standard amplification curve, and 16≤Ct≤35.
Embodiment 2: the clinical application that this test kit is provided.
Test kit of the present invention is used to complete 520 routine serum, 50 routine same person-portion plasma samples detections altogether at 3 institution of clinical trials, its positives 414 examples, negative 106 examples, with granted similar test kit reference, sample 414 example of reference reagent test positive, negative sample 106 example; The positive coincidence rate of examination reagent to HBV nucleic acid qualitative detection is 100%, and negative match-rate is 100%, and total coincidence rate is 100%, Kappa is 1; The invention provides a kind of hbv nucleic acid (DNA) immue quantitative detection reagent box (fluorescent PCR " tube method ") and contrast agents detected result consistence better, for the diagnosis of patient, treatment provide clinical secondary proof; As detected the existence of HBV virus in sample, suggestion is taken the necessary measures immediately.
Precious auspicious source biotechnology (Beijing) company limited of <110>
<120> hbv nucleic acid (DNA) immue quantitative detection reagent box (fluorescent PCR " tube method ")
<160>8
<170>PatentInversion3.3
<210>1
<211>22
<212>DNA
<213> artificial sequence
<220>
<223>HBV upstream primer sequence
<400>1
AGAGTCTAGACTCGTGGTGGAC22
<210>2
<211>23
<212>DNA
<213> artificial sequence
<220>
<223>HBV downstream primer sequence
<400>2
GAAAAAGTTRCATGGTGCTGGTG23
<210>3
<211>30
<212>DNA
<213> artificial sequence
<220>
<223>HBV fluorescent probe sequence
<400>3
ACCAATTTATGCCTACAGCCTARTACAAAG30
<210>4
<211>21
<212>DNA
<213> artificial sequence
<220>
Trip primer sequence is put in <223>
<400>4
AATTGGTCAACATGTGAAAGC21
<210>5
<211>27
<212>DNA
<213> artificial sequence
<220>
Mark downstream primer sequence in <223>
<400>5
GAATGTGGCCAAGGTTCCGTCATTTGG27
<210>6
<211>26
<212>DNA
<213> artificial sequence
<220>
Mark fluorescent probe sequence in <223>
<400>6
aatcttctaattactgtatatggaag26
<210>7
<211>256
<212>DNA
<213> artificial sequence
<220>
<223>HBV quality control product sequence
<400>7
atggagaacatcacatcaggattcctaggacccctgctcgtgttacaggcggggtttttc60
ttgttgacaagaatcctcacaataccgcagagtctagactcgtggtggacttctctcaat120
tttctagggggaactaccgtgtgtcttggccaaaattcgcagtccccaacctccaatcac180
tcaccaacctcctgtcctccaacttgtcctggttatcgctggatgtgtctgcggcgtttt240
atcatcttcctcttca256
<210>8
<211>209
<212>DNA
<213> artificial sequence
<220>
Mark plasmid sequence in <223>
<400>8
aaaatcttttcttacaagggaagtccccaattggtcaacatgtgaaagcacgtgtcatgt60
tcttacttttgtttgggtaatcttctaattactgtatatggaagatgtgaatgaagtttt120
ggtcctgaatgtggccaaggttccgtcatttggagatacgaaatcaaatctcctttaaga180
ttttgtttttataatgtgttcttccatcc209
Claims (4)
1. hbv nucleic acid (DNA) immue quantitative detection reagent box (fluorescent PCR " tube method "), utilize for a pair Auele Specific Primer of HBV nucleic acid conserved regions design, a specificity fluorescent probe, be equipped with PCR reaction solution, on quantitative real time PCR Instrument, application real-time fluorescence quantitative PCR detection technique, realized the detection by quantitative of hepatitis B virus DNA by the change of fluorescent signal, there is easy and simple to handle, use safety, advantage with low cost.
2. this test kit contains nucleic acid releasing agent, short releasing agent, encapsulant, enzyme mixation, HBV-PCR reaction solution, interior mark, negative quality control product, positive quality control product, qualitative reference product A ~ D; Nucleic acid releasing agent contains KCl, TritonX-100, Proteinase K, NaOH; Short releasing agent contains DMSO, BSA; Enzyme mixation contains Taq DNA polymerase, uridylic-N glycosylase; HBV-PCR reaction solution contains HBV conservative region primed probe, interior mark probe, MgCl
2, deoxyribonucleotide.
3. this technology adopts micro-nucleic acid releasing agent (MNRR), HBVDNA nucleic acid extraction and amplification can be carried out in a pipe, the micro-nucleic acid releasing agent getting 5 μ l adds equal-volume serum or plasma sample, repeatedly blow and beat mixing 10 times, be placed in PCR instrument 94 DEG C of cracking 10 minutes, then be down to rapidly 4 DEG C, carefully open pipe lid and add 2.5 μ l and urge releasing agent and mix, add 35 μ lPCR reaction solutions and carry out fluorescent quantitation detection.
4. examine the standard substance A ~ D of provirus serum standard panel as quantitative fluorescent PCR of four concentration gradients in institute HBV National reference in adopting, add yin and yang attribute contrast and sample to be tested simultaneous extraction, amplification, realize from sample extraction to amplification complete monitoring.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510452653.8A CN105063235A (en) | 2015-07-28 | 2015-07-28 | Kit for quickly and quantitatively detecting hepatitis b virus nucleic acid DNA (deoxyribonucleic acid) and using method of kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510452653.8A CN105063235A (en) | 2015-07-28 | 2015-07-28 | Kit for quickly and quantitatively detecting hepatitis b virus nucleic acid DNA (deoxyribonucleic acid) and using method of kit |
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|---|---|
| CN105063235A true CN105063235A (en) | 2015-11-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510452653.8A Pending CN105063235A (en) | 2015-07-28 | 2015-07-28 | Kit for quickly and quantitatively detecting hepatitis b virus nucleic acid DNA (deoxyribonucleic acid) and using method of kit |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN112921075A (en) * | 2021-03-31 | 2021-06-08 | 武汉友芝友医疗科技股份有限公司 | Hand-taking-free reagent for blood sample fluorescence PCR and application thereof |
| CN113025686A (en) * | 2021-03-12 | 2021-06-25 | 江苏吉诺思美精准医学科技有限公司 | Viral RNA releasing agent, kit and application thereof |
| CN114250325A (en) * | 2021-12-31 | 2022-03-29 | 广西壮族自治区疾病预防控制中心 | Primer, probe, kit and method for rapidly detecting 9 genotypes of hepatitis B virus |
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| CN101158634A (en) * | 2007-09-04 | 2008-04-09 | 扬子江药业集团北京海燕药业有限公司 | Hepatitis B virus (HBV) fluorescent quantificationally PCR detecting kit |
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| CN1621529A (en) * | 2003-11-27 | 2005-06-01 | 王海滨 | Micro-nucleic acid releasing agent |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN107022651B (en) * | 2017-06-02 | 2021-01-29 | 辽宁润基生物科技有限公司 | Kit for rapidly detecting hepatitis C virus nucleic acid and detection method thereof |
| CN113025686A (en) * | 2021-03-12 | 2021-06-25 | 江苏吉诺思美精准医学科技有限公司 | Viral RNA releasing agent, kit and application thereof |
| CN113025686B (en) * | 2021-03-12 | 2023-07-28 | 江苏吉诺思美精准医学科技有限公司 | Virus RNA releasing agent, kit and application thereof |
| CN112921075A (en) * | 2021-03-31 | 2021-06-08 | 武汉友芝友医疗科技股份有限公司 | Hand-taking-free reagent for blood sample fluorescence PCR and application thereof |
| CN114250325A (en) * | 2021-12-31 | 2022-03-29 | 广西壮族自治区疾病预防控制中心 | Primer, probe, kit and method for rapidly detecting 9 genotypes of hepatitis B virus |
| CN115873993A (en) * | 2021-12-31 | 2023-03-31 | 广西壮族自治区疾病预防控制中心 | Kit for detecting 9 genotypes of hepatitis B virus and application thereof |
| CN115873993B (en) * | 2021-12-31 | 2023-07-21 | 广西壮族自治区疾病预防控制中心 | Kit for detecting 9 genotypes of hepatitis B virus and application thereof |
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