CN109517876A - A kind of HPV parting detection of nucleic acids blood sample processing method - Google Patents
A kind of HPV parting detection of nucleic acids blood sample processing method Download PDFInfo
- Publication number
- CN109517876A CN109517876A CN201811267085.4A CN201811267085A CN109517876A CN 109517876 A CN109517876 A CN 109517876A CN 201811267085 A CN201811267085 A CN 201811267085A CN 109517876 A CN109517876 A CN 109517876A
- Authority
- CN
- China
- Prior art keywords
- sample
- cell
- exfoliated cell
- processing method
- class
- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of HPV parting detection of nucleic acids blood sample processing methods, belong to vitro detection technical field, solve the influence that will receive haemocyte when detecting containing blood sample, the technical issues of inhibiting the extraction of HPVDNA, lead to result false negative.The present invention classifies according to the how many pairs of cervical exfoliated cell samples containing red blood cell, and exfoliated cell preservative fluid is added and is pre-processed, under the premise of keeping cervical exfoliated cell form and complete nucleic acid, red blood cell is set sufficiently to suspend and disperse, the red blood cell in sample is washed away, the signal value of the Globin of sample is greater than 150, guarantees experiment success rate, the generation for avoiding false negative result improves the accuracy of testing result.
Description
Technical field
The present invention relates to vitro detection technical field more particularly to a kind of HPV parting detection of nucleic acids blood sample processing sides
Method.
Background technique
Cervical carcinoma is located at the second of China's female malignant morbidity and the death rate, has 465000 new hair cancer to suffer from every year
Person.Research finds the detectable HPV in 90% or so cervical cancer patient, and HPV is human papilloma virus (human
Papilloma virus), be the pathogen for leading to cervical carcinoma, HPV points for 27 kinds of hypotypes (6,11,16,18,26,31,33,35,
39,40,42,43,44,45,51,52,53,55,56,58,59,61,66,68,81,82,83), wherein HPV-16 and HPV-18
It is high-risk-type, easily causes the invasive tumour of vagina lower section, therefore, cervical carcinoma can be prevented well by carrying out the detection of HPV parting.
In the prior art, it generallys use streaming fluorescent hybridization technology and detects 27 kinds of HPV hypotypes, if there is any HPV hypotype
The signal of probe is greater than 150, that is, can determine whether that the corresponding hypotype of the probe is positive, be otherwise feminine gender.However, working as cervical exfoliated cell
When sample state contains blood, this influence that will receive haemocyte when detecting containing blood sample inhibits the extraction of HPV DNA, leads
Result false negative is caused, gently then the failure of an experiment, patient need resampling, cause the waste of personnel and reagent cost, increase patient and adopt
The pain of sample, erroneous judgement that is heavy then causing result are delayed patient's best occasion for the treatment.
Summary of the invention
The present invention in view of the above technical problems, provides a kind of HPV parting detection of nucleic acids blood sample processing method, reduces red
Influence of the cell to HPV parting testing result improves the success rate and accuracy rate of experiment.
To achieve the goals above, the invention provides the following technical scheme:
A kind of HPV parting detection of nucleic acids blood sample processing method, comprising the following steps:
(1) cervical exfoliated cell is acquired, cervical exfoliated cell sample is classified:
Erythrocyte number is less than 0.01 × 1012A/every liter of sample is A class sample；
Erythrocyte number is in (0.01~0.1) × 1012A/every liter of sample is B class sample；
Erythrocyte number is in (0.1~3) × 1012A/every liter of sample is C class sample；
Erythrocyte number is greater than 3 × 1012A/every liter of sample is D class sample；
(2) it after sample classification, is pre-processed according to how much addition exfoliated cell preservative fluids of red blood cell, processing method:
A class sample is directly used in experiment；
B class sample is used to test after mixing with exfoliated cell preservative fluid, the B class sample and the exfoliated cell preservative fluid
Volume ratio be 550~650:350~450；
C class sample is used to test after mixing with exfoliated cell preservative fluid, the C class sample and the exfoliated cell preservative fluid
Volume ratio be 350~450:550~650；
D class sample is used to test after mixing with exfoliated cell preservative fluid, the D class sample and the exfoliated cell preservative fluid
Volume ratio be 150~250:750~850.
Preferably, the method for counting of red blood cell are as follows: sample to be tested concussion is diluted after mixing with isotonic diluent liquid, and it is thin to instill blood
Born of the same parents' tally, counting red corpuscles quantity, conversion obtain the erythrocyte number contained in sample under the microscope.
Preferably, the exfoliated cell preservative fluid is the acetate buffer containing isopropanol, and pH value is 5~6.
Preferably, the exfoliated cell preservative fluid is that the cast-off cells of Shanghai Toujing Life Sci. & Tech. Co., Ltd.'s production are protected
Liquid storage.
Above-mentioned HPV parting detection of nucleic acids blood sample processing method, pretreated sample is carried out as follows
Detection:
S1, the EP containing cervical exfoliated cell sample is taken to manage, 13200rpm 5~10min of high speed centrifugation inhales and abandons supernatant；
S2,200 μ l cast-off cells nucleic acid releasing agents are added, sufficiently oscillation mixes, and places 100 DEG C of 15~20min of dry bath；
S3,13200rpm 10~15min of high speed centrifugation take 5 μ l supernatants to be added in amplified reaction pipe, and upper machine expands 1.5h；
S4, it draws in 3 μ l supernatants, 22 μ l microballoon hybridization solutions of addition, hybridizes 40min；
S5, be added 75 μ l streptavidin-phycoerythrin solution, 48 DEG C of 15~20min of incubation, using flow type analyzer into
Row detection.
Cast-off cells nucleic acid releasing agent described in preferably step S2 is Shanghai Toujing Life Sci. & Tech. Co., Ltd.'s production
Cast-off cells nucleic acid releasing agent.
Compared with prior art, the invention has the benefit that
HPV parting detection of nucleic acids blood sample processing method of the invention, falls off carefully according to how much uterine neck containing red blood cell
Born of the same parents' sample is classified, and exfoliated cell preservative fluid is added and is pre-processed, and is keeping cervical exfoliated cell form and nucleic acid complete
Under the premise of whole, red blood cell is made sufficiently to suspend and disperse, wash away the red blood cell in sample, the signal value of the Globin of sample is greater than
150, guarantee experiment success rate, avoids the generation of false negative result, improve the accuracy of testing result.
Specific embodiment
In order to make those skilled in the art more fully understand technical solution of the present invention, below in conjunction with embodiment to this
Invention is further detailed.Exfoliated cell preservative fluid and cast-off cells nucleic acid releasing agent of the present invention is all made of
The product of Shanghai Toujing Life Sci. & Tech. Co., Ltd.'s production.
Embodiment 1
16 four class blood sample HPV samples of distribution are taken, how much are classified according to containing haemocyte, after sample classification, according to red
The saturating scape cell-preservation liquid of how much additions of cell is pre-processed, processing method: A class cervical exfoliated cell sample takes 1000 μ l to add
Enter and be directly used in experiment in 1.5mlEP pipe, B class cervical exfoliated cell sample, which takes in 650 μ l addition 1.5mlEP pipe and is added, to fall off
350 μ l of cell-preservation liquid is used to test after mixing, and C class cervical exfoliated cell sample takes 450 μ l to be added in 1.5mlEP pipe and is added
550 μ l of exfoliated cell preservative fluid is used to test after mixing, and D class cervical exfoliated cell sample takes 250 μ l to be added in 1.5mlEP pipe simultaneously
It is added after 750 μ l of exfoliated cell preservative fluid is mixed for testing.
Four class blood sample EP are taken to manage, 13200rpm high speed centrifugation 5min inhales and abandons supernatant, and 200 μ l cast-off cells nucleic acid are added and release
Agent is put, sufficiently oscillation mixes, and places 100 DEG C of dry bath 15min, 13200rpm high speed centrifugation 10min, takes 5 μ l supernatants that amplification is added
In reaction tube, upper machine expands 1h30min, draws 3 μ l supernatants and is added in 22 μ l microballoon hybridization solutions, hybridize 40min, 75 μ l chains are added
Mould Avidin-phycoerythrin solution, 48 DEG C of incubation 15min, Luminex200 flow type analyzer are detected.
Interpretation of result: directly obtaining each sample by saturating scape HPV result calculating analysis software as a result, the Globin of sample
Signal value is greater than 150, entire Success in Experiment can determine whether the probe if there is the signal of any HPV hypotype probe is greater than 150
Corresponding hypotype is positive, is otherwise feminine gender.
Embodiment 2
In addition 16 four class blood sample HPV samples of distribution are chosen, how much are classified according to containing haemocyte, after sample classification,
Pre-processed according to the saturating scape cell-preservation liquid of how much additions of red blood cell, processing method: A class cervical exfoliated cell sample takes
1000 μ l are added in 1.5mlEP pipe for testing, and B class cervical exfoliated cell sample takes 600 μ l to be added in 1.5mlEP pipe and is added
400 μ l of exfoliated cell preservative fluid is used to test after mixing, and C class cervical exfoliated cell sample takes 400 μ l to be added in 1.5mlEP pipe simultaneously
It is added after 600 μ l of exfoliated cell preservative fluid is mixed for testing, D class cervical exfoliated cell sample takes 200 μ l that 1.5mlEP pipe is added
It is interior and be added after 800 μ l of exfoliated cell preservative fluid is mixed for testing.
The above four classes blood sample EP is taken to manage, 13200rpm high speed centrifugation 10min inhales and abandons supernatant, and 200 μ l cast-off cells cores are added
Sour releasing agent, sufficiently oscillation mix, and place 100 DEG C of dry bath 20min,
13200rpm high speed centrifugation 15min takes 5 μ l supernatants to be added in amplified reaction pipe, and upper machine expands 1h30min, draws 3
μ l supernatant is added in 22 μ l microballoon hybridization solutions, hybridizes 40min, and 75 μ l streptavidin-phycoerythrin solution, 48 DEG C of incubations are added
20min, Luminex200 flow type analyzer are detected.
Interpretation of result: directly obtaining each sample by saturating scape HPV result calculating analysis software as a result, the Globin of sample
Signal value is greater than 150, entire Success in Experiment can determine whether the probe if there is the signal of any HPV hypotype probe is greater than 150
Corresponding hypotype is positive, is otherwise feminine gender.
Embodiment 3
16 four class blood sample HPV samples of distribution are still further chosen, how much are classified according to containing haemocyte, sample classification
Afterwards, it is pre-processed according to the saturating scape cell-preservation liquid of how much additions of red blood cell, processing method: A class cervical exfoliated cell sample
1000 μ l are taken to be added in 1.5mlEP pipe for testing, B class cervical exfoliated cell sample takes 550 μ l to be added in 1.5mlEP pipe and adds
Enter after 450 μ l of exfoliated cell preservative fluid is mixed for testing, C class cervical exfoliated cell sample takes 350 μ l to be added in 1.5mlEP pipe
And be added after 550 μ l of exfoliated cell preservative fluid is mixed for testing, D class cervical exfoliated cell sample takes 150 μ l that 1.5mlEP is added
In pipe and it is added after 750 μ l of exfoliated cell preservative fluid is mixed for testing.
The above four classes blood sample EP is taken to manage, 13200rpm high speed centrifugation 10min inhales and abandons supernatant, and 200 μ l cast-off cells cores are added
Sour releasing agent, sufficiently oscillation mix, and place 100 DEG C of dry bath 20min,
13200rpm high speed centrifugation 15min takes 5 μ l supernatants to be added in amplified reaction pipe, and upper machine expands 1h30min, draws 3
μ l supernatant is added in 22 μ l microballoon hybridization solutions, hybridizes 40min, and 75 μ l streptavidin-phycoerythrin solution, 48 DEG C of incubations are added
20min, Luminex200 flow type analyzer are detected.
Interpretation of result: directly obtaining each sample by saturating scape HPV result calculating analysis software as a result, the Globin of sample
Signal value is greater than 150, entire Success in Experiment can determine whether the probe if there is the signal of any HPV hypotype probe is greater than 150
Corresponding hypotype is positive, is otherwise feminine gender.
Comparative example 1
In addition 16 four class blood sample HPV samples of distribution are taken, 1.5mlEP is added in each 1000 μ l cervical exfoliated cell samples of drawing
In pipe, 13200rpm high speed centrifugation 5min inhales and abandons supernatant, and 200 μ l cast-off cells nucleic acid releasing agents are added, and sufficiently oscillation mixes,
100 DEG C of dry bath 15min, 13200rpm high speed centrifugation 10min are placed, 5 μ l supernatants is taken to be added in amplified reaction pipe, upper machine amplification
1h30min draws 3 μ l supernatants and is added in 22 μ l microballoon hybridization solutions, hybridize 40min, 75 μ l streptavidin-phycoerythrin are added
Solution, 48 DEG C of incubation 15min, Luminex200 flow type analyzer are detected.
Interpretation of result: directly obtaining each sample by saturating scape HPV result calculating analysis software as a result, the sample containing blood
The signal value of Globin is all less than 150, entire the failure of an experiment.
To sum up, blood sample carries out detection test, the signal value of sample Globin after processing method processing of the invention
Greater than 150, Success in Experiment, and the signal value of untreated blood sample experimental result sample Globin is less than 150, the failure of an experiment.
It being obtained by contrast and experiment, experiment success rate that treated is high, and it is easy to operate, it avoids causing reality because sample contains blood
Inhibition is tested, the pain of patient's resampling is reduced, avoids repeating test reagent waste, save the cost ensure that testing result
Accuracy.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation
Example documented by technical solution modify perhaps equivalent replacement of some of the technical features but these modification or
Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (6)
1. a kind of HPV parting detection of nucleic acids blood sample processing method, which comprises the following steps:
(1) cervical exfoliated cell is acquired, cervical exfoliated cell sample is classified:
Erythrocyte number is less than 0.01 × 1012A/every liter of sample is A class sample；
Erythrocyte number is in (0.01~0.1) × 1012A/every liter of sample is B class sample；
Erythrocyte number is in (0.1~3) × 1012A/every liter of sample is C class sample；
Erythrocyte number is greater than 3 × 1012A/every liter of sample is D class sample；
(2) it after sample classification, is pre-processed according to how much addition exfoliated cell preservative fluids of red blood cell, processing method:
A class sample is directly used in experiment；
B class sample is used to test after mixing with exfoliated cell preservative fluid, the body of the B class sample and the exfoliated cell preservative fluid
Product is than being 550~650:350~450；
C class sample is used to test after mixing with exfoliated cell preservative fluid, the body of the C class sample and the exfoliated cell preservative fluid
Product is than being 350~450:550~650；
D class sample is used to test after mixing with exfoliated cell preservative fluid, the body of the D class sample and the exfoliated cell preservative fluid
Product is than being 150~250:750~850.
2. HPV parting detection of nucleic acids blood sample processing method according to claim 1, the method for counting of red blood cell are as follows:
Sample to be tested concussion is diluted after mixing with isotonic diluent liquid, instills blood cell counting plate, under the microscope counting red corpuscles quantity,
Conversion obtains the erythrocyte number contained in sample.
3. HPV parting detection of nucleic acids blood sample processing method according to claim 1, which is characterized in that described is de-
Falling cell-preservation liquid is the acetate buffer containing isopropanol, and pH value is 5~6.
4. HPV parting detection of nucleic acids blood sample processing method according to claim 3, which is characterized in that described is de-
Fall the exfoliated cell preservative fluid that cell-preservation liquid is Shanghai Toujing Life Sci. & Tech. Co., Ltd.'s production.
5. HPV parting detection of nucleic acids blood sample processing method according to any one of claims 1 to 4, which is characterized in that
Pretreated sample is detected as follows:
S1, the EP containing cervical exfoliated cell sample is taken to manage, 13200rpm 5~10min of high speed centrifugation inhales and abandons supernatant；
S2,200 μ l cast-off cells nucleic acid releasing agents are added, sufficiently oscillation mixes, and places 100 DEG C of 15~20min of dry bath；
S3,13200rpm 10~15min of high speed centrifugation take 5 μ l supernatants to be added in amplified reaction pipe, and upper machine expands 1.5h；
S4, it draws in 3 μ l supernatants, 22 μ l microballoon hybridization solutions of addition, hybridizes 40min；
S5,75 μ l streptavidin-phycoerythrin solution are added, 48 DEG C of 15~20min of incubation are examined using flow type analyzer
It surveys.
6. HPV parting detection of nucleic acids blood sample processing method according to claim 5, which is characterized in that step S2 institute
The cast-off cells nucleic acid releasing agent stated is the cast-off cells nucleic acid releasing agent of Shanghai Toujing Life Sci. & Tech. Co., Ltd.'s production.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811267085.4A CN109517876A (en) | 2018-10-29 | 2018-10-29 | A kind of HPV parting detection of nucleic acids blood sample processing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811267085.4A CN109517876A (en) | 2018-10-29 | 2018-10-29 | A kind of HPV parting detection of nucleic acids blood sample processing method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109517876A true CN109517876A (en) | 2019-03-26 |
Family
ID=65773198
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811267085.4A Pending CN109517876A (en) | 2018-10-29 | 2018-10-29 | A kind of HPV parting detection of nucleic acids blood sample processing method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109517876A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363011A (en) * | 2008-09-17 | 2009-02-11 | 上海艾迪康临床检验中心有限公司 | Cervical exfoliated cell preservative fluid |
| US20130143751A1 (en) * | 2010-01-19 | 2013-06-06 | Alberto Severini | Set of Probes for the Detection and Typing of 46 Human Papillomavirus Mucosal Types |
| CN108402033A (en) * | 2018-04-10 | 2018-08-17 | 南京福怡科技发展股份有限公司 | One kind having preprocessing function cervical exfoliated cell preservative fluid and application |
-
2018
- 2018-10-29 CN CN201811267085.4A patent/CN109517876A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363011A (en) * | 2008-09-17 | 2009-02-11 | 上海艾迪康临床检验中心有限公司 | Cervical exfoliated cell preservative fluid |
| US20130143751A1 (en) * | 2010-01-19 | 2013-06-06 | Alberto Severini | Set of Probes for the Detection and Typing of 46 Human Papillomavirus Mucosal Types |
| CN108402033A (en) * | 2018-04-10 | 2018-08-17 | 南京福怡科技发展股份有限公司 | One kind having preprocessing function cervical exfoliated cell preservative fluid and application |
Non-Patent Citations (1)
| Title |
|---|
| 熊玉娟等: "液态芯片检测妇女HPV感染及基因亚型分布", 《中华临床医师杂志(电子版》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103509877B (en) | A kind of PCR kit for fluorescence quantitative for detecting PRV and application thereof | |
| CN102268488B (en) | Fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for detecting bovine viral diarrhea virus and application of kit | |
| CN107328944B (en) | A kind of blood clotting and hemagglutination-inhibition test screening technique | |
| CN101294223B (en) | Primer and probe sequence for testing type II pig circular ring virus nucleotide fragment | |
| CN108048589A (en) | The Real-time PCR specific primers and probe and detection kit and detection method of ox two-pressure humidity generator | |
| CN105403712A (en) | High performance detection kit for human urine alpha 1 acidoglycoprotein | |
| CN101963612A (en) | Detection kit for sperms and check method thereof | |
| CN104777303B (en) | Basophilic granulocyte activation and degranulation identification method | |
| CN205067292U (en) | Circulation tumor cells detect reagent box | |
| CN109517876A (en) | A kind of HPV parting detection of nucleic acids blood sample processing method | |
| CN105925703A (en) | Method for screening miRNA markers in PB (peripheral blood) of kidney cancer and kidney cancer diagnosis marker miR-210 | |
| CN107525920B (en) | Poly ion liquid magnetic nanocomposites and its application to trace enriching specificity of circulating tumor cell and detection | |
| CN101434993B (en) | Liquid phase chip system for detecting cornea common pathomycete strain and detecting method thereof | |
| CN107976354A (en) | A kind of reagent for hemocyte analyzers | |
| CN101838709A (en) | Method for performing rapid gene typing on trace human papilloma virus (HPV) | |
| CN106319028A (en) | Kit for detecting mycoplasma urealytium and human mycoplasma | |
| CN103468829A (en) | Hepatitis B virus nucleic acid detection kit | |
| CN103215389B (en) | Porcine reproductive and respiratory syndrome and porcine Japanese B encephalitis dual one-step RT-PCR (Reverse Transcription-Polymerase Chain Reaction) diagnosis kit | |
| CN104073570B (en) | For the identification of the primer pair of human adenovirus 55 type, primed probe composition and their application | |
| CN106771134B (en) | A kind of carp herpesvirusⅡtype sandwich ELISA detection kit and detection method | |
| CN101149384A (en) | Device for detecting avian influenza virus antigen and detection method | |
| CN104232793A (en) | Gene chip and kit for detecting foot-and-mouth disease virus and swine fever virus | |
| CN103849683A (en) | Urine detection kit for prostate cancer and application thereof | |
| CN101294224B (en) | Primer and probe sequence for testing pig parvoviral nucleotide fragment | |
| CN104480221B (en) | A kind of dengue fever virus detection and genotyping method of fast high-flux |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190326 |