CN109457050A - Detect primer, probe, kit and the detection method of hbv nucleic acid - Google Patents
Detect primer, probe, kit and the detection method of hbv nucleic acid Download PDFInfo
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- CN109457050A CN109457050A CN201811548294.6A CN201811548294A CN109457050A CN 109457050 A CN109457050 A CN 109457050A CN 201811548294 A CN201811548294 A CN 201811548294A CN 109457050 A CN109457050 A CN 109457050A
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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
- C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
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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
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a kind of primer, probe, kit and detection methods for detecting hbv nucleic acid.The primer pair of the detection hbv nucleic acid includes: the first primer and the second primer, and sequence is respectively as shown in SEQ ID NO.1, SEQ ID NO.2.The sequence of the probe is as shown in SEQ ID NO.3.The kit includes aforementioned primer pair and probe.The detection method of the hbv nucleic acid includes: to mix sample of nucleic acid to be detected with PCR reaction solution using sample processing device, and wrap up the mixed liquor to be formed by drop formation liquid, and being formed includes single nucleic acid or the drop not comprising nucleic acid；PCR amplification is carried out to the drop using the kit, pcr amplification product is detected.Kit Monitoring lower-cut of the invention is low, high sensitivity, and specificity is good, and detection method of the invention is simple and quick, and detection efficiency is very high, at low cost.
Description
Technical field
The present invention relates to technical field of molecular biology to particularly relate to one more particularly, to nucleic acid field is used for
The primer, probe and corresponding reagent box of highly sensitive detection hepatitis type B virus (HBV) nucleic acid of kind, and detected using the kit
The method of hbv nucleic acid.
Background technique
Hepatitis type B virus (Hepatitis B Virus, HBV) belongs to Hepadnaviridae (hepadnaviridae),
It is one of the smallest DNA virus for the infection people being currently known, while is also one of the virus for being most difficult to cure.HBV can pass through
The approach such as blood, mother and baby, property and doctor source invade human body, cause liver inflammatory lesion and multiple organ injury, are to seriously threaten people
The epidemic virus of class health.HBV belongs to Class B in China and infects disease, only just up to 1,000,000 people of neopathy patient in 2017.
The patient of infection HBV virus leads to long-term persistent infection, it is chronic for may developing if failing to find treated as soon as possible in time
Hepatitis.If cirrhosis, terminal phase liver will finally occur for the Patients with Chronic HBV Infection of about 15%-40% without intervention appropriate
Disease or even liver cancer, especially liver cancer patient about 80%-90% have HBV infection background.HBV infection person should actively carry out antiviral control
It treats, and monitors the variation of its virus load.Therefore, the quantitative detection of HBV nucleic acid high sensitivity is to the clinical conditions rational use of medicines
It is of great significance with judging prognosis.
Currently, the common detection method of HBV includes the methods of virus protein antibody test, antigen detection, detection of nucleic acids.Disease
Malicious Antigen-antibody detection often selects enzyme-linked immunosorbent assay (ELISA), i.e., specific antigen or antibody is adsorbed in solid phase
On carrier, make its in sample to be tested corresponding antibodies or antigen binding, the then antigen or antibody of enzyme label, then plus substrate
Colour developing finally calculates the content of determined antigen or antibody according to the color depth.But the viral level for having just enter into body is very low, virus
Autoantigen content is often below the Monitoring lower-cut of ELISA method, and body needs can just be generated antibody after a period of time,
Blood antibody test is negative during this, and antigen-antibody is usually that can just be detected after 3-6 weeks after infection.In recent years with
The development of molecular biology, technique of gene detection is universal in each laboratory, especially fluorescent quantitative PCR technique, not only
There is good specificity, the sensitivity of detection can also be improved by expanding viral nucleic acid, shortens the window phase of viral diagnosis.
At present both at home and abroad there are many virus detection kit based on fluorescent quantitative PCR technique, but domestic detection
Kit quantification Monitoring lower-cut is higher, and sensitivity is poor.And the highly sensitive diagnostic kit price of external import is prohibitively expensive, mentions
High health care cost.
Therefore, at present there is an urgent need to develop a kind of Monitoring lower-cut is low, the detection hbv nucleic acid of high sensitivity
Kit and detection method, to meet the requirement of clinical application and checkout and diagnosis.
Summary of the invention
The main object of the present invention aiming at the above status, provide a kind of primer for detecting hbv nucleic acid,
Probe, to overcome deficiency in the prior art.
Another main purpose of the invention is to provide a kind of kit for detecting hbv nucleic acid.
Another object of the present invention also resides in the purposes for providing aforementioned agents box in detection hbv nucleic acid.
Another object of the present invention, which also resides in, provides a kind of new method for detecting hbv nucleic acid.
For realization aforementioned invention purpose, the technical solution adopted by the present invention includes:
The embodiment of the invention provides a kind of primer pairs for detecting hbv nucleic acid comprising:
The first primer, sequence is as shown in SEQ ID NO.1；
Second primer, sequence is as shown in SEQ ID NO.2.
The embodiment of the invention also provides a kind of probes for detecting hbv nucleic acid, and the sequence of the probe is such as
Shown in SEQ ID NO. 3.
The embodiment of the invention also provides a kind of kits for detecting hbv nucleic acid comprising at least one set is drawn
Object to and at least one probe, wherein one group of primer pair includes the first primer and the second primer, the sequence of the first primer is such as
Shown in SEQ ID NO.1, the sequence of second primer is as shown in SEQ ID NO.2, wherein a probe is spy above-mentioned
Needle.
The embodiment of the invention also provides kits above-mentioned in the product of preparation detection hbv nucleic acid
Purposes.
Further, include: using the method for the product testing hbv nucleic acid
Viruses of human hepatitis B's sample of nucleic acid to be detected is provided；
It using sample processing device, mixes the sample of nucleic acid to be detected with PCR reaction solution, and makes the mixing to be formed
Liquid is wrapped up by drop formation liquid, and being formed includes single nucleic acid or the drop not comprising nucleic acid；
The primer pair and probe for being included using the kit carry out PCR amplification to the drop；
Pcr amplification product is detected.
The embodiment of the invention also provides a kind of products comprising aforementioned agents box, and the products application is in hepatitis B
The detection method of malicious nucleic acid, the detection method include:
Viruses of human hepatitis B's sample of nucleic acid to be detected is provided；
It using sample processing device, mixes the sample of nucleic acid to be detected with PCR reaction solution, and makes the mixing to be formed
Liquid is wrapped up by drop formation liquid, and being formed includes single nucleic acid or the drop not comprising nucleic acid；
The primer pair and probe for being included using the kit carry out PCR amplification to the drop；
Pcr amplification product is detected.
Compared with prior art, the invention has the advantages that
1) method of highly sensitive detection hepatitis type B virus provided by the invention, the sensitivity of testing result and specificity are more
Height, and can effectively reduce testing cost；
2) present invention makes nucleic acid solution and PCR reaction solution be wrapped in drop formation liquid in runner using centrifugal force
In, single-molecule PCR reaction system is formed, the sensitivity of detection is increased, reduces Monitoring lower-cut, be conducive to find virus as early as possible
Infection；
3) in the present invention, due in drop after treatment only include monomolecular nucleic acid template, amplification when by
Interference reduce, can to avoid generate it is uncertain as a result, improve atopic；
4) different from the kit that other are only capable of detection part common virus hypotype nucleic acid, kit provided by the invention is logical
Optimizational primer design is crossed, the detection of hbv nucleic acid is allowed to cover all known hypotypes, meanwhile, the present invention passes through
Optimizational primer design, so that the fragment length of amplified production is between 100-150bp, and annealing temperature is between 56-62 DEG C, and
The interaction between designed primer is analyzed by matching silent winged Primer Analysis online tool, it is ensured that the phase between primer
Interaction will not have an impact system；
5) present invention is by optimizational primer probe, so that only can detect this kind of virus for every kind of viral primed probe,
It will not be with human genome or viruses of human hepatitis C (HCV) nucleic acid, herpes virus hominis (EBV) nucleic acid, cytomegalovirus (CMV)
Cross reaction occurs for the common viral nucleic acid such as nucleic acid, ensure that the accuracy of positive findings.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the sample processing device used in a typical embodiments of the invention.
Fig. 2 is the drop fluorescence detection result of negative control in the embodiment of the present invention 1.
Fig. 3 is the drop fluorescence detection result of No. 1 positive sample in the embodiment of the present invention 1.
Fig. 4 is the fluorescence detection result of No. 2 positive sample drops in the embodiment of the present invention 1.
Fig. 5 is the drop fluorescence intensity testing result of reference material A in the embodiment of the present invention 2.
Fig. 6 is the drop fluorescence intensity testing result of reference material B in the embodiment of the present invention 2.
Fig. 7 is the drop fluorescence intensity testing result of reference material C in the embodiment of the present invention 2.
Fig. 8 is the drop fluorescence intensity testing result of reference material D in the embodiment of the present invention 2.
Fig. 9 is the drop fluorescence intensity testing result of HCV, CMV, EBV and HBV positive sample in the embodiment of the present invention 3.
Specific embodiment
More detailed explanation will hereafter be made to technical solution of the present invention.It is understood, however, that in model of the present invention
In enclosing, above-mentioned each technical characteristic of the invention and it is ok between each technical characteristic specifically described in below (e.g. embodiment)
It is combined with each other, to form a new or preferred technical solution.Due to space limitations, I will not repeat them here.
The present invention will be further described according to specific example, but it is only that illustrative purpose is restricted without playing
Effect.
Before being described to example, it is necessary to which some remarks explanations are provided:
The difference that will cause experimental result using the reagent of different manufacturers, different batches, belongs to normal phenomenon.
When carrying out small scale experiments, to guarantee the repeatability between parallel laboratory test, it is proposed that after configuration reagent, mix well simultaneously
Packing, to guarantee the homogeneity of each experiment reagent.
The one aspect of the embodiment of the present invention provides a kind of primer pair for detecting hbv nucleic acid comprising:
The first primer, i.e. forward primer, sequence is as shown in SEQ ID NO.1；
Second primer, i.e. reverse primer, sequence is as shown in SEQ ID NO.2.
Further, primer sequence contained by the primer pair is as follows:
| HBV-F (forward primer) | 5'-CCTCGCCTCCAAGAATGTAAGT-3' |
| HBV-R (reverse primer) | 5'-GACATTCTGCATTTTAACTATGGCTCT-3' |
It is different from the kit that other are only capable of the common HBV virus subtype nucleic acid of detection part, kit provided by the invention
It is designed by optimizational primer, this kit is allowed to cover all known HBV virus subtype nucleic acid, meanwhile, the present invention passes through
Optimizational primer design, so that the amplified production of HBV nucleic acid is between 100-150bp, and annealing temperature is between 56-62 DEG C, and
By matching silent winged Primer Analysis (thermofisher primer analyzer) online tool to the phase between designed primer
Interaction is analyzed, it is ensured that the interaction between primer will not have an impact system.
The other side of the embodiment of the present invention additionally provides a kind of probe for detecting hbv nucleic acid, the spy
The sequence of needle is as shown in SEQ ID NO.3.
Further, the sequence of the probe is as follows:
| HBV-Probe | 5'-FAM-GCCTGATAGGGTGCTTGCGA-BHQ-3' |
The present invention is by optimizational primer probe, so that only can detect this kind of virus for every kind of viral primed probe, no
It can be with human genome or viruses of human hepatitis C (HCV) nucleic acid, herpes virus hominis (EBV) nucleic acid, cytomegalovirus (CMV) core
Cross reaction occurs for the common viral nucleic acid such as acid, ensure that the accuracy of positive findings.
The other side of the embodiment of the present invention additionally provides a kind of kit for detecting hbv nucleic acid, packet
At least one set of primer pair and at least one probe are included, wherein one group of primer pair includes the first primer and the second primer, described first
The sequence of primer is as shown in SEQ ID NO.1, and the sequence of second primer is as shown in SEQ ID NO.2, wherein the probe
Sequence as shown in SEQ ID NO.3.
In some embodiments, the kit further includes viral interior label primer to, internal standard probe, wherein in the virus
Marking primer pair includes the first interior label primer (i.e. internal standard forward primer) and the second interior label primer (i.e. internal standard reverse primer), and described the
The sequence of one interior label primer as shown in SEQ ID NO.4, the sequence of second interior label primer as shown in SEQ ID NO.5,
The sequence of middle internal standard probe is as shown in SEQ ID NO.6.
Further, the internal standard forward primer and reverse primer for including in interior label primer mixed liquor in kit of the present invention
Sequence is as follows:
| Internal standard-F (internal standard forward primer) | 5'-ATTAACCTTATGTGTGACAT-3' |
| Internal standard-R (internal standard reverse primer) | 5'-CATATTCGTCCACAAAATGATTCTG-3' |
Further, in internal standard probe solution internal standard probe sequence are as follows: 5'-Cy5-5-CTGTATCGTCAAGGCACT-
BHQ-3'。
In some embodiments, the kit further includes PCR reaction solution, water and drop formation liquid.
Further, the PCR reaction solution includes PCR reaction buffer, triphosphoric acid base deoxynucleotide mixed liquor
(2.5mM dNTPs), archaeal dna polymerase, uracil dna glycosylase.
Further, the PCR reaction with buffer include 20mM potassium chloride, 50mM PH8.0Tris-HCl,
1.5mM divalent magnesium ion.
In some more specifically embodiments, the kit is specifically included: PCR reaction solution, water, negative object of reference,
Positive control, plasmid standards for quantitation and drop formation liquid.
Further, PCR reaction buffer, 2.5mM dNTPs, the mixing of target nucleotide primer are included in PCR reaction solution
Liquid, target nucleotide probe solution, interior label primer mixed liquor, internal standard probe solution, 5U/ μ L hot resistant DNA polymerase, 5U/ μ L urine are phonetic
Pyridine DNA glycosylase.
Wherein, the target nucleotide primer mixed liquor includes the first primer above-mentioned and the second primer.
Wherein, the target nucleotide probe solution includes HBV specific probe.
It wherein, include internal standard forward primer and internal standard reverse primer in the interior label primer mixed liquor.
The other side of the embodiment of the present invention additionally provides aforementioned agents box and detects hbv nucleic acid in preparation
Product in purposes.
Further, include: using the method for the product testing hbv nucleic acid
Viruses of human hepatitis B's sample of nucleic acid to be detected is provided；
It using sample processing device, mixes the sample of nucleic acid to be detected with PCR reaction solution, and makes the mixing to be formed
Liquid is wrapped up by drop formation liquid, and being formed includes single nucleic acid or the drop not comprising nucleic acid；
The primer pair and probe for being included using the kit carry out PCR amplification to the drop；
Pcr amplification product is detected.
In some embodiments, the method specifically includes:
The primer pair and probe for being included using kit above-mentioned carry out drop PCR amplification to the drop；
The fluorescence intensity of drop after PCR amplification is detected, judges whether sample of nucleic acid wraps according to the fluorescence intensity
Containing hbv nucleic acid.
Further, the sample processing device includes location hole, sample holes, reagent wells, reacting hole and connecting passage.
Present treatment device is made of PC material, long 40mm, wide 20mm, thickness 2mm, and wherein sample introduction pore volume is 50 μ L, reagent pore volume
For 50 μ L, reaction pore volume is 100 μ L.
In some more specifically embodiments, the detection method is specifically included: the examination is added in drop formation liquid
Sample of nucleic acid to be detected and PCR reaction solution are added the sample holes, make sample holes and examination under the influence of centrifugal force by agent hole
Liquid is mixed by connecting passage in agent hole, and wraps up the mixed liquor to be formed by drop formation liquid, and being formed includes single nucleic acid
Or the drop not comprising nucleic acid, and enter reacting hole.
Further, the detection method may include: to be mounted on sample processing device on centrifuge by location hole,
Reagent wells are added in drop formation liquid, the nucleic acid solution extracted and PCR reaction solution is mixed in proportion, sample holes is added.With
3000rpm is centrifuged clockwise, and sample holes are mixed with liquid in reagent wells by runner under the influence of centrifugal force, allows nucleic acid samples
It is wrapped up into drop is formed in PCR reaction solution, is finally collected into reacting hole.
Further, the drop formation liquid includes the oil phase substances such as mineral oil.
In some more specific embodiments, the method can specifically include following steps:
PCR reaction solution is taken out from refrigerator to defrosting in advance, is vortexed after melting completely and mixes simultaneously brief centrifugation.Take 10-19 μ
L reaction solution is placed in PCR pipe, and the nucleic acid samples that 1-10 μ L is newly extracted are added thereto, is mixed with pipette tips, guarantees that total amount of liquid is
20μL.It draws 20 μ L mixing liquids to be added in sample holes, draws 20 μ L drop formation liquid and be added in reagent wells.By location hole,
Processing unit is fixed in centrifugal machine adapter, should be noted and keep central symmetry during installation.It is centrifuged 3 minutes with 3000rpm,
So that nucleic acid samples and PCR reaction solution flow into runner under the influence of centrifugal force, and wrapped up in runner by drop formation liquid,
Formed only includes single nucleic acid or the drop not comprising nucleic acid.
By the droplet transfer generated in reacting hole into PCR pipe, it is put into polymerase chain reaction amplification instrument (PCR amplification instrument)
In, it is expanded by the condition of following table 1:
Table 1:PCR amplification condition
Amplified production is analyzed using 8 channel drop reading systems, first according to the blank control that template is not added
It determines background fluorescence intensity, sets more than the drop of background fluorescence intensity and measure the fluorescence of each reaction drop as positive drop
Intensity, and count positive drop number.
Nucleic acid samples volume is added by counting positive drop number and determining, then can calculate B-mode liver in nucleic acid samples
Scorching viral nucleic acid quantity.
The present invention is wrapped in nucleic acid solution and PCR reaction solution in runner in drop formation liquid using centrifugal force,
Single-molecule PCR reaction system is formed, the sensitivity of detection is increased, reduces Monitoring lower-cut, be conducive to find viral sense as early as possible
Dye；In the present invention, due to only including monomolecular nucleic acid template in drop after treatment, the interference being subject in amplification subtracts
It is few, it can be uncertain as a result, raising atopic to avoid generating.
The other side of the embodiment of the present invention additionally provides a kind of product comprising aforementioned agents box, the products application
In the detection method of hbv nucleic acid, the detection method includes:
Viruses of human hepatitis B's sample of nucleic acid to be detected is provided；
It using sample processing device, mixes the sample of nucleic acid to be detected with PCR reaction solution, and makes the mixing to be formed
Liquid is wrapped up by drop formation liquid, and being formed includes single nucleic acid or the drop not comprising nucleic acid；
The primer pair and probe for being included using the kit carry out PCR amplification to the drop；
Pcr amplification product is detected.
Further, the product includes: the primer pair for being included and probe using the kit to the drop
Carry out drop PCR amplification；
The fluorescence intensity of drop after PCR amplification is detected, judges whether sample of nucleic acid wraps according to the fluorescence intensity
Containing hbv nucleic acid.
Below in conjunction with attached drawing and several preferred embodiments the technical solution of the present invention is further explained explanation, but its
In experiment condition and setup parameter be not construed as the limitation to basic technical scheme of the present invention.And protection scope of the present invention
It is not limited to the following embodiments.
Embodiment 1
1. the present embodiment sample used includes the HBV nucleic acid positive nucleic acid solution 2, and negative nucleic acid solution 1.
The preparation of 2.PCR reaction system
(1) PCR reaction solution is taken out from refrigerator to defrosting in advance, is vortexed after melting completely and mixes simultaneously brief centrifugation；
(2) it takes 19 μ L PCR reaction solutions to be placed in PCR pipe, 1 μ L nucleic acid samples is added thereto, are mixed with pipette tips；
(3) 20 μ L mixing liquids are drawn to be added in sample holes, draws 20 μ L drop formation liquid and is added in reagent wells；
(4) by location hole, sample processing device is fixed in centrifugal machine adapter, should be noted in installation process device
When keep central symmetry；
(5) with 3000rpm centrifugation 3 minutes, so that nucleic acid samples and PCR reaction solution flow into runner under the influence of centrifugal force
In, and wrapped up in runner by drop formation liquid, being formed only includes single nucleic acid or the drop not comprising nucleic acid；
(6) by the droplet transfer generated in reacting hole into PCR pipe.
3.PCR amplification
Amplification carries out in polymerase chain reaction amplification instrument (PCR amplification instrument), and response procedures are as shown in the table:
4. interpretation of result
Amplified production is analyzed using 8 channel drop reading systems (Suzhou Hao Tong Biotechnology Co., Ltd), it is first
The fluorescence intensity for first analyzing each drop in negative control, determines background fluorescence intensity with this.It is more than again that background is glimmering by setting
The drop of luminous intensity is positive drop, measures the fluorescence intensity of each reaction drop, and counts positive drop number.Each sample
Testing result using each drop detection when order as abscissa, using the fluorescence intensity of the drop as ordinate, draw each
The scatter plot of drop fluorescence intensity.Fluorescence intensity threshold line, fluorescence intensity are determined by the background fluorescence activity that negative control determines
It is positive drop more than the drop of threshold line.
Since 1 μ L nucleic acid samples are added in this experiment, so the positive drop number of each sample detection, just represents
The HBV viral nucleic acid copy number for including in every 1 μ L nucleic acid samples.
If Fig. 2 is negative control drop fluorescence intensity testing result.
If Fig. 3 is No. 1 positive sample drop fluorescence intensity testing result, it can be deduced that HBV virus core in the nucleic acid samples
Sour carrying capacity is 436copies/ μ L.
If Fig. 4 is No. 2 positive sample drop fluorescence intensity testing results, it can be deduced that HBV virus core in the nucleic acid samples
Sour carrying capacity is 268copies/ μ L.
Embodiment 2
1. the present embodiment sample used includes, the plasmid qualitative reference product containing HBV specific sequence of 4 kinds of concentration,
Middle plasmid concentration is respectively A (10copies/ μ L), B (5copies/ μ L), C (2copies/ μ L) and D (1copies/ μ L).
The preparation of 2.PCR reaction system
(1) PCR reaction solution is taken out from refrigerator to defrosting in advance, is vortexed after melting completely and mixes simultaneously brief centrifugation；
(2) it takes 10 μ L PCR reaction solutions to be placed in PCR pipe, 10 μ L nucleic acid samples is added thereto, are mixed with pipette tips；
(3) 20 μ L mixing liquids are drawn to be added in sample holes, draws 20 μ L drop formation liquid and is added in reagent wells；
(4) by location hole, sample processing device is fixed in centrifugal machine adapter, should be noted in installation process device
When keep central symmetry；
(5) with 3000rpm centrifugation 3 minutes, so that nucleic acid samples and PCR reaction solution flow into runner under the influence of centrifugal force
In, and wrapped up in runner by drop formation liquid, being formed only includes single nucleic acid or the drop not comprising nucleic acid；
(6) by the droplet transfer generated in reacting hole into PCR pipe.
3.PCR amplification
Amplification carries out in polymerase chain reaction amplification instrument (PCR amplification instrument), and response procedures are as shown in the table:
4. interpretation of result
Amplified production is analyzed using 8 channel drop reading systems (Suzhou Hao Tong Biotechnology Co., Ltd), it is first
The fluorescence intensity for first analyzing each drop in negative control, determines background fluorescence intensity with this.It is more than again that background is glimmering by setting
The drop of luminous intensity is positive drop, measures the fluorescence intensity of each reaction drop, and counts positive drop number.Each sample
Testing result using each drop detection when order as abscissa, using the fluorescence intensity of the drop as ordinate, draw each
The scatter plot of drop fluorescence intensity.Fluorescence intensity threshold line, fluorescence intensity are determined by the background fluorescence activity that negative control determines
It is positive drop more than the drop of threshold line.
Since 10 μ L nucleic acid samples are added in this experiment, so the positive drop number of each sample detection, with regard to generation
The HBV viral nucleic acid copy number for including in the every 10 μ L nucleic acid samples of table.
Such as the drop fluorescence intensity testing result that Fig. 5 is reference material A, it can be deduced that HBV viral nucleic acid carries in the reference material
Amount is 10copies/ μ L.
Such as the drop fluorescence intensity testing result that Fig. 6 is reference material B, it can be deduced that HBV viral nucleic acid carries in the reference material
Amount is 5copies/ μ L.
Such as the drop fluorescence intensity testing result that Fig. 7 is reference material C, it can be deduced that HBV viral nucleic acid carries in the reference material
Amount is 2copies/ μ L.
Such as the drop fluorescence intensity testing result that Fig. 8 is reference material D, it can be deduced that HBV viral nucleic acid carries in the reference material
Amount is 1copies/ μ L.
Embodiment 3
1. the present embodiment sample used includes viruses of human hepatitis C (HCV) nucleic acid positive, herpes virus hominis (EBV) nucleic acid
The positive, cytomegalovirus (CMV) nucleic acid positive and HBV nucleic acid positive sample each 1.
The preparation of 2.PCR reaction system
(1) PCR reaction solution is taken out from refrigerator to defrosting in advance, is vortexed after melting completely and mixes simultaneously brief centrifugation；
(2) it takes 10 μ L PCR reaction solutions to be placed in PCR pipe, 10 μ L nucleic acid samples is added thereto, are mixed with pipette tips；
(3) 20 μ L mixing liquids are drawn to be added in sample holes, draws 20 μ L drop formation liquid and is added in reagent wells；
(4) by location hole, sample processing device is fixed in centrifugal machine adapter, should be noted in installation process device
When keep central symmetry；
(5) with 3000rpm centrifugation 3 minutes, so that nucleic acid samples and PCR reaction solution flow into runner under the influence of centrifugal force
In, and wrapped up in runner by drop formation liquid, being formed only includes single nucleic acid or the drop not comprising nucleic acid；
(6) by the droplet transfer generated in reacting hole into PCR pipe.
3.PCR amplification
Amplification carries out in polymerase chain reaction amplification instrument (PCR amplification instrument), and response procedures are as shown in the table:
4. interpretation of result
Amplified production is analyzed using 8 channel drop reading systems (Suzhou Hao Tong Biotechnology Co., Ltd), it is first
The fluorescence intensity for first analyzing each drop in negative control, determines background fluorescence intensity with this.It is more than again that background is glimmering by setting
The drop of luminous intensity is positive drop, measures the fluorescence intensity of each reaction drop, and counts positive drop number.Each sample
Testing result using each drop detection when order as abscissa, using the fluorescence intensity of the drop as ordinate, draw each
The scatter plot of drop fluorescence intensity.Fluorescence intensity threshold line, fluorescence intensity are determined by the background fluorescence activity that negative control determines
It is positive drop more than the drop of threshold line.
Since 10 μ L nucleic acid samples are added in this experiment, so the positive drop number of each sample detection, with regard to generation
The HBV viral nucleic acid copy number for including in the every 10 μ L nucleic acid samples of table.
It, can if Fig. 9 is from left to right followed successively by the drop fluorescence intensity testing result of HCV, CMV, EBV and HBV positive sample
Cross reaction does not occur with HCV, CMV, EBV viral nucleic acid to obtain, viral nucleic acid carrying capacity is in HBV positive sample
30.5copies/μL。
In conclusion kit Monitoring lower-cut of the invention is low, high sensitivity by above-mentioned technical proposal, specificity is good,
Detection method of the invention is simple and quick, and the sensitivity of testing result and specificity are higher, and can effectively reduce testing cost.
It should be appreciated that the technical concepts and features of above-described embodiment only to illustrate the invention, its object is to allow be familiar with this
The personage of item technology cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.It is all
Equivalent change or modification made by Spirit Essence according to the present invention, should be covered by the protection scope of the present invention.
Sequence table
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ctgtatcgtc aaggcact 18
Claims (10)
1. a kind of primer pair for detecting hbv nucleic acid, characterized by comprising:
The first primer, sequence is as shown in SEQ ID NO.1；
Second primer, sequence is as shown in SEQ ID NO.2.
2. a kind of probe for detecting hbv nucleic acid, which is characterized in that the sequence of the probe such as SEQ ID NO.3 institute
Show.
3. a kind of kit for detecting hbv nucleic acid, it is characterised in that including at least one set of primer pair and at least one
Probe, wherein one group of primer pair includes the first primer and the second primer, the sequence of the first primer such as SEQ ID NO.1 institute
Show, the sequence of second primer is as shown in SEQ ID NO.2, wherein a probe is probe as claimed in claim 2.
4. kit as claimed in claim 3, it is characterised in that it further include viral interior label primer to, internal standard probe, wherein institute
Viral interior label primer is stated to including the first interior label primer and the second interior label primer, the sequence of first interior label primer such as SEQ ID
Shown in NO.4, the sequence of second interior label primer is as shown in SEQ ID NO.5, the wherein sequence of internal standard probe such as SEQ ID
Shown in NO.6.
5. kit as described in claim 3 or 4, it is characterised in that further include the general components of PCR amplification detection；It is preferred that
, the general components of PCR amplification detection include PCR reaction buffer, triphosphoric acid base deoxynucleotide mixed liquor,
Archaeal dna polymerase, uracil dna glycosylase, water and drop formation liquid；It is especially preferred, the PCR reaction buffer packet
Chloride containing potassium, Tris-HCl and divalent magnesium ion.
6. use of the kit described in any one of claim 3-5 in the product of preparation detection hbv nucleic acid
On the way.
7. purposes as claimed in claim 6, which is characterized in that the method for the application product testing hbv nucleic acid
Include:
Viruses of human hepatitis B's sample of nucleic acid to be detected is provided；
It using sample processing device, mixes the sample of nucleic acid to be detected with PCR reaction solution, and makes the mixed liquor quilt to be formed
Drop formation liquid package, being formed includes single nucleic acid or the drop not comprising nucleic acid；
The primer pair and probe for being included using the kit carry out PCR amplification to the drop；
Pcr amplification product is detected.
8. detection method according to claim 7, characterized by comprising: the primer for being included using kit above-mentioned
Drop PCR amplification is carried out to the drop to probe；
The fluorescence intensity of drop after PCR amplification is detected, judges whether sample of nucleic acid includes second according to the fluorescence intensity
Hepatitis virus nucleic acid；
And/or the drop formation liquid includes oil phase substance；Preferably, the oil phase substance includes mineral oil；
And/or the sample processing device includes sample holes, reagent wells, reacting hole and connecting passage；Preferably, the inspection
Survey method includes: that the reagent wells are added in drop formation liquid, by sample of nucleic acid to be detected and PCR reaction solution be added it is described into
Sample hole mixes sample holes by connecting passage with liquid in reagent wells, and makes the mixed liquor to be formed
It is wrapped up by drop formation liquid, being formed includes single nucleic acid or the drop not comprising nucleic acid, and enters reacting hole.
9. a kind of product comprising kit described in any one of claim 3-5, the products application is in hepatitis B
The detection method of malicious nucleic acid, the detection method include:
Viruses of human hepatitis B's sample of nucleic acid to be detected is provided；
It using sample processing device, mixes the sample of nucleic acid to be detected with PCR reaction solution, and makes the mixed liquor quilt to be formed
Drop formation liquid package, being formed includes single nucleic acid or the drop not comprising nucleic acid；
The primer pair and probe for being included using the kit carry out PCR amplification to the drop；
Pcr amplification product is detected.
10. product as claimed in claim 9, characterized by comprising: the primer pair and probe for being included using the kit
Drop PCR amplification is carried out to the drop；
The fluorescence intensity of drop after PCR amplification is detected, judges whether sample of nucleic acid includes second according to the fluorescence intensity
Hepatitis virus nucleic acid.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811548294.6A CN109457050A (en) | 2018-12-18 | 2018-12-18 | Detect primer, probe, kit and the detection method of hbv nucleic acid |
| PCT/CN2019/115837 WO2020125246A1 (en) | 2018-12-18 | 2019-11-06 | Primers, probe, kit and detection method for detecting hepatitis b virus nucleic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811548294.6A CN109457050A (en) | 2018-12-18 | 2018-12-18 | Detect primer, probe, kit and the detection method of hbv nucleic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109457050A true CN109457050A (en) | 2019-03-12 |
Family
ID=65613628
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811548294.6A Withdrawn CN109457050A (en) | 2018-12-18 | 2018-12-18 | Detect primer, probe, kit and the detection method of hbv nucleic acid |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN109457050A (en) |
| WO (1) | WO2020125246A1 (en) |
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| CN109852732A (en) * | 2019-04-02 | 2019-06-07 | 重庆医科大学附属儿童医院 | The invisible hepatitis B detection kit of quantitative fluorescent PCR |
| CN109929750A (en) * | 2019-04-11 | 2019-06-25 | 石家庄禾柏生物技术股份有限公司 | A kind of drop formation device and method |
| CN110951897A (en) * | 2019-12-26 | 2020-04-03 | 苏州德思普生物科技有限公司 | Treponema pallidum detection primer and kit |
| CN111172320A (en) * | 2019-12-26 | 2020-05-19 | 苏州德思普生物科技有限公司 | Detection primer, kit and method for respiratory syncytial virus F gene |
| WO2020125246A1 (en) * | 2018-12-18 | 2020-06-25 | 苏州绘真生物科技有限公司 | Primers, probe, kit and detection method for detecting hepatitis b virus nucleic acid |
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