CN108330212A - A kind of primer sets, composition and kit for detecting hepatitis B - Google Patents
A kind of primer sets, composition and kit for detecting hepatitis B Download PDFInfo
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Abstract
The invention discloses a kind of primer sets for detecting hepatitis B, including with the forward primer of nucleotide sequence shown in SEQ ID NO.1 and the reverse primer with nucleotide sequence shown in SE ID NO.2.A kind of composition for detecting hepatitis B including the above-mentioned primer sets for detecting hepatitis B, it further include target spot probe, target spot probe has nucleotide sequence shown in SEQ ID NO.3, the two ends difference mark fluorescent group and quenching group of target spot probe.A kind of includes the above-mentioned kit for detecting hepatitis B for detecting hepatitis B composition, further includes PCR buffer solutions, dNTPs, Taq enzyme, UNG enzymes and nucleic acid extracting reagent.Using hepatitis type B virus A H, the multiple hypotype bioinformatic analysis of totally 8 types can cover 8 HBV types and each hypotype very well in S sections conserved region setting primed probe section.
Description
Technical field
The present invention relates to the detection technique fields of hepatitis type B virus (HBV), more particularly to one kind is for detecting B-type hepatitis
Primer sets, composition and the kit of poison.
Background technology
Hepatitis type B virus (HBV) is a kind of infectious virus seriously endangering global human health, and virus infection is in generation
Criticality is popular, and China belongs to hepatitis B infection endemic area.Acute and chronic HBV infection easily causes multiple organ damage,
It may lead to the generation of the diseases such as hepatic sclerosis, liver cancer, hepatic failure.Therefore, periodically to hepatitis B infected patient's body HBV carrying capacity standard
Determine that amount detection seems extremely important to the indication of the antiviral therapy of hepatitis B and monitoring.
The quantitative detection of hbv nucleic acid is hepatitis B infected in addition to for the treatment time of hepatitis B and monitoring
Virus load is also the important evidence of antiviral therapy object judgement in peripheral blood in patients.Therefore, the HBV nucleic acid of high accuracy
Quantitative detection has important clinical meaning.
Most domestic medical institutions are generally monitored using one-step method quantitative detecting reagent in peripheral blood in patients at present
HBV virus loads, such reagent is although cheap, easy to operate, but there are accuracy low, poor anti jamming capability, sensitivity
The problems such as insufficient.Specifically, one-step method quantitative detecting reagent defect：Free nucleic acid extraction purification process, the impurity in peripheral blood are difficult
Situations such as to completely remove, PCR processes being caused to be easy to happen inhibition or interference, leading to not detection；Different peripheral blood in patients are dry
It is different (blood fat, hemoglobin, medicine etc.) to disturb material concentration and type, leads to that quantitative individual difference is big, poor repeatability
It is low with accuracy；Peripheral blood causes sensitivity low without viral nucleic acid concentration process.
Low-level virus load (is less than 10 in3IU/mL) being susceptible to can not detect or quantitative concentrations deviation is big
The problems such as, at the same in low-level virus load be antiviral therapy object judgement critical section.Domestic large and medium-sized doctor thus
Treat the COBAS AmpliPrep COBAS that mechanism then mainly applies antiviral therapy object judgement import reagent Roche
TaqMan HBV Test, this method has many advantages, such as that automation, accuracy is high, the range of linearity is wide, reproducible, but it is used
Closed equipment and reagent, somewhat expensive, the reasons such as patient burden is big cause to be difficult to clinically extensive use at present.It is clinical
On also have the high quick HBV nucleic acid quantifications detection reagent in part, carried using column or magnetic bead extracting method carry out nucleic acid concentration purifying, it is glimmering
Light PCR is quantitatively detected, but since that there are noise backgrounds is high for reagent, leads to that sensitivity is low, accuracy is not high.
The quantitative detecting reagent defect of other reagents containing extraction purification：Probe sequence, which designs long (>=25bp), makes fluorescence
Apart from excessive, quenching group can not effectively absorb fluorescence signal, keep entire reaction system background higher for group and quenching group,
Eventually lead to reagent sensitivity decline, while long probe sequence is easy to make probe itself to fold, cause annealing stage without
Method is that accuracy declines effectively with template matches.Since probe TM is (when TM is two sections of complementary base sequence unwindings 50%
Temperature) generally with primer TM differences at 5-10 DEG C, therefore corresponding primer sequence is also in 22bp or more, while there is also primers
It itself folds and template combines the problems such as being obstructed.
Invention content
It is an object of the invention to propose a kind of primer sets for detecting hepatitis B, have quantitative precision high
Feature.
Another object of the present invention is to propose a kind of composition for detecting hepatitis B, there is quantitative precision
High feature.
Another object of the present invention is to propose a kind of kit for detecting hepatitis B, there is quantitative precision
High feature.
For this purpose, the present invention uses following technical scheme：
A kind of primer sets for detecting hepatitis B, including the forward direction with nucleotide sequence shown in SEQ ID NO.1
Primer and reverse primer with nucleotide sequence shown in SEQ ID NO.2.
Further, one or more of nucleotide sequence of at least one of forward primer and reverse primer thymus gland
Pyrimidine bases replace with uracil base.
A kind of composition for detecting hepatitis B including the above-mentioned primer sets for detecting hepatitis B, also
Including target spot probe, target spot probe has nucleotide sequence shown in SEQ ID NO.3, and two ends of target spot probe are marked respectively
Remember fluorophor and quenching group.
Further, further include internal standard probe, internal standard probe is with nucleotide sequence shown in SEQ ID NO.4, internal standard spy
The two ends difference mark fluorescent group and quenching group of needle, the fluorophor that target spot probe and internal standard probe mark are different.
Further, one or more of nucleotide sequence of at least one of target spot probe and internal standard probe thymus gland
Pyrimidine bases replace with uracil base.
A kind of includes the above-mentioned kit for detecting hepatitis B for detecting hepatitis B composition, is also wrapped
Include PCR buffer solutions, dNTPs, Taq enzyme, UNG enzymes and nucleic acid extracting reagent.
Further, primer, probe, PCR buffer solutions, dNTPs, Taq enzyme, UNG enzymes be mixed and added into no DNase and
RNase purified waters become PCR reaction premixed liquids, and the formula of PCR reaction premixed liquids is as follows：
10 μ L of PCR buffer solutions；
100mM dNTPs(ATCGU)0.27-1.35μL；
50 μM of forward primer 0.25-1.5 μ L；
50 μM of reverse primer 0.25-1.5 μ L；
50 μM of target spot probe 0.05-0.8 μ L；
50 μM of internal standard probe 0.05-0.8 μ L；
Taq enzyme 0.5-1 μ L；
UNG enzyme 0.02-0.15 μ L；
It is added without DNase and RNase purified waters to 20 μ L of final volume.
Further, nucleic acid extracting reagent includes 450 μ L of cell pyrolysis liquid, 40 μ L of magnetic bead mixed liquor, the first cleaning solution
500 μ L, the second cleaning solution 500 μ L and eluent 30-120 μ L；
Cell pyrolysis liquid includes 2.5M guanidinium isothiocyanates, 10% triton X-100 and 15% isopropanol；
Magnetic bead mixed liquor includes that super suitable magnetic nanoparticle, Proteinase K, 62.5copy/ μ L internal standards plasmids and 50% are sweet
Oil；
First cleaning solution includes 1M guanidinium isothiocyanates and 50% absolute ethyl alcohol；
Second cleaning solution includes TRIS/ sodium chloride and 50% absolute ethyl alcohol；
Eluent is no DNase and RNase deionized waters.
Beneficial effects of the present invention are：
1, it using hepatitis type B virus A-H multiple hypotype bioinformatic analysis of totally 8 types, is set in S sections conserved region
Determine primer section, 8 HBV types and each hypotype can be covered very well.
2, primer chooses high CG sections, can effectively shorten sequence length (≤20bp) while improving sequence TM values, subtracts
Few primer itself folds, and primer is made effectively to arrange in pairs or groups with template；Simultaneously because the reduction of primer sequence base, makes reagent quantitative mark
The influence of quasi- product and detection sample environment difference (interfering nucleic acid, interference albumen etc.) is preferably minimized, and primer is in wider interference ring
It remains to keep stable template joint efficiency to be expanded under border, effectively improves the accuracy of reagent quantitative.
3, it using hepatitis type B virus A-H multiple hypotype bioinformatic analysis of totally 8 types, is set in S sections conserved region
Determine primed probe section, 8 HBV types and each hypotype can be covered very well.
4, probe chooses high CG sections, since probe length (≤20bp) is reduced, reduces probe itself and folds, make probe
Effectively arrange in pairs or groups with template；Probe length shortening simultaneously makes quenching group more effectively absorb fluorescence signal, effectively reduces entire reaction
The autofluorescent background noise of system improves reagent sensitivity and accuracy.
5, using magnetic bead and Proteinase K mixed liquor as the dilution and preservation of kit internal standard plasmid, in magnetic bead and albumen
In enzyme K mixed liquors, digested since Proteinase K can carry out protein-based enzyme system effective decompose, internal standard plasmid can be effective
The degradation of DNA enzymatic is avoided, the stability of the accuracy and preservation of internal standard plasmid concentration is improved.
6, kit of the invention monitors false negative using competitive internal standard, and internal standard uses primer pair identical with target spot
Sequence, amplified production length is identical as target spot amplified production length, while CG contents having the same, therefore can be to greatest extent
Identical amplification efficiency is kept with target spot, reaches best monitoring false negative effect.
7, kit of the invention is to adapt to compared with short primer probe sequence and reduce reaction system internal composition due to pure
The disturbed condition caused by reasons such as degree, use most simple system formulation (PCR buffer solutions, dNTPs, primed probe, go DNase and
RNase purified waters, Taq enzyme and UNG enzymes) any additive is not added.
Description of the drawings
Fig. 1 is the amplification curve diagram of each one multiple holes of concentration template of the embodiment of the present invention 1；
Fig. 2 is the range of linearity and related coefficient of quantitative concentrations logarithm and theoretical concentration logarithm in the embodiment of the present invention 1
Figure；
Fig. 3 is 180IU/mL concentration template wherein 10 multiple holes amplification curve diagrams in the embodiment of the present invention 4；
Fig. 4 is 90IU/mL wherein 10 multiple holes amplification curve diagrams in the embodiment of the present invention 4；
Fig. 5 is 45IU/mL wherein 10 multiple holes amplification curve diagrams in the embodiment of the present invention 4；
Fig. 6 is 20IU/mL wherein 10 multiple holes amplification curve diagrams in the embodiment of the present invention 4；
Fig. 7 is 20 multiple holes precision figure of 10000IU/mL concentration in the embodiment of the present invention 5；
Fig. 8 is 20 multiple holes precision figure of 1000IU/mL concentration in the embodiment of the present invention 5；
Fig. 9 is 20 multiple holes precision figure of 100IU/mL concentration in the embodiment of the present invention 5.
Specific implementation mode
Below in conjunction with the accompanying drawings and the technical solution that further illustrates the present invention of specific implementation mode.
A kind of primer sets for detecting hepatitis B, including the forward direction with nucleotide sequence shown in SEQ ID NO.1
Primer and reverse primer with nucleotide sequence shown in SEQ ID NO.2.Wherein, as shown in table 1, SEQ ID NO.1 institutes
Show that the length of nucleotide sequence is 16bp, GC% 63%；The length of nucleotide sequence shown in SEQ ID NO.2 is 17bp,
GC% is 59%.
Table 1
| Title | Base sequence (5 ' -3 ') | GC% | Length |
| SEQ ID NO.1 | GATGTGTCTGCGGCGT | 63% | 16 |
| SEQ ID NO.2 | CGGGCAACATACCTTGG | 59% | 17 |
Primer pair is the areas specific amplification HBV S gene.Using the hepatitis type B virus A-H multiple hypotype lifes of totally 8 types
Object bioinformatics analysis sets primer section in S sections conserved region, can cover 8 HBV types and each hypotype very well.
Primer chooses high CG sections, can effectively shorten sequence length (≤20bp) while improving sequence TM values, reduction
Primer itself folds, and primer is made effectively to arrange in pairs or groups with template；Simultaneously because the reduction of primer sequence base, makes reagent quantitative standard
The influence of product and detection sample environment difference (interfering nucleic acid, interference albumen etc.) is preferably minimized, and primer is in wider interference environment
Under remain to keep stable template joint efficiency to be expanded, effectively improve the accuracy of reagent quantitative.
In other embodiments, forward primer and reverse primer can be modified with method known in this field, packet
It includes and methylates, replaces modification between one or more natural nucleotides and nucleotide by natural nucleus glycoside analog, in nucleotide
The addition of in sequence or both ends one or more nucleotide residue, groups replace one or more of sequence nucleotide residue
Change other nucleotide residue etc. into.
In other embodiments, one in the nucleotide sequence of at least one of forward primer and reverse primer or
Multiple thymine alkali bases replace with uracil base.Primer pair after modification still has preferable quantitative precision.
The present invention also provides a kind of including the above-mentioned primer sets for detecting hepatitis B for detecting B-type hepatitis
The composition of poison, further includes target spot probe, and target spot probe has a nucleotide sequence shown in SEQ ID NO.3, and the two of target spot probe
A end difference mark fluorescent group and quenching group.Wherein, the length of nucleotide sequence shown in SEQ ID NO.3 is 19bp,
GC% is 58%.
Using hepatitis type B virus A-H multiple hypotype bioinformatic analysis of totally 8 types, set in S sections conserved region
Primed probe section can cover 8 HBV types and each hypotype very well.
Probe chooses high CG sections, since probe length (≤20bp) is reduced, reduces probe itself and folds, probe is made to have
Effect is arranged in pairs or groups with template；Probe length shortening simultaneously makes quenching group more effectively absorb fluorescence signal, effectively reduces entire reactant
The autofluorescent background noise of system improves reagent sensitivity and accuracy.
Further, which further includes internal standard probe, and internal standard probe has SEQ ID
Nucleotide sequence shown in NO.4, two ends difference mark fluorescent group and quenching group of internal standard probe, target spot probe and interior
The fluorophor for marking probe label is different.Wherein, the length of nucleotide sequence shown in SEQ ID NO.4 is 19bp, and GC% is
58%.
Table 2
| Title | Base sequence (5 ' -3 ') | GC% | Length |
| SEQ ID NO.3 | CCTGCTGCTATGCCTCATC | 58% | 19 |
| SEQ ID NO.4 | CGTCAGACCACTCCTACAC | 58% | 19 |
Fluorophor can be, but not limited to be FAM, ROX, CY5, HEX, JOE, CY3, NED, TAMRA, TAXAS, RED,
Any one of VIC, TET；Quenching group can be, but not limited to be any one of BHQ, TAMRA, MGB, DABCYL.
Target spot probe and internal standard probe can be modified with method known in this field, including be methylated, by natural
Modification between the one or more natural nucleotides of nucleoside analog substitution and nucleotide, in nucleotide sequence or the addition of both ends
One or more of sequence nucleotide residue is substituted for other nucleotide by one or more nucleotide residues, group
Residue etc..
Specifically, at least one of target spot probe and internal standard probe are through the modification that methylates；Target spot probe and internal standard are visited
One or more of nucleotide sequence of at least one of needle thymine alkali bases replace with uracil base.After modification
Target spot probe and internal standard probe still have preferable quantitative precision.
The present invention also provides it is a kind of including it is above-mentioned for detect hepatitis B composition for detecting hepatitis B
Kit, further include PCR buffer solutions, dNTPs, Taq enzyme, UNG enzymes and nucleic acid extracting reagent.
The kit of the present invention is to adapt to compared with short primer probe sequence and reduce reaction system internal composition due to purity
Etc. disturbed condition caused by reasons, the most simple system formulation of use do not add any additive.
Further, primer, probe, PCR buffer solutions, dNTPs, Taq enzyme, UNG enzymes be mixed and added into no DNase and
RNase purified waters become PCR reaction premixed liquids, and the formula of PCR reaction premixed liquids is as follows：
10 μ L of PCR buffer solutions；
100mM dNTPs(ATCGU)0.27-1.35μL；
50 μM of forward primer 0.25-1.5 μ L；
50 μM of reverse primer 0.25-1.5 μ L；
50 μM of target spot probe 0.05-0.8 μ L；
50 μM of internal standard probe 0.05-0.8 μ L；
Taq enzyme 0.5-1 μ L；
UNG enzyme 0.02-0.15 μ L；
It is added without DNase and RNase purified waters to 20 μ L of final volume.
The PCR reaction premixed liquids use simplest system formulation, adapt to compared with short primer probe sequence and reduce reaction
System internal composition due to purity etc. caused by disturbed condition, ensure quantitative accuracy.
Further, nucleic acid extracting reagent includes 450 μ L of cell pyrolysis liquid, 40 μ L of magnetic bead mixed liquor, the first cleaning solution
500 μ L, the second cleaning solution 500 μ L and eluent 30-120 μ L；
Cell pyrolysis liquid includes 2.5M guanidinium isothiocyanates, 10% triton X-100 and 15% isopropanol；
Magnetic bead mixed liquor includes that super suitable magnetic nanoparticle, Proteinase K, 62.5copy/ μ L internal standards plasmids and 50% are sweet
Oil；
First cleaning solution includes 1M guanidinium isothiocyanates and 50% absolute ethyl alcohol；
Second cleaning solution includes TRIS/ sodium chloride and 50% absolute ethyl alcohol；
Eluent is no DNase and RNase deionized waters.
The biological sample that nucleic acid extraction purifying is carried out using above-mentioned nucleic acid extracting reagent includes that the tissue from human body is even
Slurry, cell, whole blood, serum, blood plasma, swab soak etc..
Using magnetic bead and Proteinase K mixed liquor as the dilution and preservation of kit internal standard plasmid, in magnetic bead and protease
In K mixed liquors, digested since Proteinase K can carry out protein-based enzyme system effective decompose, internal standard plasmid can effectively be kept away
Exempt from the degradation of DNA enzymatic, improves the stability of the accuracy and preservation of internal standard plasmid concentration.
Embodiment 1
One, reagent prepares as follows：
1, national plasmid standards for quantitation (National Reference Standard forHBV DNA lot numbers： 300022-
201601).National plasmid standards for quantitation a concentration of 1.0 × 108IU/mL.Then it is diluted to conjunction with the negative and clear serum of appearance
Suitable concentration.
2, negative quality-control product：The negative and clear serum of appearance.
3, critical positive quality control product and strong positive quality-control product.Using the negative and clear serum of appearance to HBV full-length genomes
Plasmid is diluted, and assignment is carried out using the standard items that can be traced to the source to national plasmid standards for quantitation.
4, plasmid standards for quantitation A-D：HBV full-length genome plasmids are diluted using negative and appearance clear serum, are made
Its concentration is 107-102Between using can trace to the source to national plasmid standards for quantitation standard items carry out assignment.
Two, clinical sample method for extracting nucleic acid.
Nucleic acid extraction step includes (1)-(4), specific as follows：
(1) 450 μ L cell pyrolysis liquids, 40 μ L magnetic beads mixed liquor, 250 μ L samples, mixing 2 are added in 1.5mL centrifuge tubes
It is stored at room temperature 10 minutes after minute.Of short duration centrifugation is carried out, magnetic bead, all solution of reject are adsorbed.
(2) 500 the first cleaning solutions of μ L are added, mixing is stored at room temperature 2 minutes after 2 minutes.Of short duration centrifugation is carried out, magnetic is adsorbed
Pearl, all solution of reject.
(3) 500 the second cleaning solutions of μ L are added, mixing is stored at room temperature 2 minutes after 2 minutes.That of short duration centrifugation this time is inhaled
Attached magnetic bead, all solution of reject.
(4) room temperature dries magnetic bead 80 μ L eluents is added after ten minutes, and mixing is stored at room temperature 5 minutes after 2 minutes.It carries out
Of short duration centrifugation adsorbs magnetic bead, collects purified DNA solution.
Three, fluorescence quantitative PCR reaction solution is prepared
PCR reaction solution：Table 3
| Title | 1 test |
| PCR reaction premixed liquids | 20μL |
| HBV DNA | 30μL |
PCR reaction premixed liquid formulas：Table 4
| Ingredient names | Concentration | Single reaction amount ranges |
| PCR buffer solutions | 5× | 10μL |
| dNTPs(ATCGU) | 100mM | 0.27μL |
| Forward primer | 50μM | 0.25μL |
| Reverse primer | 50μM | 0.25μL |
| Target spot probe | 50μM | 0.05μL |
| Internal standard probe | 50μM | 0.05μL |
| Taq enzyme | 5U/μL | 0.5μL |
| UNG enzymes | 1U/μL | 0.02μL |
| Without DNase and RNase purified waters | / | Add to 20 μ L of final volume |
Wherein, forward primer has nucleotide sequence shown in SEQ ID NO.1；Reverse primer has SEQ ID NO.2 institutes
The nucleotide sequence shown；Target spot probe has nucleotide sequence shown in SEQ ID NO.3；Internal standard probe has SEQ ID NO.4
Shown nucleotide sequence.The two ends difference mark fluorescent group and quenching group of target spot probe, two ends of internal standard probe
Mark fluorescent group and quenching group, the fluorophor that target spot probe and internal standard probe mark are different respectively at end.
Four, reaction condition is as shown in table 5.
Table 5
Five, the linear accuracy experiment of kit
Using negative serum by high level clinical sample (via the COBAS AmpliPrep COBAS TaqMan of Roche Holding Ag
HBV Test, Version2.0 kits are quantified) be diluted to 400000000IU/mL, 200000000IU/mL,
20000000IU/mL、2000000IU/mL、200000IU/mL、20000IU/mL、 2000IU/mL、200IU/mL、100IU/
ML, 50IU/mL, 20IU/mL, 15IU/mL, 7.5IU/mL, each concentration do 3 repetitions, pass through following standard judging result
Accuracy：Sample quantitative concentrations absolute deviation is no more than ± 0.3log, and log concentration CV% is no more than 10%.
Table 6
In table 6：B=︱ M-T ︱；B：Absolute deviation；M：Measured concentration logarithm；T：Theoretical concentration logarithm.As a result such as 6 institute of table
Show, 20 to 4 in the range of linearity × 108IU/mL log concentrations absolute deviation is no more than ± 0.3log, and each concentration C V% is equal
No more than 10%.
Fig. 1 be 400000000IU/mL, 200000000IU/mL, 20000000IU/mL, 2000000IU/mL,
200000IU/mL、20000IU/mL、2000IU/mL、200IU/mL、100IU/mL、50IU/mL、 20IU/mL、15IU/mL、
7.5IU/mL templates, 3 multiple holes of each concentration, wherein the amplification curve diagram of one multiple holes of each concentration.It will be seen from figure 1 that
Each concentration template has preferable amplification rate.
Fig. 2 is the range of linearity and related coefficient of quantitative concentrations logarithm and theoretical concentration logarithm, quantitatively dense from Fig. 2
Degree logarithm has good linear relationship with theoretical concentration logarithm.
It follows that carrying out sample using the kit of the present embodiment quantifies accuracy in detection height.
Embodiment 2
The PCR reaction solution formula of the present embodiment is as shown in table 7.
Table 7
| Ingredient names | Concentration | Single reaction amount ranges |
| PCR buffer solutions | 5× | 10μL |
| dNTPs(ATCGU) | 100mM | 0.81μL |
| Forward primer | 50μM | 0.875μL |
| Reverse primer | 50μM | 0.875μL |
| Target spot probe | 50μM | 0.4μL |
| Internal standard probe | 50μM | 0.4μL |
| Taq enzyme | 5U/μL | 0.75μL |
| UNG enzymes | 1U/μL | 0.15μL |
| Without DNase and RNase purified waters | / | Add to 20 μ L of final volume |
| HBV DNA | / | 30μL |
| total | / | 50μL |
Use negative serum by high level clinical sample (via sieve using the method fluorescence quantitative PCR detection in embodiment 1
COBAS AmpliPrep COBAS TaqMan HBV Test, the Version2.0 kits of family name company are quantified) dilution
To 400000000IU/mL, 200000000IU/mL, 20000000IU/mL, 2000000IU/mL, 200000IU/mL,
20000IU/mL, 2000IU/mL, 200IU/mL, 100IU/mL, 50IU/mL, 20IU/mL, 15IU/mL, 7.5IU/mL, each
Concentration does 3 repetitions.Pass through the accuracy of following standard judging result：Sample quantitative concentrations absolute deviation be no more than ±
0.3log, log concentration CV% are no more than 10%.
Table 8
In table 8：B=︱ M-T ︱；B：Absolute deviation；M：Measured concentration logarithm；T：Theoretical concentration logarithm.As a result such as 8 institute of table
Show, 20 to 4 in the range of linearity × 108IU/mL log concentrations absolute deviation is no more than ± 0.3log, and each concentration C V% is equal
No more than 10%.It follows that carrying out sample using the kit of the present embodiment quantifies accuracy in detection height.
Embodiment 3
The PCR reaction solution formula of the present embodiment is as shown in table 9.
Table 9
| Ingredient names | Concentration | Single reaction amount ranges |
| PCR buffer solutions | 5× | 10μL |
| dNTPs(ATCGU) | 100mM | 1.35μL |
| Forward primer | 50μM | 1.5μL |
| Reverse primer | 50μM | 1.5μL |
| Target spot probe | 50μM | 0.8μL |
| Internal standard probe | 50μM | 0.8μL |
| Taq enzyme | 5U/μL | 1μL |
| UNG enzymes | 1U/μL | 0.15μL |
| Without DNase and RNase purified waters | / | Add to 20 μ L of final volume |
| HBV DNA | / | 30μL |
| total | / | 50μL |
Use negative serum by high level clinical sample (via sieve using the method fluorescence quantitative PCR detection in embodiment 1
COBAS AmpliPrep COBAS TaqMan HBV Test, the Version2.0 kits of family name company are quantified) dilution
To 400000000IU/mL, 200000000IU/mL, 20000000IU/mL, 2000000IU/mL, 200000IU/mL,
20000IU/mL, 2000IU/mL, 200IU/mL, 100IU/mL, 50IU/mL, 20IU/mL, 15IU/mL, 7.5IU/mL, each
Concentration does 3 repetitions.Pass through the accuracy of following standard judging result：Sample quantitative concentrations absolute deviation be no more than ±
0.3log, log concentration CV% are no more than 10%.
Table 10
In table 10：B=︱ M-T ︱；B：Absolute deviation；M：Measured concentration logarithm；T：Theoretical concentration logarithm.As a result such as table 10
It is shown, 20 to 4 in the range of linearity × 108IU/mL log concentrations absolute deviation is no more than ± 0.3log, and each concentration C V%
No more than 10%.It follows that carrying out sample using the kit of the present embodiment quantifies accuracy in detection height.
Embodiment 4 determines kit quantification limit (LOQ) and limit of identification (LOD)
The PCR reaction solution formula of the present embodiment is as shown in table 11.
Table 11
| Ingredient names | Concentration | Single reaction amount ranges |
| PCR buffer solutions | 5× | 10μL |
| dNTPs(ATCGU) | 100mM | 0.72μL |
| Forward primer | 50μM | 1μL |
| Reverse primer | 50μM | 1μL |
| Target spot probe | 50μM | 0.2μL |
| Internal standard probe | 50μM | 0.2μL |
| Taq enzyme | 5U/μL | 0.75μL |
| UNG enzymes | 1U/μL | 0.08μL |
| Without DNase and RNase purified waters | / | Add to 20 μ L of final volume |
| HBV DNA | / | 30μL |
| total | / | 50μL |
Use negative serum by HBV DNA countries quantitative criterion using the method fluorescence quantitative PCR detection in embodiment 1
Product are diluted to 180IU/mL, 90IU/mL, 45IU/mL, 20IU/mL, 15IU/mL, 7.5IU/mL, and each concentration does 25 weights
It is multiple.Pass through the accuracy of following standard judging result：Sample quantitative concentrations absolute deviation is no more than ± 0.3log, log concentration
CV% is no more than 10%.
Table 12
In table 12：B=︱ M-T ︱；B：Absolute deviation；M：Measured concentration logarithm；T：Theoretical concentration logarithm.As a result such as table 12
It is shown, 20 to 4 in the range of linearity × 108IU/mL log concentrations absolute deviation is no more than ± 0.3log, and each concentration C V%
No more than 10%.
Table 13
As shown in Table 13, the quantitative limit (LOQ) of the kit of the present embodiment is 20IU/mL, and limit of identification (LOD) is low
In 7.5IU/mL.
Fig. 3-6 is respectively 10 multiple holes amplification curves of 180IU/mL, 90IU/mL, 45IU/mL, 20IU/mL concentration template
Figure, it is known that each concentration template has preferable amplification rate, multiple multiple holes to have good stability.
5 kit Precision Experiment of embodiment
The PCR reaction solution formula of the present embodiment is as shown in table 14.
Table 14
Use negative serum by HBV DNA countries quantitative criterion using the method fluorescence quantitative PCR detection in embodiment 1
Product are diluted to 10000IU/mL, 1000IU/mL, 100IU/mL, and each concentration does 20 repetitions, calculates each concentration samples
The average value of result logarithm is quantitatively detected, and calculates the precision of each concentration samples.Pass through following standard judging result
Accuracy：The coefficient of variation (CV) of same concentration pattern detection result logarithm is not higher than 5%.
Table 15
In table 15,C.V：The coefficient of variation；SD：Standard deviation；Measured concentration logarithm
Average.As a result as shown in Table 15, the sample coefficient of variation (CV) of three concentration is respectively less than 5%, and it is preferable to illustrate that system has
Detect precision.Fig. 7-9 is 20 multiple holes precision of 10000IU/mL, 1000IU/mL, 100IU/mL concentration respectively.
The kit Precision Experiment of 6 present invention of embodiment
The PCR reaction solution formula of the present embodiment is as shown in table 16.
Table 16
| Ingredient names | Concentration | Single reaction amount ranges |
| PCR buffer solutions | 5× | 10μL |
| dNTPs(ATCGU) | 100mM | 1.35μL |
| Forward primer | 50μM | 1.5μL |
| Reverse primer | 50μM | 1.5μL |
| Target spot probe | 50μM | 0.8μL |
| Internal standard probe | 50μM | 0.8μL |
| Taq enzyme | 5U/μL | 1μL |
| UNG enzymes | 1U/μL | 0.15μL |
| Without DNase and RNase purified waters | / | Add to 20 μ L of final volume |
| HBV DNA | / | 30μL |
| total | / | 50μL |
Use negative serum by HBV DNA countries quantitative criterion using the method fluorescence quantitative PCR detection in embodiment 1
Product are diluted to 10000IU/mL, 1000IU/mL, 100IU/mL, and each concentration does 20 repetitions, calculates each concentration samples
The average value of result logarithm is quantitatively detected, and calculates the precision of each concentration samples.Pass through following standard judging result
Accuracy：The coefficient of variation (CV) of same concentration pattern detection result logarithm is not higher than 5%.
Table 17
In table 17,C.V：The coefficient of variation；SD：Standard deviation；Measured concentration logarithm
Average.As a result as shown in table 17, the sample coefficient of variation (CV) of three concentration is respectively less than 5%, and it is preferable to illustrate that system has
Detect precision.
The technical principle of the present invention is described above in association with specific embodiment.These descriptions are intended merely to explain the present invention
Principle, and limiting the scope of the invention cannot be construed in any way.Based on the explanation herein, this field
Technical staff would not require any inventive effort the other specific implementation modes that can associate the present invention, these modes are all
It will fall under the scope of the present invention.
<110>You Te medical science and technologies Co., Ltd of Foshan City
<120>A kind of primer sets, composition and kit for detecting hepatitis B
<160>4
<210>1
<211>16
<212> DNA
<213>Artificial sequence
<400>1
gatgtgtctg cggcgt 16
<210>2
<211>17
<212> DNA
<213>Artificial sequence
<400>2
Cgggcaacataccttgg 17
<210>3
<211>19
<212> DNA
<213>Artificial sequence
<400>3
cctgctgcta tgcctcatc 19
<210>4
<211>19
<212> DNA
<213>Artificial sequence
<400>4
cgtcagacca ctcctacac 19
Claims (9)
1. a kind of primer sets for detecting hepatitis B, which is characterized in that comprising with nucleotides sequence shown in SEQ ID NO.1
The forward primer of row and reverse primer with nucleotide sequence shown in SEQ ID NO.2.
2. according to claim 1 for detecting the primer sets of hepatitis B, which is characterized in that the forward primer and anti-
One or more of nucleotide sequence at least one of primer thymine alkali bases replace with uracil base.
3. including the combination for detecting hepatitis B as claimed in claim 1 or 2 for detecting the primer sets of hepatitis B
Object, which is characterized in that further include target spot probe, the target spot probe has nucleotide sequence shown in SEQ ID NO.3, the target
The two ends difference mark fluorescent group and quenching group of point probe.
4. according to claim 3 for detecting the composition of hepatitis B, which is characterized in that further include internal standard probe or
The reverse sequence of target spot probe, the internal standard probe have SEQ ID NO.4 shown in nucleotide sequence, the two of the internal standard probe
A end difference mark fluorescent group and quenching group, the fluorophor that the target spot probe and internal standard probe mark are different.
5. according to claim 4 for detecting the composition of hepatitis B, which is characterized in that the target spot probe and interior
It is through the modification that methylates to mark at least one of probe.
6. according to claim 4 for detecting the composition of hepatitis B, which is characterized in that the target spot probe and interior
One or more of the nucleotide sequence of at least one of mark probe thymine alkali bases replace with uracil base.
7. including the kit for detecting hepatitis B for detecting hepatitis B composition described in claim 4,
It is characterized in that, further includes PCR buffer solutions, dNTPs, Taq enzyme, UNG enzymes and nucleic acid extracting reagent.
8. for detecting hepatitis B kit described in claim 7, it is characterised in that：The primer, probe, PCR buffer solutions,
DNTPs, Taq enzyme, UNG enzymes, which are mixed and added into no DNase and RNase purified waters, becomes PCR reaction premixed liquids, the PCR reactions
The formula of premixed liquid is as follows：
10 μ L of PCR buffer solutions；
100mM dNTPs(ATCGU)0.27-1.35μL；
50 μM of forward primer 0.25-1.5 μ L；
50 μM of reverse primer 0.25-1.5 μ L；
50 μM of target spot probe 0.05-0.8 μ L；
50 μM of internal standard probe 0.05-0.8 μ L；
Taq enzyme 0.5-1 μ L；
UNG enzyme 0.02-0.15 μ L；
It is added without DNase and RNase purified waters to 20 μ L of final volume.
9. for detecting hepatitis B kit described in claim 7, it is characterised in that：The nucleic acid extracting reagent includes cell
450 μ L of lysate, 40 μ L of magnetic bead mixed liquor, 500 μ L of the first cleaning solution, the second cleaning solution 500 μ L and eluent 30-120 μ L；
The cell pyrolysis liquid includes 2.5M guanidinium isothiocyanates, 10% triton X-100 and 15% isopropanol；
The magnetic bead mixed liquor includes super suitable magnetic nanoparticle, Proteinase K, 62.5copy/ μ L internal standards plasmids and 50% glycerine；
First cleaning solution includes 1M guanidinium isothiocyanates and 50% absolute ethyl alcohol；
Second cleaning solution includes TRIS/ sodium chloride and 50% absolute ethyl alcohol；
The eluent is no DNase and RNase deionized waters.
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| CN111304368A (en) * | 2020-03-19 | 2020-06-19 | 艾康生物技术(杭州)有限公司 | Method, oligonucleotide and kit for detecting novel coronavirus |
| CN113249526A (en) * | 2021-06-30 | 2021-08-13 | 瑞博奥(广州)生物科技股份有限公司 | PCR reaction sequence combination for detecting hepatitis B virus and kit thereof |
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| CN110499392A (en) * | 2019-08-21 | 2019-11-26 | 佛山市优特医疗科技有限公司 | A kind of primer sets for detecting Ebola virus, composition and kit |
| CN111304368A (en) * | 2020-03-19 | 2020-06-19 | 艾康生物技术(杭州)有限公司 | Method, oligonucleotide and kit for detecting novel coronavirus |
| CN113249526A (en) * | 2021-06-30 | 2021-08-13 | 瑞博奥(广州)生物科技股份有限公司 | PCR reaction sequence combination for detecting hepatitis B virus and kit thereof |
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Application publication date: 20180727 |