CN109161588A - For detecting primer sets, probe, kit and the detection method of hepatitis B virus YMDD motif area medicament-resistant mutation - Google Patents
For detecting primer sets, probe, kit and the detection method of hepatitis B virus YMDD motif area medicament-resistant mutation Download PDFInfo
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Abstract
Primer sets, probe, kit and the detection method that the present invention provides a kind of for detecting hepatitis B virus YMDD motif area medicament-resistant mutation comprising for detecting the first specific primer sequence of the mutational site rtM204I, the second specific primer sequence for detecting the mutational site rtM204V and for detecting the mutational site rtL180M third specific primer sequence.A possibility that susceptibility of kit provided by the invention is high, and quickly, stability is good, closed detection for detection, reduces Final pollution.
Description
Technical field
The present invention relates to technical field of molecular biology, belong to external diagnosis reagent field medicament-resistant mutation detection method, tool
Body is related to a kind of primer sets for detecting hepatitis B virus YMDD motif area medicament-resistant mutation, probe, kit and detection
Method.
Background technique
Treatment chronic hepatitis B mainly uses interferon or nucleoside analog at present, such as Lamivudine, adefovirdipivoxil, Ganciclovir
Deng interfering hbv replication to achieve the purpose that treatment.Lamivudine (Lamivudine), Chinese name He Puting are that oral karyon is phonetic
Pyridine nucleoside analog was researched and developed successfully and was produced in 1992 by GlaxoSmithKline PLC company, is entered within 1998 China and is gone through to use
In clinic, Lamivudine is convenient to take, adverse reaction is few, easy for patients to accept, is most effective and safe suppression generally acknowledged at present
The nucleosides similar medicine of virus replication processed, the appearance of the medicine make Anti-HBV activity treatment that qualitative leap have occurred, and make to be not suitable for α-interference
Extract for treating or the unresponsive patient for the treatment of have new therapeutic choice.
The duplication of HBV gene group will just can be carried out by reverse transcription mechanism, be initially formed rna replicon intermediate, and rna replicon
The formation of intermediate needs the effect of reverse transcriptase just to be carried out, but the azymia proofreading function, is easy in process of reverse-transcription
Mispairing, which occurs, leads to gene mutation, in HBV gene group the catalyzed combination position of reverse transcriptase polymerase have one it is highly conserved
YMDD motif, i.e. tyrosine-methionine-asparatate-asparatate, Lamivudine can be specifically bound with it,
It is that minus-strand dna and termination DNA chain extension effectively inhibit HBV DNA replication dna by inhibiting pregenome RNA reverse transcription.
YMDD motif is necessary to polymerase activity, is the high conserved region of HBV reverse transcriptase, the HBV after variation is to drawing
The sensibility of meter Fu Ding declines, and so as to cause treatment failure, even results in that sb.'s illness took a turn for the worse.If medicament-resistant mutation continues to use after occurring
Medicine not only increases the unnecessary financial burden of patient and causes the waste of social resources, it is also possible to which there are serious epidemiology
Harm.Clinically whole therapeutic scheme, the rational use of medicines are exchanged in the variation of detection YMDD motif and improving curative effect, there is important guidance to anticipate
Justice.
The medicament-resistant mutation generated during Lomivudine on Hepatitis B is reported in recent years is concentrated mainly on 552 amino acids
YMDD → YIDD/YVDD, i.e., the YMDD motif area HBV occur base substitute G743T or A741G, i.e. Atg → Att/Gtg,
What is interesting is in some medicament-resistant mutations in addition to these base mutations are also often accompanied by other base mutations, as M552V mutation is total adjoint
The L528M mutation in the area P gene B, exist simultaneously may be related with M552V and M552I drug resistance power for two kinds of mutation.It is external real
Test the strong and weak sequence for showing various mutant drug-resistants are as follows: M552I > L528M+M552V > M552V > L528M, at present it has been proposed that
These mutation are divided into I group (M552V/L528M) and II group (M552I).
According to new nomenclature, the variable position and type of the relevant HBV persister of Lamivudine are rtM204I/V (C
Area) ± rtL180M (area B), experiment in vitro shows that rtL180M can not only restore the duplication energy of C region mutation strain (rtM204I/V)
Power, and the tolerance degree to nucleoside analogue drugs can be increased.Two kinds of mutation combinations are prompted to occur more prominent than single form
Become the drug resistance that may more increase virus.
The detection method of medicament-resistant mutation is mainly include the following types: mass spectral analysis, fluorescence polarization, PCR- peptide nucleic acid, limit at present
Property fragment length polymorphic method RFLP processed, Line probe assay method LiPA, quantitative fluorescent PCR melting curve analysis method, PCR are anti-
To Dot hybridization.Mass spectral analysis, fluorescence polarization are very high or cumbersome to technology and equipment requirement, are unfavorable for vast base doctor
Institute carries out application.
The advantages of fluorescent PCR is to be quick on the draw, is specific high: with the special Taqman probe of template sequence in primer spy
The specificity of the PCR further increased on the basis of different；One specific product of every amplification only discharges the fluorescent dye of a molecule,
Instrument detection is specific amplified as a result, non-specific product does not influence detection signal, effectively improves the specificity of detection.
There are many fluorophors of different wave length to available so that Taqman sonde method may be implemented to detect in same pipe it is more
Weight PCR, reduces cost and also improves efficiency avoiding the influence that fluorescent dye reacts PCR with accuracy.
RNase H2 is a kind of enzyme of single-minded identification RNA-DNA heterozygote, and can specific recognition single rna base and DNA
The site of template complementation.This patent is combined using RNase H2 and round pcr develops a kind of novel hepatitis type B virus
YMDD motif area medicament-resistant mutation detection method does not inquire temporarily at present this method applying to hepatitis B virus YMDD motif area
The relevant technologies in medicament-resistant mutation detection.
Summary of the invention
In view of this, the present invention takes full advantage of the advantages of Taqman probe technique, in conjunction with the characteristics of RNase H2, use
Double enzyme systems are directed to HBV YMDD drug-tolerant gene mutation rtM204I/V (area C) ± rtL180M (area B) design mode specific probe,
Each probe marks different fluorescent dyes to detect in different wave length, to improve the mesh for detecting low Resistance mutation gene sensitivity
's.
In a first aspect, provide it is a kind of for detecting the primer sets of hepatitis B virus YMDD motif area medicament-resistant mutation,
Including for detecting the first specific primer sequence of the mutational site rtM204I, second for detecting the mutational site rtM204V
Specific primer sequence and for detecting the mutational site rtL180M third specific primer sequence；First specificity is drawn
Object sequence includes nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2；Second specific primer sequence includes
Nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.3；The third specific primer sequence includes SEQ ID
Nucleotide sequence shown in NO.4 and SEQ ID NO.5.
Preferably, nucleotide sequence shown in SEQ ID NO.2, nucleotide sequence, SEQ shown in SEQ ID NO.3
The end 3' of nucleotide sequence shown in ID NO.4 is respectively connected with spacerarm.
Second aspect provides a kind of for detecting the probe of hepatitis B virus YMDD motif area medicament-resistant mutation, packet
It includes: the first probe sequence corresponding with the mutational site rtM204I/V, and the second probe corresponding with the mutational site rtL180M
Sequence；5 ' the ends label of first probe sequence and the second probe sequence has, and 3 ' ends label has base
Group；First probe sequence includes: nucleotide sequence shown in SEQ ID NO.6；Second probe sequence includes: SEQ
Nucleotide sequence shown in ID NO.7.
Preferably, the fluorophor includes FAM fluorophor, and the quenching group includes quenching group BHQ1.
The third aspect, provide it is a kind of for detecting the kit of hepatitis B virus YMDD motif area medicament-resistant mutation,
It include: DNA profiling, first specific primer sequence~third specific primer sequence, first probe sequence and
Two probe sequences, enzyme mixation, PCR reaction premixed liquid and dNTPs.
Preferably, the PCR reaction premixed liquid includes:
(1) the first PCR reaction premixed liquid: comprising: which PCR buffer, 4 kinds of dNTPs, the first probe sequence and first are special
Specific primer sequence；
(2) the 2nd PCR reaction premixed liquids: comprising: which PCR buffer, 4 kinds of dNTPs, the first probe sequence and second are special
Specific primer sequence；
(3) the 3rd PCR reaction premixed liquids: comprising: which PCR buffer, 4 kinds of dNTPs, the second probe sequence and third are special
Specific primer sequence；
The enzyme mixation includes: RNase H2 and thermal starting Taq archaeal dna polymerase, and the RNase H2 enzyme and Taq
Archaeal dna polymerase concentration ratio is 1:50~1:500.
Preferably, the kit further include: negative quality-control product and positive quality control product.
Preferably, the negative quality-control product includes the normal human serum of the YMDD mutation feminine gender by inactivation treatment；It is described
Positive quality control product includes the positive human serum of the YMDD rtM204I+rtL180M mutation by inactivation treatment.
Preferably, in the enzyme mixation, RNase H2 enzyme and Taq archaeal dna polymerase concentration ratio are 1:250.
Fourth aspect, provide it is a kind of for hepatitis B virus YMDD motif area medicament-resistant mutation detection method, including with
Lower step:
(1) sample extraction: the HBV DNA profiling in serum is extracted；
(2) PCR amplification detects: the HBV DNA extracted using step (1) utilizes kit as claimed in claim 6 as template
PCR amplification is carried out, by the first PCR reaction premixed liquid~the 3rd PCR reaction premixed liquid respectively with enzyme mixation according to mass ratio 36:
It after 0.5 is made into mixed liquor, is added in corresponding reacting hole, carries out PCR amplification after the DNA profiling of extraction is then added, and
Do negative control hole and Positive control wells；The pcr amplification reaction condition are as follows: 95 DEG C initial denaturation 5 minutes, then 95 DEG C denaturation 10
Second, 60 DEG C of annealing extend 45 seconds, totally 35 recycle；
(3) result judgement: according to the signal value of each fluorescence channel to determine whether there is amplification, and determined according to amplification
With the presence or absence of mutation.
The beneficial effects of the present invention are: the present invention develops one kind for detecting hepatitis B virus YMDD motif area drug resistance
Primer sets, probe, kit and the detection method of mutation have used two-phase enzyme system RNase H2 and archaeal dna polymerase skill
Art, due to being changed on the basis of general primer using double enzyme system amplification, i.e., at the end 3' of primer with one
RNA base enables RNase H2 to play a role instead of DNA base, devise for the mutational site rtM204I/V,
The primed probe in the mutational site rtL180M, using fluorescent PCR, closed detection, a possibility that reducing Final pollution, in addition,
Compared to PCR revert dot blot hybridization, detection time can be shortened half, and efficiency significantly improves.
Detailed description of the invention
Fig. 1 show the amplification curve of the mutational site the rtM204I positive；
Fig. 2 show the amplification curve of the mutational site the rtM204V positive；
Fig. 3 show the amplification curve of the mutational site the rtL180M positive；
Fig. 4 show sample rtM204V mutation, rtL180M sports positive amplification curve.
Specific embodiment
Below in conjunction with specific embodiment to provided by the invention a kind of for hepatitis B virus YMDD motif area drug resistance
The kit of mutation is further described.The embodiments described below is exemplary, for explaining only the invention, and cannot
It is interpreted as limitation of the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.Reality as used in the following examples
It tests material unless otherwise specified, is that market is commercially available, and W is A+T degeneracy base, lowercase a, c represent ribonucleic acid
Base, capitalization represent DNA base.
Embodiment one
The present embodiment devises the primer sets for detecting hepatitis B virus YMDD motif area medicament-resistant mutation comprising uses
Draw in detection the first specific primer sequence of the mutational site rtM204I, for detecting second specificity in the mutational site rtM204V
Object sequence and for detecting the mutational site rtL180M third specific primer sequence；Specifically, first specific primer
Sequence includes nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2；Second specific primer sequence includes
Nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.3；The third specific primer sequence includes SEQ ID
Nucleotide sequence shown in NO.4 and SEQ ID NO.5；
Preferably, nucleotide sequence shown in SEQ ID NO.2, nucleotide sequence, SEQ shown in SEQ ID NO.3
The end 3' of nucleotide sequence shown in ID NO.4 is respectively connected with spacerarm x, can specifically in conjunction with template sequence, and
It can specific amplified after RNase H2 shearing.
Embodiment two
The present embodiment designs a kind of for detecting the probe of hepatitis B virus YMDD motif area medicament-resistant mutation comprising:
The first probe sequence corresponding with the mutational site rtM204I/V, and the second probe sequence corresponding with the mutational site rtL180M
Column；5 ' the ends label of first probe sequence and the second probe sequence has, and 3 ' ends label has；
First probe sequence includes: nucleotide sequence shown in SEQ ID NO.6；Second probe sequence includes: SEQ ID
Nucleotide sequence shown in NO.7.
Preferably, the fluorophor includes FAM fluorophor, and the quenching group includes quenching group BHQ1.
Embodiment three
The present embodiment devises a kind of for detecting the kit of hepatitis B virus YMDD motif area medicament-resistant mutation, packet
It includes: in the first specific primer sequence~third specific primer sequence, embodiment two in DNA profiling, embodiment one
One probe sequence and the second probe sequence, enzyme mixation, PCR reaction premixed liquid, dNTPs, negative quality-control product and positive quality control
Product.
Specifically, the PCR reaction premixed liquid includes:
(1) the first PCR reaction premixed liquid: comprising: which PCR buffer, 4 kinds of dNTPs, the first probe sequence and first are special
Specific primer sequence；Preferably, the concentration of first probe sequence and the first specific primer sequence is 0.25 μM；
(2) the 2nd PCR reaction premixed liquids: comprising: which PCR buffer, 4 kinds of dNTPs, the first probe sequence and second are special
Specific primer sequence；Preferably, the concentration of first probe sequence and the second specific primer sequence is 0.25 μM；
(3) the 3rd PCR reaction premixed liquids: comprising: which PCR buffer, 4 kinds of dNTPs, the second probe sequence and third are special
Specific primer sequence；Preferably, the concentration of second probe sequence and third specific primer sequence is 0.25 μM；
The enzyme mixation includes: RNase H2 and thermal starting Taq archaeal dna polymerase, and the RNase H2 enzyme and Taq
Archaeal dna polymerase concentration ratio is 1:50~1:500 (preferably 1:250)；
And the negative quality-control product includes the normal human serum of the YMDD mutation feminine gender by inactivation treatment；The positive matter
Control product include the positive human serum of the YMDD rtM204I+rtL180M mutation by inactivation treatment.
Kit in the present embodiment unlike general reagent box, the present invention using double enzyme system amplification,
That is therefore RNase H2 and the double enzyme systems of Taq archaeal dna polymerase change, i.e., in primer on the basis of general primer
One, the end 3' RNA base is instead of DNA base, such RNase H2 competence exertion effect.The invention patent is passed through to sequence
The optimization design of analysis and primed probe, finishing screen have selected and can dash forward to for rtM204I for one group shown in the invention patent
Conjugate the primed probe of point, the mutational site rtM204V, the mutational site rtL180M.In order to guarantee the reliability of detection system, this
Patent of invention also introduces positive and negative quality-control product.
Example IV
A kind of method for the detection of hepatitis B virus YMDD motif area medicament-resistant mutation is present embodiments provided, it is specific
Include the following steps；
(1) the HBV DNA template in hepatitis type B virus serum sample sample extraction: is extracted using DNA extracting solution；
Detailed process includes: concentrate 100 μ L, 12000rpm to be added into hepatitis type B virus serum centrifugation 10 minutes；
Abandon supernatant；The mixing of 25 μ L lysis buffers is added, 100 DEG C crack for 10 minutes；Last 12000rpm is centrifuged 10 minutes, and supernatant is
HBV DNA profiling used.
(2) PCR amplification detect: using step (1) extract HBV DNA as template, using the kit in embodiment three into
The corresponding augmentation detection of row；
Specifically, by the first PCR reaction premixed liquid~the 3rd PCR reaction premixed liquid respectively with enzyme mixation according to volume ratio
36:0.5 is made into the first mixed liquor~third mixed liquor, then each 36 μ L of the first mixed liquor~third mixed liquor is added to accordingly
Reacting hole in, be then respectively adding the 4 μ l of HBV DNA profiling of extraction, establish 40 μ L PCR reaction systems carry out PCR reaction,
A negative control hole and a Positive control wells are done simultaneously.PCR reaction condition be 95 DEG C initial denaturation 5 minutes, then 95 DEG C change
Property 10 seconds, 60 DEG C of annealing extend 45 seconds, totally 35 circulation；
(3) result judgement: according to the signal value of each fluorescence channel to determine whether there is amplification, and determined according to amplification
With the presence or absence of mutation, specifically, have amplification represent the corresponding detection site of the fluorescence channel be it is positive, i.e., with the presence of being mutated,
Without amplification, then illustrate the corresponding detection site mutation in the channel lower than this kit detection limit.
It should be noted that the technical characteristic in the various embodiments described above can carry out any combination, and the technology being composed
Scheme all belongs to the scope of protection of the present invention.
The mutational site the rtM204I positive sample that measures according to the above method, the mutational site rtM204V positive sample,
The amplification curve of the mutational site rtL180M positive sample is respectively as shown in attached drawing 1~3.
By for the detection of kit Mr. Yu's serum sample of hepatitis B virus YMDD motif area medicament-resistant mutation, use
DNA extracting solution extracts the HBV DNA profiling in serum sample, and step is detected according to the above method, detection gained amplification curve
As shown in Fig. 4, Fig. 4 shows that sample rtM204V sports the positive, and rtL180M sports the positive, kit test result
It is consistent with sequencing result.
Further, the serum sample for choosing 100 HBV patients carries out hepatitis B virus YMDD base using kit
The test of sequence area medicament-resistant mutation, the kit test result of above-mentioned clinical sample is compareed with sequencing result, match rate up to 98.3%,
Clinical sample verification result is good.Kit provided by the invention and detection method can fast and accurately be directed to hepatitis B
Virus YMDD motif area medicament-resistant mutation is detected, and accuracy in detection and detection efficiency are high, for clinically hepatitis B patient reality
When adjustment therapeutic scheme, the rational use of medicines and improve curative effect have important directive significance.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of for detecting the primer sets of hepatitis B virus YMDD motif area medicament-resistant mutation, it is characterised in that: it includes using
Draw in detection the first specific primer sequence of the mutational site rtM204I, for detecting second specificity in the mutational site rtM204V
Object sequence and for detecting the mutational site rtL180M third specific primer sequence；
First specific primer sequence includes nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2；
Second specific primer sequence includes nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.3；
The third specific primer sequence includes nucleotide sequence shown in SEQ ID NO.4 and SEQ ID NO.5.
2. primer sets as described in claim 1, it is characterised in that: nucleotide sequence shown in SEQ ID NO.2, SEQ ID
Nucleotide sequence shown in NO.3, nucleotide sequence shown in SEQ ID NO.4 the end 3' be respectively connected with spacerarm.
3. a kind of for detecting the probe of hepatitis B virus YMDD motif area medicament-resistant mutation, it is characterised in that: the probe packet
It includes: the first probe sequence corresponding with the mutational site rtM204I/V, and the second probe corresponding with the mutational site rtL180M
Sequence；
5 ' the ends label of first probe sequence and the second probe sequence has, and 3 ' ends label has base
Group；
First probe sequence includes: nucleotide sequence shown in SEQ ID NO.6；
Second probe sequence includes: nucleotide sequence shown in SEQ ID NO.7.
4. probe as claimed in claim 3, it is characterised in that: the fluorophor includes FAM fluorophor, described that base is quenched
Group includes quenching group BHQ1.
5. a kind of for detecting the kit of hepatitis B virus YMDD motif area medicament-resistant mutation, it is characterised in that: include: DNA
First visits described in first specific primer sequence described in template, claim 1~third specific primer sequence, claim 2
Needle sequence and the second probe sequence, enzyme mixation, PCR reaction premixed liquid and dNTPs.
6. kit as claimed in claim 5, it is characterised in that: the PCR reaction premixed liquid includes:
(1) the first PCR reaction premixed liquid: comprising: PCR buffer, 4 kinds of dNTPs, the first probe sequence and the first specificity
Primer sequence；
(2) the 2nd PCR reaction premixed liquids: comprising: PCR buffer, 4 kinds of dNTPs, the first probe sequence and the second specificity
Primer sequence；
(3) the 3rd PCR reaction premixed liquids: comprising: PCR buffer, 4 kinds of dNTPs, the second probe sequence and third specificity
Primer sequence；
The enzyme mixation includes: RNase H2 and thermal starting Taq archaeal dna polymerase, and the RNase H2 enzyme and Taq DNA
Polymerase concentration ratio is 1:50~1:500.
7. kit as claimed in claim 6, it is characterised in that: the kit further include: negative quality-control product and positive matter
Control product.
8. kit as claimed in claim 7, it is characterised in that: the feminine gender quality-control product includes the YMDD by inactivation treatment
It is mutated negative normal human serum；The positive quality control product includes the YMDD rtM204I+rtL180M mutation by inactivation treatment
Positive human serum.
9. kit as claimed in claim 6, it is characterised in that: in the enzyme mixation, RNase H2 enzyme and Taq DNA are poly-
Synthase concentration ratio is 1:250.
10. a kind of detection method for hepatitis B virus YMDD motif area medicament-resistant mutation, which is characterized in that including following step
It is rapid:
(1) sample extraction: the HBV DNA profiling in serum is extracted；
(2) PCR amplification detects: the HBV DNA extracted using step (1) is carried out as template using kit as claimed in claim 6
PCR amplification, by the first PCR reaction premixed liquid~the 3rd PCR reaction premixed liquid respectively with enzyme mixation according to weight ratio 36:0.5
It after being made into mixed liquor, is added in corresponding reacting hole, carries out PCR amplification after the DNA profiling of extraction is then added, and do one
A negative control hole and a Positive control wells；The pcr amplification reaction condition are as follows: 95 DEG C initial denaturation 5 minutes, then 95 DEG C change
Property 10 seconds, 60 DEG C of annealing extend 45 seconds, totally 35 circulation；
(3) result judgement: according to the signal value of each fluorescence channel to determine whether there is amplification, and determined whether according to amplification
There are mutation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110846426A (en) * | 2019-12-13 | 2020-02-28 | 华南农业大学 | Fluorescent PCR (polymerase chain reaction) primer group and kit for rapidly detecting salmonella pullorum |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234368A (en) * | 2018-08-13 | 2019-01-18 | 武汉千麦医学检验所有限公司 | A method of for hepatitis B virus YMDD motif area medicament-resistant mutation |
| CN109234367A (en) * | 2018-08-13 | 2019-01-18 | 武汉千麦医学检验所有限公司 | A kind of kit for hepatitis B virus YMDD motif area medicament-resistant mutation |
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2018
- 2018-09-29 CN CN201811145051.8A patent/CN109161588A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234368A (en) * | 2018-08-13 | 2019-01-18 | 武汉千麦医学检验所有限公司 | A method of for hepatitis B virus YMDD motif area medicament-resistant mutation |
| CN109234367A (en) * | 2018-08-13 | 2019-01-18 | 武汉千麦医学检验所有限公司 | A kind of kit for hepatitis B virus YMDD motif area medicament-resistant mutation |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110846426A (en) * | 2019-12-13 | 2020-02-28 | 华南农业大学 | Fluorescent PCR (polymerase chain reaction) primer group and kit for rapidly detecting salmonella pullorum |
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