CN105624332B - Triple real-time fluorescence quantitative PCR detection methods of three kinds of retrovirus of monkey - Google Patents
Triple real-time fluorescence quantitative PCR detection methods of three kinds of retrovirus of monkey Download PDFInfo
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Abstract
The invention discloses a kind of triple real-time fluorescence quantitative PCR detection methods of three kinds of retrovirus of monkey.Select the gag gene of monkey immunodeficiency virus SIV, the 5'LTR gene of monkey reverse D type virus SRV1, the gag gene conserved region sequence of monkey T lymphocyte taxis virus I-form STLV1 is as detection target sequence, synthesize its DNA fragmentation, and separately design three pairs of primer pairs of synthesis and 3 probes: SIV: upper primer SI-F, lower primer SI-R, probe SI-P, SRV1: upper primer SR-F, lower primer SR-R, probe SR-P, STLV1: upper primer ST-F, lower primer ST-R, probe ST-P, establish triple real-time fluorescence quantitative PCR detection methods, accurate qualitative detection can be carried out to three kinds of monkey retrovirus.The invention further relates to detection kit, have the advantages that detection specificity is high, accuracy is high, sensitivity is good.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of triple real-time fluorescences of three kinds of retrovirus of monkey
Quantitative PCR detecting method.
Background technique
Monkey immunodeficiency virus (Simian immunodeficiency virus, SIV), also referred to as cercopithecus aethiops virus
(African Green Monkey virus) is a kind of retrovirus that can influence at least 33 kinds African Primates.SIV with
There is higher homology in gene order with inhibition of HIV, after non-human primates taint with SIV, the morbidity machine of similar AIDS occurs
The lesion of the damage of immune system and multisystem multiple organ tissue, SIV, can occur after virus infection in system and clinical symptoms performance
It is distributed in rhesus monkeys relatively broad, can be involved multiple systems including immune system.
Monkey D type retrovirus (simian type-Dretro virus, SRV) belongs to Retroviridae, D type reverse transcription
Tobamovirus is all that monkey acquired immunity lacks with monkey immunodeficiency virus (simian immunodeficiency virus, SIV)
Fall into a kind of Causative virus of syndrome (SAIDS).The SRV having now been found that has 5 serotypes: SRV-1, and 2,3,4,5.SRV master
The target cell wanted is lymphocyte, lymphocyte can be caused to exhaust after infection, induces immune deficiency.Various SRV has it
Attribute and group can make 80% or more monkey morbidity lethal once being passed to susceptible monkey group.
Monkey T lymphocyte leukemia virus (simian T-lymphotropic virus, STLV1) is white with people's T lymph
Blood disease viral (human T-lymphotropic virus, HTLV) is referred to as the thermophilic T lymphocyte virus (primate of primate
T-lymphotropic virus, PTLV), belong to Epsilonretrovirus ε.Monkey T lymphocyte taxis virus is initially from Asia
It is separated to in African monkey body, artificial propagation monkey and wild monkey can be infected by STLV1, but show without clinical symptoms.By STLV1
There is anemia similar with the people infected by HTLV in the cercopithecus aethiops of infection, and the heredity of the HTLV of STLV1 and people are same
Source property is up to 90-95%, and there are some researches prove China is wild and raising rhesus serum is widely present STLV1 antibody.STLV1 virus
The immune system of main infringement monkey, causes the lesion or immunity function disorder of immune organ, so that interference experiment is studied, it is SPF
One of several viruses that monkey must exclude.
Multiple fluorescence quantitative PCR technology is the new technology to grow up on the basis of quantitative fluorescent PCR, it is emphasized
Multiple targets are expanded in the same reaction system simultaneously.Have multinomial research recently and multiple quantitative PCR technology is applied to virus
Detection field.Weyer CT etc. establishes triple real-time fluorescences of the qualitative detection to 9 kinds of serotype molecule partings of African horse sickness
PCR method；Haines FJ etc. establishes while detecting classic swine fever and African swine fever multiplex real-time reverse transcriptase PCR method；Perhaps
The triple real time fluorescent PCR methods established while detecting three kinds of kinds of fish Rhabdovirus such as bright are built, as the result is shown multiplex PCR system
It is essentially identical in terms of sensitivity and specificity with single amplification.But up to the present, report there is no to have while detecting
The kit of SIV, SRV1 and STLV1.
For retrovirus, immunosupress is generated by the animals of such virus infection, is caused special in serum
Property antibody level it is relatively low, cause antibody test false negative phenomenon occur, real-time quantitative PCR passes through directly to the cause of disease of infection animal
Body nucleic acid specific fragment is expanded, the Strength Changes of fluorescence signal in detection amplification, to reach testing goal.This method
Advantage is, directly detects to the nucleic acid of retrovirus, improves the specificity of detection；And for retrovirus core
The amplification and amplification of sour specific fragment, improve the sensibility of detection.
According to target gene kind inner height, conservative, inter-species high special selection principle, compares by sequence B LAST and analyzes,
The gag gene of SIV, the 5'LTR gene of SRV1, STLV1 gag gene as detection target gene ideal candidate.The present invention
These three genes are chosen as target gene, design primer and probe, experiment screening is carried out to the multipair primer combination of probe of design
Optimization, obtains best primer combination of probe, establishes triple real-time fluorescence PCR detection methods.
Summary of the invention
Based on the above shortcomings, the purpose of the present invention is to provide a kind of the triple glimmering in real time of three kinds of retrovirus of monkey
Fluorescent Quantitative PCR detection method and its primer special, probe and corresponding kit is provided based on this method.The kit can be used for
The detection of monkey retrovirus.More particularly to the monkey immunodeficiency virus (SIV) of test experience monkey, monkey retrovirus simultaneously
(SRV1) and viral 1 type (STLV1) of monkey T lymphocyte taxis.
A kind of primer pair of the detection method that three kinds of triple real-time fluorescence quantitative PCRs of retrovirus of monkey can be detected simultaneously and
Probe,
Monkey immunodeficiency virus SIV:
Upper primer SI-F: as shown in sequence table Seq.No1,
Lower primer SI-R: as shown in sequence table Seq.No2,
Probe SI-P: as shown in sequence table Seq.No3；
Monkey reverses D type virus SRV1:
Upper primer SR-F: as shown in sequence table Seq.No4,
Lower primer SR-R: as shown in sequence table Seq.No5,
Probe SR-P: as shown in sequence table Seq.No6；
Monkey T lymphocyte taxis virus I-form STLV1:
Upper primer ST-F: as shown in sequence table Seq.No7,
Lower primer ST-R: as shown in sequence table Seq.No8,
Probe ST-P: as shown in sequence table Seq.No9.
The present invention also has following technical characteristic:
1, a kind of positive of detection method that can detect three kinds of triple real-time fluorescence quantitative PCRs of retrovirus of monkey simultaneously is right
According to the preparation method of product, include the following steps,
The preparation of step (1) pcr template: the synthesis of SIV, SRV1, STLV1 conserved region gene sequence.
Step (2) is using each sequence synthesized in step (1) as template, and the above primer SI-F, lower primer SI-R, above draw respectively
Object SR-F, lower primer SR-R, upper primer ST-F, lower primer ST-R are primer, carry out the PCR amplification of target gene；
Step (3) positive reference substance pMD18-SIV, the preparation of pMD18-SRV1, pMD18-STLV1:
In step (2), the PCR reaction system of target gene is as follows:
Primer each 15 μ L of 2 μ L, 2 × Taq MasterMix, 5 μ L of template, ultrapure water complement to 30 μ L.
The reaction condition of PCR are as follows: 50 DEG C of 30min of reverse transcription；95 DEG C of 5min thermal startings；95 DEG C of 15s, 55 DEG C of 25s, 72 DEG C are prolonged
15s is stretched, carries out 40 circulations altogether；72 DEG C of extension 10min；
In step (3), the preparation process of positive reference substance includes the following steps: gained PCR product point in step (2)
It is not connect with cloning vector pMD-18-T carrier, converts E. coli competent DH5 α, obtain positive colony bacterial strain, and prepare matter
Grain pMD18-SIV, pMD18-SRV1, pMD18-STLV1 is as positive reference substance.
2, a kind of positive control that can detect three kinds of triple real-time fluorescence quantitative PCRs of retrovirus of monkey simultaneously as described above
Product, pMD18-SIV, as shown in sequence table Seq.No10；PMD18-SRV1, as shown in sequence table Seq.No11；pMD18-
STLV1, as shown in sequence table Seq.No12.
3, a kind of detection method that can detect three kinds of triple real-time fluorescence quantitative PCRs of retrovirus of monkey simultaneously, PCR reaction
System is as follows:
12.5 μ L of RT-PCR reaction solution, 0.5 0.5 μ L of μ L, RTMIX of Taq enzyme, (200nmol/L is wrapped primer mixed liquor respectively
It is 1:1:1 containing three kinds of viral specific primer ratios) totally 3 μ L, (100nmol/L separately includes three kinds of viruses to probe mixed liquor
Specificity fluorescent probe, ratio 1:1:1) totally 3 μ L, the 5 μ L of sample nucleic acid of extraction remove RNAase as reaction template
Free water complements to 25 μ L；
The reaction condition of PCR are as follows: 50 DEG C of 30min of reverse transcription；95 DEG C of 5min thermal startings, later with 95 DEG C of 15s, 55 DEG C of 25s,
40 circulations are carried out altogether.
4, a kind of detection kit that can detect three kinds of triple real-time fluorescence quantitative PCRs of retrovirus of monkey simultaneously, packet
It is as follows to include reagent,
Primer pair and probe:
Monkey immunodeficiency virus SIV:
Upper primer SI-F: as shown in sequence table Seq.No1,
Lower primer SI-R: as shown in sequence table Seq.No2,
Probe SI-P: as shown in sequence table Seq.No3；
Monkey reverses D type virus SRV1:
Upper primer SR-F: as shown in sequence table Seq.No4,
Lower primer SR-R: as shown in sequence table Seq.No5,
Probe SR-P: as shown in sequence table Seq.No6；
Monkey T lymphocyte taxis virus I-form STLV1:
Upper primer ST-F: as shown in sequence table Seq.No7,
Lower primer ST-R: as shown in sequence table Seq.No8,
Probe ST-P: as shown in sequence table Seq.No9；
Positive reference substance:
PMD18-SIV, as shown in sequence table Seq.No10,
PMD18-SRV1: as shown in sequence table Seq.No11,
PMD18-STLV1: as shown in sequence table Seq.No12；
RT-PCR premix needed for negative controls ddH2O, Viral nucleic acid extraction reagent, real-time fluorescence quantitative PCR amplification
Liquid and Taq enzyme.
It is an advantage of the present invention that tri- kinds of viruses of SIV, SRV1 and STLV1 can be detected simultaneously, directly to retrovirus
Nucleic acid is detected, and the specificity of detection is improved；And amplification and amplification for retroviral nucleic acid specific fragment, it improves
The sensibility of detection.
Detailed description of the invention
Fig. 1 is three kinds of recombination positive plasmid PCR qualification result figures.
Fig. 2 is SIV viral nucleic acid standard items real-time fluorescence quantitative RT-PCR canonical plotting.
Fig. 3 is SRV1 viral nucleic acid standard items real-time fluorescence quantitative RT-PCR canonical plotting.
Fig. 4 is STLV1 viral nucleic acid standard items real-time fluorescence quantitative RT-PCR canonical plotting.
Fig. 5 is the triple qRT-PCR sensitivity tests result figures of SIV.
Fig. 6 is the triple qRT-PCR sensitivity tests result figures of SRV1.
Fig. 7 is the triple qRT-PCR sensitivity tests result figures of STLV1.
Fig. 8 is the triple qRT-PCR specific test result figures of SIV, SRV1 and STLV1.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
The synthesis of target gene
The sequence in three kinds of viruses announced according to GenBank determines the gag base with SIV in conjunction with bioinformatics means
Cause, the gag gene conserved region sequence of the 5'LTR gene of SRV1, STLV1 synthesize its DNA fragmentation, gene as detection target sequence
Synthesis is completed by the raw work biotechnology service in Shanghai.Virus gene sequence is as shown in table 1.
1: three kind of viral DNA sequence dna of table
Embodiment 2
The synthesis of specific primer
According to the three of synthesis kinds of viral gene orders, specific primer is synthesized, sequence is as shown in table 2.
Table 2: specific primer sequence
Embodiment 3
The preparation of positive plasmid
1, PCR amplification target gene
(1) SIV gene is expanded:
PCR reaction system: primer each 15 μ L of 2 μ L, 2 × Taq MasterMix, 5 μ L of template, ultrapure water complement to 30 μ L.
The reaction condition of PCR are as follows: 50 DEG C of 30min of reverse transcription；95 DEG C of 5min thermal startings, later with 95 DEG C of 15s, 55 DEG C of 25s,
72 DEG C of extension 15s carry out 40 circulations altogether；72 DEG C of extension 10min.
(2) amplification of SRV1, STLV gene
PCR system and reaction condition are as described in (1).
2, the preparation of positive plasmid
To be about containing SIV gag gene, 5 ' LTR gene of SRV1 and STLV1gag gene PCR amplified production respectively
95-139bp segment is cloned into pMD18T carrier (Takara) respectively, and specific operation process is as follows:
(1) following DNA solution is prepared in microcentrifugal tube, full dose is 5 μ L.
pMD18-T Vector 1μL
1 μ L of target gene PCR product
ddH2O 3μL
(2) Solution I of 5 μ L (equivalent) is added.
(3) 16 DEG C of reaction 30min.
(4) the ice bath 30min into 100 μ L DH5 α competent cells is added in full dose (10 μ L).
(5) 42 DEG C of heating 45s, then 1min is placed in ice.
(6) 890 μ LSOC culture mediums, 37 DEG C of shaken cultivation 60min are added.
(7) it is cultivated on the L- Agar Plating containing X-Gal, IPTG, Amp, forms single colonie.Counting white,
Blue colonies.
(8) white colony is selected, the length scale of Insert Fragment in PCR method confirmation carrier is used.As a result as shown in Figure 1.
(9) the positive colony recombinant bacterial strain Multiplying culture for going out screening and identification extracts recombinant plasmid.Recombinant plasmid is distinguished
It is named as pMD18-SIV, pMD18-SRV1, pMD18-STLV1.
(10) plasmid concentration is measured using ultraviolet specrophotometer, according to conventional cut-and-try formula: C=[(N × 6.02 ×
1023)/(MW×109) plasmid copy number is calculated, wherein N is plasmid concentration, and unit is ng/ μ L, and C is that every microlitre of plasmid extraction is molten
Contained target gene copy number in liquid；MW is cloned plasmids molecular weight.
Embodiment 2
The foundation of triple real-time fluorescence PCR detection methods
Probe is designed according to three kinds of viral gene orders, sequence is as follows:
SIV (monkey immunodeficiency virus) gene probe:
SI-P:5’-(FAM)–CTCCTCTGCCGCTAGATGGTG-(BHQ1-3)-3’
SRV1 (monkey reverses D type virus) gene probe:
SR-P:5’-(CY5)-CTCCAGGTTTCCTACTGTTGGT-(BHQ2-3)-3’
STLV (monkey T lymphocyte taxis virus I-form) gene probe:
ST-P:5’-(HEX)-ACAGGCCATTAAGCAA-(BHQ1-3)-3’
Each component concentration in real-time fluorescence PCR reaction system is changed using single-factor concentration gradient experimental method one by one, with excellent
Change real-time fluorescence PCR reaction system, finally determine triple real-time fluorescence PCRs while three kinds of viral reaction systems of detection are as follows:
One-step method: 12.5 μ L of RT-PCR reaction solution, Taq enzyme 0.5 μ L, RTMIX 0.5 μ L, primer mixed liquor (200nmol/
L, separately including three kinds of viral specific primer ratios is 1:1:1) totally 3 μ L, (100nmol/L is separately included probe mixed liquor
Three kinds of viral specificity fluorescent probes, ratio 1:1:1) totally 3 μ L, the 5 μ L of sample nucleic acid of extraction are gone as reaction template
RNAase Free water complements to 25 μ L.
The reaction condition parameter of PCR are as follows: 50 DEG C of 30min of reverse transcription；95 DEG C of 5min thermal startings, later with 95 DEG C of 15s, 55 DEG C
25s carries out 40 circulations altogether.It is detected using ABI7500 quantitative fluorescent PCR.
Embodiment 3
The Virus Standard product for having demarcated copy number are subjected to doubling dilution, concentration is respectively 108-102Copies/mL is carried out
PCR amplification, using CT value as ordinate, establishes standard curve using viral nucleic acid copy number as abscissa.As shown in Figure 2,3, 4, three
The standard curve of kind viral nucleic acid is respectively provided with good sheerly relationship.SIV, SRV1 and the amplification efficiency (E) of STLV1 and related
Coefficient (r2) is respectively as follows: 98.908%, 0.989；95.115%, 0.990；96.465%, 0.998.
Embodiment 4
The sensitivity test of method
Cloned plasmids containing target viral gene are demarcated, copy number is adjusted to SIV (108copies/mL)、SRV1
(108) and STLV1 (10 copies/mL8Copies/mL gradient dilution (10 is carried out after) respectively3-108Copies/ml), each dilute
Degree of releasing does 3 in parallel, and reaction condition presses the reference value after above-mentioned optimization and carries out fluorescence quantitative RT-RCR reaction.Using CT value as vertical
Coordinate draws standard curve by abscissa PCR instrument of samples copy number automatically.The results show that being set as 40 when will test CT thresholding
When a cycle values, then the triple real-time fluorescent quantitative RT-PCR methods established can detect that the lower limit of each viral nucleic acid sample is equal
It can reach 103Copies/ml illustrates that sensibility is good.As a result as shown in Fig. 5, Fig. 6, Fig. 7.
Embodiment 5
Specific test
Respectively with containing SIV, STLV1, SRV1, b virus of monkey (BV) and monkey measles virus (MV) positive serum or whole blood
Sample is detected as referring to the fluorescence quantitative RT-RCR for carrying out target viral, while observation caliber product and feminine gender are bent according to whether there is or not amplifications
Line generates, and the specificity of this method is verified with this.The results show that 3 kinds of viruses can specifically identify corresponding template, not with its
Cross reaction occurs for its reference virus, illustrates specific preferable.As a result as shown in Figure 8.
Embodiment 6
Repetitive test
By each Virus Sample (10 of different diluted concentrations6、105、104) SIV, SRV1, STLV1 carry out according to above-mentioned condition
Fluorescence quantitative RT-RCR detection, each dilution of every kind of virus are repeated 6 times, and calculate separately CT value, standard deviation (s) and variation lines
Number (CV).For the detection CT value standard of the respective sample of different nucleic acid concentrations between 1.12-2.3, the coefficient of variation is below 1%, says
It is bright that there is preferable repeatability.
Embodiment 7
Practical application
SIV, SRV1 and STLV1 have been carried out to 18 parts of samples of acquisition using triple real-time fluorescence RT-PCR methods of foundation
The screening of nucleic acid detects 1 part of SIV, 0 part of SRV1,2 parts of STLV1 altogether.It is carried out using substance real-time fluorescence PCR detection method
Verifying, coincidence rate 100%, display the method for the present invention have good reliability and practicability.
Embodiment 8
The assembling of kit
Negative controls are ddH in kit2O, and include that viral nucleic acid DNA extracts reagent, real-time fluorescence quantitative PCR expands
The reagents such as RT-PCR premixed liquid, Taq enzyme, primer and probe needed for increasing.
Claims (3)
1. a kind of primer pair and spy of the detection method that can detect three kinds of triple real-time fluorescence quantitative PCRs of retrovirus of monkey simultaneously
Needle, which is characterized in that
Monkey immunodeficiency virus SIV:
Upper primer SI-F: as shown in sequence table Seq ID No.1,
Lower primer SI-R: as shown in sequence table Seq ID No.2,
Probe SI-P: as shown in sequence table Seq ID No.3；
Monkey reverses D type virus SRV1:
Upper primer SR-F: as shown in sequence table Seq ID No.4,
Lower primer SR-R: as shown in sequence table Seq ID No.5,
Probe SR-P: as shown in sequence table Seq ID No.6；
Monkey T lymphocyte taxis virus I-form STLV1:
Upper primer ST-F: as shown in sequence table Seq ID No.7,
Lower primer ST-R: as shown in sequence table Seq ID No.8,
Probe ST-P: as shown in sequence table Seq ID No.9.
2. a kind of positive reference substance for the detection method that can detect three kinds of triple real-time fluorescence quantitative PCRs of retrovirus of monkey simultaneously
Preparation method, include the following steps,
The preparation of step (1) pcr template: with the gag gene conserved region of the gag gene of SIV, the 5'LTR gene of SRV1, STLV1
Sequence is as detection target sequence, the above primer SI-F of difference, lower primer SI-R, upper primer SR-F, lower primer SR-R, upper primer
ST-F, lower primer ST-R are primer, carry out the PCR amplification of target gene；The PCR reaction system of target gene is as follows: primer each 2
μ L, 2 × Taq MasterMix 15 μ L, 5 μ L of template, ultrapure water complement to 30 μ L；The reaction condition of PCR are as follows: 50 DEG C of reverse transcription
30min；95 DEG C of 5min thermal startings；95 DEG C of 15s, 55 DEG C of 25s, 72 DEG C of extension 15s carry out 40 circulations altogether；72 DEG C of extensions
10min；
Step (2) positive reference substance pMD18-SIV, the preparation of pMD18-SRV1, pMD18-STLV1: by gained in step (1)
PCR product is connect with cloning vector pMD-18-T carrier respectively, converts E. coli competent DH5 α, obtains positive colony bacterium
Strain, and plasmid pMD18-SIV is prepared, pMD18-SRV1, pMD18-STLV1 is as positive reference substance；It is characterized in that,
Wherein, pMD18-SIV, as shown in sequence table Seq ID No.10；PMD18-SRV1, such as sequence table Seq ID No.11 institute
Show；PMD18-STLV1, as shown in sequence table Seq ID No.12.
3. a kind of detection kit that can detect three kinds of triple real-time fluorescence quantitative PCRs of retrovirus of monkey simultaneously, feature
It is, including reagent is as follows:
Primer pair and probe:
Monkey immunodeficiency virus SIV:
Upper primer SI-F: as shown in sequence table Seq ID No.1,
Lower primer SI-R: as shown in sequence table Seq ID No.2,
Probe SI-P: as shown in sequence table Seq ID No.3；
Monkey reverses D type virus SRV1:
Upper primer SR-F: as shown in sequence table Seq ID No.4,
Lower primer SR-R: as shown in sequence table Seq ID No.5,
Probe SR-P: as shown in sequence table Seq ID No.6；
Monkey T lymphocyte taxis virus I-form STLV1:
Upper primer ST-F: as shown in sequence table Seq ID No.7,
Lower primer ST-R: as shown in sequence table Seq ID No.8,
Probe ST-P: as shown in sequence table Seq ID No.9；
Positive reference substance:
PMD18-SIV, as shown in sequence table Seq ID No.10,
PMD18-SRV1: as shown in sequence table Seq ID No.11,
PMD18-STLV1: as shown in sequence table Seq ID No.12；
Negative controls ddH2O, Viral nucleic acid extraction reagent, real-time fluorescence quantitative PCR amplification needed for RT-PCR premixed liquid and
Taq enzyme.
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| CN106636461B (en) * | 2016-09-21 | 2018-08-14 | 广东省实验动物监测所 | A kind of detection primer group and method that can be detected simultaneously and distinguish SIV, SRV, STLV |
| CN106244730B (en) * | 2016-09-21 | 2018-08-14 | 广东省实验动物监测所 | RT-LAMP detection primers group, kit and the detection method of the thermophilic T lymphotropic virus type Is of monkey |
| CN107653345B (en) * | 2017-11-08 | 2021-04-27 | 中国人民解放军军事医学科学院野战输血研究所 | Real-time fluorescence quantitative PCR method for simian B virus, universal primer, MGB probe and kit thereof |
| CN108490173A (en) * | 2018-03-10 | 2018-09-04 | 广东省实验动物监测所 | A kind of multiple liquid-phase chip detection method and kit |
| CN111304370B (en) * | 2020-04-22 | 2023-05-26 | 广东省实验动物监测所 | SIV primer and probe for rapidly detecting monkey immunodeficiency virus, kit and use method thereof |
| CN113025753B (en) * | 2021-03-26 | 2023-05-26 | 中国人民解放军军事科学院军事医学研究院 | Real-time fluorescent quantitative PCR method for universal TaqMan-MGB probe for monkey B virus |
| CN115323076B (en) * | 2022-08-05 | 2023-06-30 | 武汉珈创生物技术股份有限公司 | Primer probe composition, kit and detection method for detecting monkey D-type retrovirus |
| CN115852058A (en) * | 2022-12-23 | 2023-03-28 | 苏州药明检测检验有限责任公司 | Primer and probe combination for detecting T cell lymphotropic virus of monkey and application thereof |
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