CN107488746A - A kind of method and its special complete reagent for detecting HTLV - Google Patents
A kind of method and its special complete reagent for detecting HTLV Download PDFInfo
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- CN107488746A CN107488746A CN201710821933.0A CN201710821933A CN107488746A CN 107488746 A CN107488746 A CN 107488746A CN 201710821933 A CN201710821933 A CN 201710821933A CN 107488746 A CN107488746 A CN 107488746A
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Abstract
The invention discloses a kind of method and its special complete reagent for detecting HTLV.Reagent set includes primer pair HTLV and probe HTLV；Primer pair HTLV is made up of primer HTLV F and primer HTLV R；Target sequences of the primer pair HTLV in HTLV genomes contains special RNA fragments；The nucleotide sequence for the specific DNA fragment that special RNA fragment reverse transcriptions obtain is as shown in the sequence 1 of sequence table；Probe HTLV is the single strand dna of 20 30 nucleotides compositions, identical with the partial sector in specific DNA fragment.It is demonstrated experimentally that detecting HTLV 1 or HTLV 2 using the reagent set, specificity is good, and sensitivity is higher (sensitivity reaches 100copies/ reaction systems).Competitive internal standard of the internal standard as target nucleic acids in reagent set simultaneously, can be used as object of reference, for preventing the generation of false negative result.The present invention has important application value.
Description
Technical field
The present invention relates to field of medical examination, and in particular to a kind of method of HTLV and its special of detecting
Reagent set.
Background technology
Human t lymphotropic virus (Human T-cell lymphotropic virus, HTLV) is 1980
The first human retrovirus that American Gallo has found from skin-type t cell lymphoma patient's body first, by genome
And serological reaction can be divided into 1 type (HTLV-1) and 2 types (HTLV-2).The virus is spherical particles, and inside is by RNA nucleoprotein
And 20 outer face body protein capsid compositions are centered around, outermost layer has capsule structure, and surface inserting has glycoprotein.The whole world at present
About 1000 to 2,000 ten thousand people infect HTLV, and in the Beijing in China, Guangxi, Jiangxi, Xinjiang, Liuzhou, Hefei and Sichuan etc. 10
Multiple provinces and cities have found HTLV cases of infection, while localized fine scale prevalence also occur in some coastal areas.HTLV is most
Adult T-cell leukemia, tropical spastic paralysis and the generation of other related neurological disorders can be caused eventually.
HTLV route of transmission mainly has Transfusion Transmission, breast milk to propagate and spread through sex intercourse three kinds, and effective curative so far
Thing and preventative vaccine are not yet succeeded in developing.Therefore, unique positive method of controlling is exactly timely discovery HTLV the infected, and is adopted
Measure is taken to cut off its route of transmission.
More conventional detection method is to use ELISA method detection HTLV antibody at present, other to also have indirect immunofluorescence
Method, Western blot and recombinant immune marking etc..But research is shown and not all HTLV the infected can produce antibody, and
Remained in blood preparation in unicellular infection, therefore Serologic detection has some false positives and a Problem of False Negative, specificity compared with
Difference, it can not solve the above problems well.
The content of the invention
The technical problems to be solved by the invention are how to detect HTLV.
In order to solve the above technical problems, present invention firstly provides detection HTLV reagent set, it may include primer pair
HTLV and probe HTLV；The primer pair HTLV can be made up of primer HTLV-F and primer HTLV-R；The primer pair HTLV exists
Target sequence in HTLV genomes contains special RNA fragments；The specific DNA fragment that the special RNA fragment reverse transcriptions obtain can
For following y1) or y2)：
Y1) the single strand dna shown in the sequence 1 of sequence table；
Y2 sequence 1 by the substitution of one or several nucleotides and/or missing and/or addition and) had into phase with sequence 1
The DNA molecular of congenerous；
The probe HTLV can be the single strand dna that 20-30 nucleotides form, and in the specific DNA fragment
Partial sector is identical.
The primer HTLV-F can be following a1) or a2)：
A1) the single strand dna in sequence table shown in sequence 2；
A2 sequence 2 by the substitution of one or several nucleotides and/or missing and/or addition and) had into phase with sequence 2
The single strand dna of congenerous.
The primer HTLV-R can be following b1) or b2)：
B1) the single strand dna in sequence table shown in sequence 3；
B2 sequence 3 by the substitution of one or several nucleotides and/or missing and/or addition and) had into phase with sequence 3
The single strand dna of congenerous.
The probe HTLV can be following c1) or c2)：
C1) the single strand dna in sequence table shown in sequence 4；
C2 sequence 4 by the substitution of one or several nucleotides and/or missing and/or addition and) had into phase with sequence 4
The single strand dna of congenerous.
The end of the probe HTLV can have fluorescence labeling.
Mark fluorescent group and group can be quenched respectively in the both ends of the probe HTLV.
5 ' the ends of the probe HTLV can have FAM fluorescence labelings, and 3 ' ends can have TAMRA fluorescence labelings.
The reagent set can be specifically made up of the primer pair HTLV and the probe HTLV.
In the reagent set, the primer HTLV-F, the primer HTLV-R and the probe HTLV mol ratio tool
Body can be 2.5：2.5：1.
In above-mentioned reagent set, the primer HTLV-F, the primer HTLV-R and the probe HTLV measurer body can
It is as follows：6.25 μM of primer HTLV-F, 6.25 μM of primer HTLV-R and 2.5 μM of probe HTLV.
Any of the above-described reagent set may also include internal standard and internal standard detection probe；Single strand RNA molecule is designated as in described；
The DNA fragmentation that the internal standard reverse transcription obtains can be following z1) or z2)：
Z1) the single strand dna shown in the sequence 5 of sequence table；
Z2 sequence 5 by the substitution of one or several nucleotides and/or missing and/or addition and) had into phase with sequence 5
The DNA molecular of congenerous；
The internal standard detection probe can be the single strand dna of 20-30 nucleotides composition, be obtained with the internal standard reverse transcription
To DNA fragmentation in partial sector it is identical.
The internal standard detection probe can be following d1) or d2)：
D1) the single strand dna in sequence table shown in sequence 6；
D2 sequence 6 by the substitution of one or several nucleotides and/or missing and/or addition and) had into phase with sequence 6
The single strand dna of congenerous.
The end of the internal standard detection probe can have fluorescence labeling.
Mark fluorescent group and group can be quenched respectively in the both ends of the internal standard detection probe.
5 ' ends of the internal standard detection probe can have JOE fluorescence labelings, and 3 ' ends can have TAMRA fluorescence labelings.
The reagent set can specifically be examined by the primer pair HTLV, the probe HTLV, the internal standard and the internal standard
Probing pin forms.
Any of the above-described reagent set may also include working standard；The working standard is single strand RNA molecule；Institute
It is the specific DNA fragment to state the DNA fragmentation that working standard reverse transcription obtains.
Any of the above-described reagent set may also include negative control.The negative control can be without the normal of HTLV RNA
Human plasma.
Any of the above-described reagent set may also include reference material.The reference material can be the human plasma of the RNA containing HTLV-1
Or the human plasma of the RNA containing HTLV-2.
The reagent set can specifically be detected by the primer pair HTLV, the probe HTLV, the internal standard, the internal standard
Probe, the working standard, the negative control and reference material composition.
The preparation method of any of the above-described reagent set, including by the primer in any of the above-described reagent set
The step of HTLV-F, the primer HTLV-R and the probe HTLV are individually packed.
Kit containing any of the above-described reagent set falls within protection scope of the present invention.
D1) or d2) or d3) or d4) fall within protection scope of the present invention：
D1) any of the above-described reagent set is preparing the application in being used to detect HTLV kit；
D2) any of the above-described reagent set or the kit whether contain in testing sample is detected or it is doubtful containing
Application in HTLV；
D3) any of the above-described reagent set is preparing the application in being used to identify HTLV kit；
D4) any of the above-described reagent set or the kit identify virus to be measured whether be or whether candidate is
Application in HTLV.
The present invention also protect a kind of detection testing sample whether the method containing HTLV, can be A1) or A2)：
A1) using the total serum IgE of testing sample as template, real-time quantitative PCR detection is carried out with any of the above-described reagent set,
Then judged as follows：If the reagent set can realize the real-time quantitative PCR to the total serum IgE, testing sample
In contain or doubtful containing HTLV；If the reagent set can not realize the real-time quantitative PCR to the total serum IgE, to be measured
Do not contained in sample or doubtful do not contain HTLV；
A2) specific DNA fragment, Ran Houjin whether are contained in the DNA fragmentation that the total serum IgE reverse transcription of detection testing sample obtains
Row is following to be judged：If the DNA fragmentation that the total serum IgE reverse transcription of testing sample obtains contains specific DNA fragment, in testing sample
Contain or doubtful containing HTLV；If the DNA fragmentation that the total serum IgE reverse transcription of testing sample obtains does not contain specific DNA fragment,
Testing sample does not contain or doubtful does not contain HTLV；
The specific DNA fragment can be following y1) or y2)：
Y1) the single strand dna shown in the sequence 1 of sequence table；
Y2 sequence 1 by the substitution of one or several nucleotides and/or missing and/or addition and) had into phase with sequence 1
The DNA molecular of congenerous.
The present invention also protect it is a kind of identify virus to be measured whether the method for being HTLV, can be B1) or B2)：
B1) using viral RNA to be measured as template, real-time quantitative PCR detection is carried out with any of the above-described reagent set, so
After judged as follows：If the reagent set can realize the real-time quantitative PCR to the RNA, virus to be measured be or
Candidate is HTLV；If the reagent set can not realize the real-time quantitative PCR to the RNA, virus to be measured is not or waited
Choosing is not HTLV；
B2) detect in the DNA fragmentation that viral RNA reverse transcriptions to be measured obtain whether contain specific DNA fragment, then carry out
It is following to judge：If containing specific DNA fragment in the DNA fragmentation that viral RNA reverse transcriptions to be measured obtain, virus to be measured be or
Candidate is HTLV；If specific DNA fragment, virus to be measured are not contained in the DNA fragmentation that viral RNA reverse transcriptions to be measured obtain
It is not or candidate is not HTLV；
The specific DNA fragment can be following y1) or y2)：
Y1) the single strand dna shown in the sequence 1 of sequence table；
Y2 sequence 1 by the substitution of one or several nucleotides and/or missing and/or addition and) had into phase with sequence 1
The DNA molecular of congenerous.
Any of the above-described HTLV is HTLV-1 or HTLV-2.
Above, " reagent set can realize the real-time quantitative PCR to the total serum IgE " or " reagent set
The real-time quantitative PCR to the RNA can be realized " concretely：The total serum IgE or the RNA HTLV detection curves are sun
Property amplification curve, HTLV copy number is more than 100 and internal standard detection curve is positive amplification curve.
Above, " reagent set can not realize the real-time quantitative PCR to the total serum IgE " or " reagent set
The real-time quantitative PCR to the RNA can not be realized " concretely：The total serum IgE or the RNA HTLV detection curves are the moon
Property amplification curve and internal standard detection curve is positive amplification curve.
In any of the above-described described method, the reaction system of described " carrying out real-time quantitative PCR detection " may include：Template,
Tris-HCl buffer solutions, KCl, dATP, dGTP, dCTP, dUTP, MgCl2, reverse transcriptase, Taq archaeal dna polymerases, UNG enzymes, draw
Thing is to HTLV, probe HTLV and internal standard detection probe.The template can be the total serum IgE of testing sample or viral RNA to be measured.
In any of the above-described described method, the reaction system of described " carrying out real-time quantitative PCR detection " specifically can by template,
Tris-HCl buffer solutions, KCl, dATP, dGTP, dCTP, dUTP, MgCl2, reverse transcriptase, Taq archaeal dna polymerases, UNG enzymes, draw
Thing forms to HTLV, probe HTLV and internal standard detection probe.The template can be the total serum IgE or to be measured viral of testing sample
RNA。
In any of the above-described described method, the reaction system of described " carrying out real-time quantitative PCR detection " concretely system
First.25 μ L systems first are made up of 10 μ L templates and 15 μ L RT-PCR reaction solutions.The template can be testing sample total serum IgE or
Viral RNA to be measured.
RT-PCR reaction solutions can be KCl containing 100mM, 0.4mM dATP, 0.4mM dGTP, 0.4mM dCTP, 0.4mM
dUTP、6mM MgCl2, 3U/ μ L reverse transcriptases, 2U/ μ L Taq archaeal dna polymerases, 1U/ μ L UNG enzymes, 6.25 μM of primer HTLV-
F, pH8.3,20mMTris-HCl buffering of 6.25 μM of primer HTLV-R, 2.5 μM of probe HTLV and 2.5 μM of internal standard detection probes
Liquid.
In any of the above-described described method, the response procedures of described " carrying out real-time quantitative PCR detection " are concretely：37℃
10min；50℃15min；95℃2min；95 DEG C of 15sec, 60 DEG C of 45sec, 40 circulations.
Above, the testing sample can be test plasma.
It is demonstrated experimentally that HTLV, specific good, the higher (sensitivity of sensitivity are detected using reagent set provided by the invention
Reach 100copies/ reaction systems).Use simultaneously in reagent set provided by the invention, competition of the internal standard as target nucleic acids
Property internal standard, can be used as object of reference, for preventing the generation of false negative result, have interior target sample by detecting addition, can be with
Whether suppressed understand whole real-time quantitative PCR detection architecture, be easy to prompt false negative.The present invention applies valency with important
Value.
Brief description of the drawings
Fig. 1 is sensitivity experiment result.
Fig. 2 is the experimental result of detection reference material.
Fig. 3 is specificity experiments result.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average.
QIAamp Viral RNA Mini Kit kits are the product of QIAGEN companies.The high big extraction reagent kit of pure plasmid
For the product of TIANGEN Biotech (Beijing) Co., Ltd., catalog number DP116.Carrier pGEM-T is Promega companies
Product, production code member A362A.T7 rna polymerase and DNase I are the product of Takara companies, and article No. is respectively
2540 and 2270.
Embodiment 1, detect HTLV reagent set preparation
First, the preparation of RT-PCR reaction solutions
The present inventor passes through many experiments, by HTLV-1 and HTLV-2 nucleotide sequence being compared point
Analysis, screen the conserved sequence that a segment length is 173bp (its nucleotide sequence after transcribing is as shown in sequence 1 in sequence table).
According to the conserved sequence, primer HTLV-F, primer HTLV-R, probe HTLV and internal standard detection probe are designed and synthesized.It is each to draw
In the nucleotide sequence of thing and probe, W=A/T, R=A/G, Y=C/T, M=A/C, K=T/G.
RT-PCR reaction solutions are by Tris-HCl buffer solutions, KCl, dATP, dGTP, dCTP, dUTP, MgCl2, reverse transcriptase,
Taq archaeal dna polymerases, UNG enzymes, primer pair HTLV (being made up of primer HTLV-F and primer HTLV-R), probe HTLV and internal standard inspection
Probing pin forms.
RT-PCR reaction solutions are KCl containing 100mM, 0.4mM dATP, 0.4mM dGTP, 0.4mM dCTP, 0.4mM
dUTP、6mM MgCl2, 3U/ μ L reverse transcriptases, 2U/ μ L Taq archaeal dna polymerases, 1U/ μ L UNG enzymes, 6.25 μM of primer HTLV-
F, pH8.3,20mMTris-HCl buffering of 6.25 μM of primer HTLV-R, 2.5 μM of probe HTLV and 2.5 μM of internal standard detection probes
Liquid.
Primer HTLV-F：5 '-CTGTCCAGAGCAYCARMTC-3 ' (sequence 2 in sequence table).
Primer HTLV-R：5 '-GAGKCGAGGGATAAGGWAYTG-3 ' (sequence 3 in sequence table).
Probe HTLV：5 '-FAM-ATCGATGGACGCGTTRTCRGCTC-TAMRA-3 ' (sequence 4 in sequence table).
Internal standard detection probe：5 '-JOE-AGACCCAGTTTGGAAAGGACCAGC-TAMRA-3 ' (sequence 6 in sequence table).
Probe HTLV 5 ' ends have FAM fluorescence labelings, and 3 ' ends have TAMRA fluorescence labelings.
5 ' ends of internal standard detection probe have JOE fluorescence labelings, and 3 ' ends have TAMRA fluorescence labelings.
2nd, the preparation of HTLV reagent set is detected
HTLV reagent set is detected by RT-PCR reaction solutions, working standard, negative control, internal standard and reference material group
Into.
1st, working standard
(1) double chain DNA molecule in chemical synthesis composition sequence table shown in sequence 1 is used.
(2) double chain DNA molecule that step (1) synthesizes is connected with carrier pGEM-T, obtains recombinant plasmid.
(3) recombinant plasmid for obtaining step (2) imports bacillus coli DH 5 alpha, obtains recombination bacillus coli.
(4) monoclonal for the recombination bacillus coli for obtaining step (3) is inoculated in 5mL containing 100 μ g/mL ampicillins
In LB culture mediums, then 37 DEG C, 190rpm shaken cultivation 12h, bacterium solution is obtained.
(5) bacterium solution for taking step (4) to obtain, according to the operating procedure of the big extraction reagent kit of high pure plasmid, plasmid is extracted.
(6) plasmid for taking step (5) to obtain, in-vitro transcription is first carried out using t7 rna polymerase, then using DNase I
Digested to remove DNA, obtain RNA solution.
The RNA solution that step (6) obtains is diluted with the water of deoxyribonuclease, obtaining RNA copy numbers is
105Copies/ μ L RNA solution 1,104Copies/ μ L RNA solution 2, RNA copy numbers is 103Copies/ μ L RNA solution
3rd, RNA copy numbers are 102Copies/ μ L RNA solution 4 and RNA copy numbers is 101Copies/ μ L RNA solution 5.
RNA solution 1, RNA solution 2, RNA solution 3, RNA solution 4 and RNA solution 5 are working standard.
2nd, negative control
Negative control is the human normal plasma without HTLV RNA.
3rd, internal standard
(1) using the double chain DNA molecule shown in sequence 5 in chemical synthesis composition sequence table, (sequence 5 and sequence 1 are only
One difference is：The nucleotide sequence in internal standard detection probe region is different).
(2) double chain DNA molecule that step (1) synthesizes is connected with carrier pGEM-T, obtains recombinant plasmid.
(3) recombinant plasmid for obtaining step (2) imports bacillus coli DH 5 alpha, obtains recombination bacillus coli.
(4) monoclonal for the recombination bacillus coli for obtaining step (3) is inoculated in 5mL containing 100 μ g/mL ampicillins
In LB culture mediums, then 37 DEG C, 190rpm shaken cultivation 12h, bacterium solution is obtained.
(5) bacterium solution for taking step (4) to obtain, according to the operating procedure of the big extraction reagent kit of high pure plasmid, plasmid is extracted.
(6) plasmid for taking step (5) to obtain, in-vitro transcription is first carried out using t7 rna polymerase, then using DNase I
Digested to remove DNA, obtain RNA solution.
The RNA solution that step (6) obtains is diluted with the water of deoxyribonuclease, obtaining RNA copy numbers is
105Copies/ μ L internal standard.
It should be noted that competitive internal standard of the internal standard as target nucleic acids, can be used as object of reference, for preventing false the moon
Property result generation, have interior target sample by detecting addition, it will be appreciated that whether whole real-time quantitative PCR detection architecture suppressed
System, it is easy to prompt false negative.
4th, reference material
The human plasma of the RNA containing HTLV-1 is provided by Tianjin blood disease hospital, and supplier's informed consent.
The human plasma of the RNA containing HTLV-2 is provided by Tianjin blood disease hospital, and supplier's informed consent.
The foundation of embodiment 2, detection HTLV method
First, the extraction of the total serum IgE of test plasma
(1) test plasma is taken, adds 10 μ L internal standards, mixes, obtains mixed liquor.
(2) mixed liquor is taken, RNA is extracted according to the operating procedure of QIAamp Viral RNA Mini Kit kits, obtains
The total serum IgE of test plasma.
2nd, reaction system is prepared
Reaction system first is 25 μ L, is made up of the total serum IgE and 15 μ L RT-PCR reaction solutions of 10 μ L test plasmas.
Reaction system second (negative control) is 25 μ L, by the total serum IgE and 15 μ L RT-PCR reaction solution groups of 10 μ L normal plasmas
Into.The step of total serum IgE for preparing normal plasma is：No HTLV RNA human normal plasma is taken, adds 10 μ L internal standards, mixes, obtains
To mixed liquor；Mixed liquor is taken, RNA is extracted according to the operating procedure of QIAamp Viral RNA Mini Kit kits, obtains dense
Spend the total serum IgE of the normal plasma for 10ng/ μ L.
Reaction system third (reference material) is 25 μ L, by the total serum IgE (RNA of hypotype containing the HTLV-1 blood plasma of 10 μ L positive blood plasma
The blood plasma total serum IgE of total serum IgE or the RNA of hypotype containing HTLV-2) and 15 μ L RT-PCR reaction solutions composition.
The step of blood plasma total serum IgE for preparing hypotype containing HTLV-1 RNA is：The human plasma of the RNA containing HTLV-1 is taken, adds 10 μ
L internal standards, mix, obtain mixed liquor；Mixed liquor is taken, according to the operating procedure of QIAamp Viral RNA Mini Kit kits
RNA is extracted, the blood plasma total serum IgE for the RNA of hypotype containing HTLV-1 that concentration is 10ng/ μ L is obtained after dilution.
According to above-mentioned steps, " human plasma of the RNA containing HTLV-1 " is replaced with into " human plasma of the RNA containing HTLV-2 ", its
Its step is constant, and the blood plasma total serum IgE for the RNA of hypotype containing HTLV-2 that concentration is 10ng/ μ L is obtained after dilution.
Reaction system fourth (working standard) is 25 μ L, and by 10 μ L working standards, (RNA solution 1, RNA solution 2, RNA are molten
Liquid 3, RNA solution 4 or RNA solution 5) and 15 μ L RT-PCR reaction solutions composition.
3rd, real-time quantitative PCR detects
Each reaction system that step 2 is prepared is subjected to real-time quantitative PCR detection on real-time fluorescence PCR instrument.React bar
Part：37℃10min；50℃15min；95℃2min；95 DEG C of 15sec, 60 DEG C of 45sec, 40 circulations.FAM is gathered at 60 DEG C to lead to
The fluorescence signal in road and JOE passages.Wherein, FAM Air conduct measurements HTLV, JOE Air conduct measurement internal standard.
4th, result judges
After completing step 3, threshold line is adjusted to more than the amplification line of background signal and negative control, system is according to anti-
The standard curve of system fourth preparation and CT values is answered to calculate the copy number of HTLV in each sample automatically.Using copy number as horizontal seat
Mark, fluorescent value is ordinate, draws amplification curve.Then make the following judgment：
The 1st, if the HTLV detection curves of the total serum IgE of test plasma are negative amplification curve and internal standard detection curve is positive
Amplification curve, then HTLV-1 or HTLV-2 (i.e. true negative) is not contained in test plasma；
The 2nd, if the HTLV detection curves of the total serum IgE of test plasma are positive amplification curve, HTLV copy number be 100 with
Upper and internal standard detection curve is positive amplification curve, then contains HTLV-1 and/or HTLV-2 in test plasma；
The 3rd, if the HTLV detection curves of the total serum IgE of test plasma are positive amplification curve, HTLV copy number is less than
100, and internal standard detection curve is positive amplification curve, then HTLV-1 and/or HTLV-2 nucleic acid be present in the total serum IgE of test plasma
Pollution is, it is necessary to recheck；
If the 4, internal standard detection curve is negative amplification curve, suppression or PCR amplifications in the total serum IgE of test plasma be present
Reaction exists abnormal, it is necessary to recheck.
Positive amplification curve is " S types " amplification curve.Negative amplification curve is non-" S types " amplification curve.
In negative control, HTLV detection curves are negative amplification curve and internal standard detection curve is positive amplification curve.
In reference material, HTLV detection curves are positive amplification curve and internal standard detection curve is positive amplification curve.
Embodiment 3, sensitivity experiment
First, reaction system is prepared
Reaction system a is 25 μ L, by 10 μ L RNA solutions (RNA solution 1, RNA solution 2, RNA solution 3, RNA solution 4 or
RNA solution 5) and 15 μ L RT-PCR reaction solutions composition.
Reaction system second of the reaction system second with step 2 in embodiment 2.
Reaction system third of the reaction system third with step 2 in embodiment 2.
Reaction system fourth of the reaction system fourth with step 2 in embodiment 2.
2nd, real-time quantitative PCR detects
With step 3 in embodiment 2.
3rd, result judges
With step 4 in embodiment 2.
HTLV detection curves are shown in that (1 is that RNA solution 1,2 is that RNA solution 2,3 is that RNA solution 3,4 is that RNA solution 4,5 is to Fig. 1
RNA solution 5).As a result show, the sensitivity for the reagent set detection HTLV that embodiment 1 provides is 100copies/ reaction systems.
Embodiment 4, the test experience of reference material
First, reaction system is prepared
Reaction system b is 25 μ L, and by the total serum IgE of 10 μ L positive blood plasma, (RNA of hypotype containing HTLV-1 blood plasma total serum IgE contains
HTLV-2 hypotypes RNA blood plasma total serum IgE) and 15 μ L RT-PCR reaction solutions composition.
The step of blood plasma total serum IgE for preparing hypotype containing HTLV-1 RNA is：The human plasma of the RNA containing HTLV-1 is taken, adds 10 μ
L internal standards, mix, obtain mixed liquor；Mixed liquor is taken, according to the operating procedure of QIAamp Viral RNA Mini Kit kits
RNA is extracted, the blood plasma total serum IgE for the RNA of hypotype containing HTLV-1 that concentration is 10ng/ μ L is obtained after dilution.
According to above-mentioned steps, " human plasma of the RNA containing HTLV-1 " is replaced with into " human plasma of the RNA containing HTLV-2 ", its
Its step is constant, and the blood plasma total serum IgE for the RNA of hypotype containing HTLV-2 that concentration is 10ng/ μ L is obtained after dilution.
Reaction system second of the reaction system second with step 2 in embodiment 2.
Reaction system fourth of the reaction system fourth with step 2 in embodiment 2.
2nd, real-time quantitative PCR detects
With step 3 in embodiment 2.
3rd, result judges
With step 4 in embodiment 2.
HTLV detection curves are shown in that (HTLV-1 is the RNA of hypotype containing HTLV-1 blood plasma total serum IgE to Fig. 2, and HTLV-2 is containing HTLV-2
Hypotype RNA blood plasma total serum IgE).As a result show, the reagent set provided using embodiment 1 can detect HTLV-1 and HTLV-2.
Embodiment 5, specificity experiments
First, reaction system is prepared
Reaction system c is 25 μ L, by total serum IgE (HTLV-1 total serum IgE, HTLV-2 total serum IgE, the HIV of 10 μ L samples to be tested
Total serum IgE, HCV total serum IgE, the total serum IgE of influenza virus, the total serum IgE of measles virus, HBV total serum IgE, HPV total serum IgE or list
The total serum IgE of pure herpesviral) and 15 μ L RT-PCR reaction solutions composition.HIV, HCV, influenza virus, measles virus, HBV, HPV and
The Strain of herpes simplex virus provides by No. 302 Hospital, CPLA.
Reaction system second of the reaction system second with step 2 in embodiment 2.
Reaction system third of the reaction system third with step 2 in embodiment 2.
Reaction system fourth of the reaction system fourth with step 2 in embodiment 2.
2nd, real-time quantitative PCR detects
With step 3 in embodiment 2.
3rd, result judges
With step 4 in embodiment 2.
HTLV detection curves are shown in that (other viruses are HIV, HCV, influenza virus, measles virus, HBV, HPV and simple blister to Fig. 3
Exanthema virus).As a result show, the reagent set provided using embodiment 1 detects HTLV, has very high specificity.
Embodiment 6, application
First, the acquisition of the total serum IgE of sample to be tested
Sample to be tested amounts to 30 parts.Sample to be tested 1 to sample to be tested 28 is to have identified to contain HTLV (HTLV-1 in blood plasma
Or HTLV-2) people venous blood 2mL.Sample to be tested 29 and sample to be tested 30 are to have identified the people that HTLV is not contained in blood plasma
Venous blood 2mL.
The 1st, sample to be tested 1 to sample to be tested 30 is added to the sterile collection tube containing EDTA or sodium citrate anticoagulant respectively
In, gently overturn and mix, then room temperature, 2000rpm centrifugations 5min.
2nd, after completing step 1, upper plasma is collected, precipitation is transferred to another sterile centrifugation tube.
3rd, after completing step 2,10 μ L internal standards are added into the sterile centrifugation tube, mixes, obtains mixed liquor.
4th, after completing step 3, mixed liquor is taken, according to the operating procedure of QIAamp Viral RNA Mini Kit kits
RNA is extracted, obtains the total serum IgE of sample to be tested.
2nd, reaction system is prepared
Reaction system d is 25 μ L, is made up of the total serum IgE and 15 μ L RT-PCR reaction solutions of 10 μ L samples to be tested.
Reaction system second of the reaction system second with step 2 in embodiment 2.
Reaction system third of the reaction system third with step 2 in embodiment 2.
Reaction system fourth of the reaction system fourth with step 2 in embodiment 2.
3rd, real-time quantitative PCR detects
With step 3 in embodiment 2.
4th, result judges
With step 4 in embodiment 2.
As a result show, sample to be tested 1 is accredited as in blood plasma to sample to be tested 28 contains HTLV, sample to be tested 29 and to be measured
Sample 30, which is accredited as in blood plasma, does not contain HTLV, completely the same with actual effect.
Result above shows that the reagent set provided using embodiment 1 is detected to the testing sample containing HTLV, ties
Fruit is accurately and reliably.
<110>Beijing Fuan bio tech ltd of China
<120>A kind of method and its special complete reagent for detecting HTLV
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 173
<212> DNA
<213>Artificial sequence
<220>
<223>
<220>
<221> misc_feature
<222>（107）
<223> W is A or T
<220>
<221> misc_feature
<222>（54）
<223> Y is T or C
<220>
<221> misc_feature
<222>（57,88,91,105）
<223> R is A or G
<220>
<221> misc_feature
<222>（58,120）
<223> M is A or C
<400> 1
ctatgttcca cccgcctaca tcgacatgcc ctcctggcca cctgtccaga gcaycarmtc 60
acctgggacc ccatcgatgg acgcgttrtc rgctctcctc tccartwcct tatccctcgm 120
ctcccctcct tccccaccca gagaacctca aggaccctca aggtccttac ccc 173
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<220>
<221> misc_feature
<222>（17）
<223> M is A or C
<220>
<221> misc_feature
<222>（13）
<223> Y is T or C
<220>
<221> misc_feature
<222>（16）
<223> R is A or G
<400> 2
ctgtccagag caycarmtc 19
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<220>
<221> misc_feature
<222>（17）
<223> W is A or T
<220>
<221> misc_feature
<222>（19）
<223> Y is T or C
<220>
<221> misc_feature
<222>（58,120）
<223> K is T or G
<400> 3
gagkcgaggg ataaggwayt g 21
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<220>
<221> misc_feature
<222>（16,19）
<223> R is A or G
<400> 4
atcgatggac gcgttrtcrg ctc 23
<210> 5
<211> 174
<212> DNA
<213>Artificial sequence
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<222>（108）
<223> W is A or T
<220>
<221> misc_feature
<222>（54）
<223> Y is T or C
<220>
<221> misc_feature
<222>（57,106）
<223> R is A or G
<220>
<221> misc_feature
<222>（58,121）
<223> M is A or C
<400> 5
ctatgttcca cccgcctaca tcgacatgcc ctcctggcca cctgtccaga gcaycarmtc 60
acctgggacc ccagacccag tttggaaagg accagctcct ctccartwcc ttatccctcg 120
mctcccctcc ttccccaccc agagaacctc aaggaccctc aaggtcctta cccc 174
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 6
agacccagtt tggaaaggac cagc 24
Claims (10)
1. HTLV reagent set is detected, including primer pair HTLV and probe HTLV；The primer pair HTLV is by primer HTLV-F
Formed with primer HTLV-R；Target sequences of the primer pair HTLV in HTLV genomes contains special RNA fragments；It is described special
The specific DNA fragment that RNA fragment reverse transcriptions obtain is following y1) or y2)：
Y1) the single strand dna shown in the sequence 1 of sequence table；
Y2 sequence 1 by the substitution of one or several nucleotides and/or missing and/or addition and) had into identical work(with sequence 1
The DNA molecular of energy；
The probe HTLV is the single strand dna of 20-30 nucleotides composition, with the part area in the specific DNA fragment
Duan Xiangtong.
2. reagent set as claimed in claim 1, it is characterised in that：
The primer HTLV-F is following a1) or a2)：
A1) the single strand dna in sequence table shown in sequence 2；
A2 sequence 2 by the substitution of one or several nucleotides and/or missing and/or addition and) had into identical work(with sequence 2
The single strand dna of energy；
The primer HTLV-R is following b1) or b2)：
B1) the single strand dna in sequence table shown in sequence 3；
B2 sequence 3 by the substitution of one or several nucleotides and/or missing and/or addition and) had into identical work(with sequence 3
The single strand dna of energy；
The probe HTLV is following c1) or c2)：
C1) the single strand dna in sequence table shown in sequence 4；
C2 sequence 4 by the substitution of one or several nucleotides and/or missing and/or addition and) had into identical work(with sequence 4
The single strand dna of energy.
3. reagent set as claimed in claim 1 or 2, it is characterised in that：The end of the probe HTLV has fluorescence labeling.
4. the reagent set as described in claims 1 to 3 is any, it is characterised in that：The reagent set also includes internal standard and internal standard
Detection probe；Single strand RNA molecule is designated as in described；The DNA fragmentation that the internal standard reverse transcription obtains is following z1) or z2)：
Z1) the single strand dna shown in the sequence 5 of sequence table；
Z2 sequence 5 by the substitution of one or several nucleotides and/or missing and/or addition and) had into identical work(with sequence 5
The DNA molecular of energy；
The internal standard detection probe is the single strand dna of 20-30 nucleotides composition, is obtained with the internal standard reverse transcription
Partial sector in DNA fragmentation is identical.
5. reagent set as claimed in claim 4, it is characterised in that：The internal standard detection probe is following d1) or d2)：
D1) the single strand dna in sequence table shown in sequence 6；
D2 sequence 6 by the substitution of one or several nucleotides and/or missing and/or addition and) had into identical work(with sequence 6
The single strand dna of energy.
6. the reagent set as described in claim 1 to 5 is any, it is characterised in that：The reagent set also includes working standard；
The working standard is single strand RNA molecule；The DNA fragmentation that the working standard reverse transcription obtains is the specific DNA piece
Section.
7. the kit containing any reagent set of claim 1 to 6.
8.d1) or d2) d3) or d4)：
D1) any reagent set of claim 1 to 6 is preparing the application in being used to detect HTLV kit；
D2) kit described in any reagent set of claim 1 to 6 or claim 7 in testing sample is detected whether
Contain or the doubtful application containing in HTLV；
D3) any reagent set of claim 1 to 6 is preparing the application in being used to identify HTLV kit；
D4) kit described in any reagent set of claim 1 to 6 or claim 7 identify virus to be measured whether be
Or whether candidate is the application in HTLV.
9. a kind of detection testing sample whether the method containing HTLV, be A1) or A2)：
A1) using the total serum IgE of testing sample as template, real-time quantitative PCR is carried out with any reagent set of claim 1 to 6
Detection, is then judged as follows：If the reagent set can realize the real-time quantitative PCR to the total serum IgE, to be measured
Contain in sample or doubtful containing HTLV；If the reagent set can not realize the real-time quantitative PCR to the total serum IgE,
Do not contained in testing sample or doubtful do not contain HTLV；
A2) whether contain specific DNA fragment in the DNA fragmentation that the total serum IgE reverse transcription of detection testing sample obtains, then carry out such as
Lower judge：If the DNA fragmentation that the total serum IgE reverse transcription of testing sample obtains contains specific DNA fragment, contain in testing sample
Or doubtful contain HTLV；If the DNA fragmentation that the total serum IgE reverse transcription of testing sample obtains does not contain specific DNA fragment, to be measured
Sample does not contain or doubtful does not contain HTLV；
The specific DNA fragment is following y1) or y2)：
Y1) the single strand dna shown in the sequence 1 of sequence table；
Y2 sequence 1 by the substitution of one or several nucleotides and/or missing and/or addition and) had into identical work(with sequence 1
The DNA molecular of energy.
10. it is a kind of identify virus to be measured whether the method for being HTLV, be B1) or B2)：
B1) using viral RNA to be measured as template, real-time quantitative PCR inspection is carried out with any reagent set of claim 1 to 6
Survey, then judged as follows：If the reagent set can realize the real-time quantitative PCR to the RNA, virus to be measured
For or candidate be HTLV；If the reagent set can not realize the real-time quantitative PCR to the RNA, virus to be measured is not
Or candidate is not HTLV；
B2) detect in the DNA fragmentation that viral RNA reverse transcriptions to be measured obtain whether contain specific DNA fragment, then carry out as follows
Judge：If containing specific DNA fragment in the DNA fragmentation that viral RNA reverse transcriptions to be measured obtain, virus to be measured is or candidate
For HTLV；If not containing specific DNA fragment in the DNA fragmentation that viral RNA reverse transcriptions to be measured obtain, virus to be measured is not
Or candidate is not HTLV；
The specific DNA fragment is following y1) or y2)：
Y1) the single strand dna shown in the sequence 1 of sequence table；
Y2 sequence 1 by the substitution of one or several nucleotides and/or missing and/or addition and) had into identical work(with sequence 1
The DNA molecular of energy.
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| CN201710821933.0A CN107488746A (en) | 2017-09-13 | 2017-09-13 | A kind of method and its special complete reagent for detecting HTLV |
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| CN201710821933.0A CN107488746A (en) | 2017-09-13 | 2017-09-13 | A kind of method and its special complete reagent for detecting HTLV |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107988400A (en) * | 2017-12-18 | 2018-05-04 | 中国人民解放军军事医学科学院微生物流行病研究所 | Detect the reagent set of staphylococcus haemolyticus |
| CN113136453A (en) * | 2020-01-19 | 2021-07-20 | 上海爱萨尔生物科技有限公司 | Primer for specifically detecting HTLV-II proviral DNA and application thereof |
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| CN107988400A (en) * | 2017-12-18 | 2018-05-04 | 中国人民解放军军事医学科学院微生物流行病研究所 | Detect the reagent set of staphylococcus haemolyticus |
| CN113136453A (en) * | 2020-01-19 | 2021-07-20 | 上海爱萨尔生物科技有限公司 | Primer for specifically detecting HTLV-II proviral DNA and application thereof |
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Application publication date: 20171219 |