CN108130385A - A kind of human cytomegalovirus kit for detecting nucleic acid - Google Patents
A kind of human cytomegalovirus kit for detecting nucleic acid Download PDFInfo
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- CN108130385A CN108130385A CN201711447814.XA CN201711447814A CN108130385A CN 108130385 A CN108130385 A CN 108130385A CN 201711447814 A CN201711447814 A CN 201711447814A CN 108130385 A CN108130385 A CN 108130385A
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The present invention relates to a kind of kits of highly sensitive detection human cytomegalovirus nucleic acid.It expands the viral specific nucleic acids sequence using pair of primers, and passes through two fluorescence probe detection human cytomegalovirus nucleic acid, human cytomegalovirus nucleic acid in the highly sensitive qualitative or quantitative detection whole blood of energy, blood plasma, urine, milk sample.Kit is easy to operate quickly, testing result is sensitive and accurate, can be as effective auxiliary detection means of human cytomegalovirus infection.
Description
Technical field
The present invention relates to a kind of human cytomegalovirus nucleic acid fluorescent PCR detection kits, belong to biotechnology.
Background technology
Human cytomegalovirus（Human Cytomegalic Virus, HCMV) belong to herpetoviridae, it is human herpes
Maximum a kind of virus in viral group, diameter 200nm, core are bifilar linear DNA, there is typical herpesviral structure.HCMV
It can only be proliferated, and cannot be grown in other zooblasts in people's fibroblast culture, proliferation is very slow, first to detach
Cellular pathogenic effect (CPE) could occur in 1 wheat harvesting period of need, lead to cell rounding, expand, cell and core gigantism, around core
There is large-scale eosinophilic inclusion, thus also known as cytomegalic inclusion disease (Cytomegalic inclusion disease,
CID).People's HCMV infection is a kind of systemic infection syndrome, is clinically involved for multi viscera, and complicated symptoms are various, most
For subclinical infection, the retinitis, hepatitis, pneumonia, encephalitis, colitis, monocytosis,mononucleosis, decrease of platelet can occur for minority
The multiple organs lesions such as property purpura.HCMV infection is very universal, and in worldwide prevalence, infection rate is very high in crowd, flourishing and hair
National infection rate is respectively 45~50% and more than 90% in exhibition.HCMV viruses can hide in vivo for a long time, once immunity of organisms
Decline, virus will be activated and cause a disease, and particularly to leukaemia, immunosuppressive patient, latent herpesviral activates to be formed
Recurrent infection causes transplant organ necrosis and endangers patients ' lives, threatens human health when serious.Further, since giant cell is sick
Poison can cause stillborn foetus, miscarriage, premature labor through intrauterine infection, may also lead to congenital malformation, thus HCMV infection can also influence aristogenesis
Good child-rearing and quality of the people.
Clinical labororatory's HCMV infection detection generally uses the culture of goldstandard virus purification, serology and detection of nucleic acids side
Method, and respectively have advantage and disadvantage.Since HCMV growth cycles are long（30 days）, virus purification cultivation be not suitable for clinical labororatory use.
Serologic detection detects viral associated proteins using antigen-antibody reaction, has the advantages that detection is quick, simple, easily operated,
But detection sensitivity is not high, and since antibody generates " window phase " after infection, also there are certain hysteresis qualitys for detection.Detection of nucleic acids skill
In art, particularly fluorescence PCR method have many advantages, such as specificity and high sensitivity, can quantify, is easy to operate, cost it is relatively low, energy
The presence of HCMV nucleic acid in detection whole blood, blood plasma, serum, urine and milk equal samples, as HCMV infection detection
Auxiliary tool promotes and applies.
The present invention is based on above-mentioned technical backgrounds, it is therefore an objective to the highly sensitive detection human cytomegalovirus-specific primer of design screening
Probe develops HCMV nucleic acid fluorescent PCR kits, can realize the qualitative or quantitative detection of high sensitivity of HCMV viral nucleic acids, be
The auxiliary diagnosis of clinical HCMV infection or treatment provide foundation.
Invention content
The present invention provides a kind of fluorescent PCR kit of highly sensitive detection human cytomegalovirus nucleic acid, which is characterized in that institute
Kit is stated using a pair of of human cytomegalovirus amplimer and two fluorescence probes, pair of primers has SEQ ID NO：1 He
SEQ ID NO：2 sequence, two Fluorescence Fluorescence probes have SEQ ID NO：3 and SEQ ID NO：4 sequences and fluorescence probe
5 ' ends mark identical fluorescein or fluorescent dye reporter group, 3 ' end mark fluorescent quenching groups.Kit uses SEQ ID
NO：1~4 primer and fluorescence probe are aided with the fluorescence of Tris-HCl, KCl, dNTPs, Taq archaeal dna polymerase and UNG enzymes composition
PCR reaction systems, can highly sensitive qualitative or quantitative detection whole blood, blood plasma, urine, human cytomegalovirus nucleic acid in milk sample.
Kit is easy to operate quickly, testing result is sensitive and accurate, can be as effective auxiliary detection means of human cytomegalovirus infection.
Technical solution
By to human cytomegalovirus gene sequence queries in GenBank, with molecular biology primed probe design software to each
Design is compared in the gene order in kind of source, and according to the principle that TaqMan fluorescent PCRs design, preferably a pair of of HCMV is specific
It is highly conserved in upstream and downstream primer amplification viral genome can early gene（IE2）Middle UL122 regions
139bp segment target sequences, two that on detection target sequence location non-overlapping copies or complementation are devised between upstream and downstream primer are glimmering
Light probe A, B, wherein fluorescence probe A and the complementation of target sequence antisense strand, fluorescence probe B and target sequence positive-sense strand are complementary, therefore one
Secondary PCR cycle reaction can generate 2 fluorescent moleculars, substantially increase detection sensitivity.4 primer and probe nucleotide sequences
（5’ -> 3’）It is as follows.
Sense primer：AgAgAgCgATTggTgTTgCg, sequence are denoted as SEQ ID NO：1.
Downstream primer：CCgAgTTggACAACgAgAAgg, sequence are denoted as SEQ ID NO：2.
Fluorescence probe A：ATgCggCTCACCTCgTCAATCTTg, sequence are denoted as SEQ ID NO：3, middle probe 5 ' is held
Flag F AM（6-carboxy-fluorescein）Fluorescent reporter group, 3 ' end label BHQ（Black hole quencher）It quenches
Go out group.
Fluorescence probe B：AAgAACACCCCCTTCTgCACACCC, sequence are denoted as SEQ ID NO：4, middle probe 5 ' is held
Flag F AM fluorescent reporter groups, 3 ' end label BHQ quenching groups.
Two end of fluorescence probe 5 ' requirements mark identical fluorescein or fluorescent dye reporter group, in addition to FAM fluorescent bases
Group, can also select JOE（carboxy-dichloro-dimethoxyfluorescein）、VIC、HEX（carboxy-
hexachloro-fluorescein）、ROX（5-Carboxy-X-rhodamine）、Cy3（Cyanine 3）、Cy5（Cyanine
5）Fluorophors are waited, 3 ' ends can also select TAMRA in addition to marking BHQ quenching groups（Carboxy-tetra-methyl-
rhodamine）Wait quenching groups.
Between present invention design HCMV pair of primers, there is very close Tm values, and fluorescence between two fluorescence probes
Probe Tm values are higher than 5~10 DEG C of primer Tm, without apparent primer dimer or hairpin structure between primer probe sequence, protect
The specificity and high efficiency of amplification have been demonstrate,proved, has been conducive to improve kit detection sensitivity.
The amplification reaction system is respectively as follows into being grouped as：10mM Tris-HCl（pH8.3）, 50mM KCl, 0.2~
0.5mM dNTPs, 1.5~5 mM MgCl2, each 0.1~0.5 μM of primer and fluorescence probe（SEQ ID NO：1~4）, 1~5 U
Taq archaeal dna polymerases and 0.01~1 U UNG enzymes, the 10 μ l of template of extraction, 30 μ l of total reaction volume.More specifically, amplification is anti-
Answer each ingredient of liquid final concentration of：Tris-HCl（pH8.3）10 mM, KCl 50 mM, dATP, dGTP, dCTP, dUTP each 0.2
MM, MgCl23.5 mM, pair of primers（SEQ ID NO：3 and SEQ ID NO：4）Each 0.3 μM, two fluorescence probes（SEQ ID
NO：3 and SEQ ID NO：4）Each 0.2 μM, 2 U of Taq archaeal dna polymerases, 0.5 U of UNG enzymes.
The HCMV kit for detecting nucleic acid is as follows using amplification program：According to 94 DEG C after 50 DEG C of 2min, 94 DEG C of 5min
10sec, 60 DEG C of 45sec cyclic amplifications 45 times acquire FAM channel wavelength fluorescence signals at 60 DEG C in circulation step.
It is linear that the quality-control product of the HCMV kit for detecting nucleic acid includes 1 negative control, 1 positive control and 4
The viral nucleic acid quantitative calibration product of gradient concentration, 4 calibration object HCMV nucleic acid concentrations are respectively 5.0x107 copies/ml、
5.0x106 copies/ml、5.0x105 copies/ml、5.0x104Copies/ml, calibration object is contains SEQ ID NO：1
The TA cloned plasmids of~2 amplification target sequences.Positive control is the Escherichia coli containing the cloned plasmids.
The HCMV kit for detecting nucleic acid, using the viral DNA extracts reagent based on paramagnetic particle method nucleic acid extraction, in nucleic acid
Sample nucleic acid automation extraction is realized on extraction apparatus.
A pair of of the HCMV primers and two fluorescence probes preferably obtained by above-mentioned design is aided with Fluorescence PCR its
Its component and kit reagent box, the present invention provides the kits of real-time fluorescence quantitative PCR detection, optimize PCR reactions
System and amplification program.Certain this research field technical staff can adjust PCR reaction systems according to the general requirement of fluorescent PCR
And program, also the same purpose that can realize detection.
Advantageous effect
The present invention is grown up according to the primed probe and technical solution of above-mentioned design by optimizing reaction system and amplification condition development
Cytomegalovirus nucleic acid fluorescent PCR immue quantitative detection reagent boxes, main feature are as follows：
（1）Based on improved TaqMan probe Fluorescence PCR assay, two and amplification target sequence are devised between one couple of PCR primers
Column position non-overlapping copies or the fluorescence probe of complementation breach traditional TaqMan probe fluorescent PCR one cycle reaction and generate one
The limitation of a fluorescent molecular, each PCR cycle of target sequence DNA molecular can generate two fluorescent moleculars, substantially increase examination
Agent box detects HCMV nucleic acid sensitivity.
（2）Tm values approach and without secondary structure is intersected, ensure that virus between the HCMV primers of design, between fluorescence probe
The specificity and high efficiency of amplification are conducive to improve the sensitivity of kit detection HCMV nucleic acid.
（3）Result false positive caused by dUTP-UNG enzyme systems in amplification system can avoid gene-amplification product pollution,
Improve the accuracy of kit testing result.
（4）Kit extracts sample nucleic acid, suitable for whole blood, blood plasma, serum, urine, milk using paramagnetic particle method automation
Equal samples type, extraction detection is easy to operate quick, can report testing result in 2 hours, improve work efficiency.
The These characteristics of kit are HCMV primed probes using specific designs and are combined with Fluorescence PCR assay
Using and directly cause.From the principle, kit of the present invention HCMV clinical samples detection in by expanding real-time fluorescence PCR
Increase the analysis of fluorescence signal, judge presence and the virus load of viral nucleic acid specific fragment, technical scheme of the present invention design
Rationally, the highly sensitive qualitative or quantitative detection of human cytomegalovirus is can be widely applied to, it can be as the effective auxiliary of HCMV infection
Help detection means.
Specific embodiment
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make the present invention by common technical knowledge various technical changes or modification, and such equivalent forms equally fall within the application institute
Attached claims limited range.
Embodiment 1：Human cytomegalovirus kit for detecting nucleic acid primed probe designs
It is excellent using molecular biology primer-design software according to the HCMV gene orders inquired in NCBI GenBank databases
The primer probe sequence for selecting acquisition is as shown in table 1, and HCMV primer amplifications are 139bp segments on virus IE2 genes.
The kit primed probe that table 1 designs
The synthesis of more than primed probe commission Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Embodiment 2：Human cytomegalovirus kit for detecting nucleic acid is prepared
Kit reaction buffer prepares 32 person-portion kits reaction buffering voluntarily to prepare, by 2 each component concentration of table and volume
Liquid, it is 20 μ l that the reaction buffer of preparation, which each reacts packing dosage, and total reaction volume is 30 μ l after adding in 10 μ l of template.
2 kit reaction buffer of table prepares each group partial volume
Kit negative control uses normal person's negative plasma；Quantitative calibration product are the clone that target sequence segment is expanded containing HCMV
Plasmid is diluted to 4 concentration as kit quantification calibration object by the use of TE buffer solutions：5.0x107 copies/ml、5.0x106
copies/ml、5.0x105 copies/ml、5.0x104copies/ml；Positive control is the large intestine containing above-mentioned cloned plasmids
Bacillus, employment negative plasma are diluted to a concentration of 5.0x103 copies/ml。
The paramagnetic particle method virus that kit viral DNA extracts reagent is developed using Shanghai Xingyao Medical Technology Development Co., Ltd.
DNA extraction kit, it includes 5 magnetic bead, lysate, Proteinase K, cleaning buffer solution, elution buffer components, and when use tries
Agent box negative control, positive control, sample extract nucleic acid using the kit.
Kit amplification program is：Expand after 50 DEG C of 2min, 94 DEG C of 5min according to 94 DEG C of 10sec, 60 DEG C of 45sec cycles
Increase 45 times, acquire FAM channel wavelength fluorescence signals in circulation step at 60 DEG C.
Included by the HCMV kit for detecting nucleic acid components of above-mentioned preparation：Reaction buffer, negative control, positive control
And 4 quantitative calibration product, it is desirable that -20 DEG C of storage and transport.
Embodiment 3：The measure of kit detection sensitivity
（1）Viral DNA extracts
It learns from else's experience clinical identification, HCMV positive blood plasma known to concentration, HCMV concentration is 2x10 in sample5copies/ml）.So
Normal person's negative plasma is used afterwards by 10 times of continuous gradient dilutions of the mixing sample to 2x104 copies/ml、2x103
copies/ml、2x102 copies/ml、2x101Copies/ml draws above-mentioned viral dilution, kit negative control, sun
Property each 400 μ l of control, the paramagnetic particle method viral DNA extracts reagent being used together in embodiment 2 carries out virus on instrument for extracting nucleic acid
DNA is extracted, each 50 μ l of nucleic acid that last each sample obtains.
（2）Fluorescent PCR detects
Kit reaction buffer in embodiment 2 is taken out into freeze thawing, mixing, of short duration centrifugation from -20 DEG C of refrigerators, by 20 μ l/ person-portions
Reaction buffer is dispensed into PCR reaction tubes, be then respectively adding the kit negative control of said extracted, positive control,
The template of viral dilution extraction and each 10 μ l of quantitative calibration product without nucleic acid extraction.Each reaction pipe volume is 30 μ
Above-mentioned reaction tube is put on ABI7500 fluorescent PCR instrument by l, is set each reaction tube sample type and is set quantitative calibration product dense
Angle value reacts 2 minutes according to 50 DEG C, then keeps the temperature 5 minutes for 94 DEG C, then recycled 45 times within 45 seconds by 94 DEG C 10 seconds → 60 DEG C（
60 DEG C of acquisition FAM channel wavelength fluorescence signals）Response procedures operation.According to instrument software and kit explanation after amplification
Book analyzes and determines experimental result.
（3）Interpretation of result
Kit negative control testing result is negative for HCMV, and positive control testing result is positive for HCMV, in FAM channels by dense
Angle value sets HCMV quantitative calibration product magnitudes, and calibration object detection linearly dependent coefficient is all higher than 0.98, Pass Test Quality Control requirement.
Observe 2x104 copies/ml、2x103 copies/ml、2x102 copies/ml、2x101The sample of copies/ml concentration
Product gradient is repeated 5 times detection respectively, and HCMV results are the positive, thus may determine that kit is sensitive to the detection of HCMV nucleic acid
Degree can reach 2x101Copies/ml can realize the highly sensitive qualitative or quantitative detection of HCMV nucleic acid.
Embodiment 4：Application of the kit in people's CID patient's sample HCMV detections
（1）Viral DNA extracts
Take clinical 25, the HCMV infection sample collected（Each 5 of whole blood, blood plasma, serum, urine, milk sample）、Epstein-
Barr viruses infect whole blood sample 5, and the paramagnetic particle method disease in embodiment 2 is used together with kit negative control, positive control
Malicious DNA extracts reagents carry out viral DNA extraction on instrument for extracting nucleic acid, and each 400 μ l of sample volume obtain core after extraction
Each 50 μ l of acid.
（2）Fluorescent PCR testing result
Kit in embodiment 2 is expanded into part（Reaction buffer）Freeze thawing, mixing, of short duration centrifugation are taken out from -20 DEG C of refrigerators, is pressed
Reaction buffer is dispensed into PCR reaction tubes by 20 μ l/ person-portions, be then respectively adding said extracted kit negative control,
Positive control, the template of sample extraction and each 10 μ l of quantitative calibration product without nucleic acid extraction.Each reaction pipe volume is
Above-mentioned reaction tube is put on ABI7500 fluorescent PCR instrument by 30 μ l, is set each reaction tube sample type and is set quantitative calibration
Product concentration value reacts 2 minutes according to 50 DEG C, then keeps the temperature 5 minutes for 94 DEG C, then recycled 45 times within 45 seconds by 94 DEG C 10 seconds → 60 DEG C
（FAM channel wavelength fluorescence signals are acquired at 60 DEG C）Response procedures operation.According to instrument software and kit after amplification
Specification analyzes and determines experimental result, it is known that this 25 samples（Whole blood, blood plasma, serum, urine, milk sample each 2）It is
HCMV nucleic acid is positive and 5 Epstein-barr viruses infection whole blood samples are negative for HCMV, and it is various to illustrate that kit is suitable for
The detection of HCMV in type sample, accuracy and specificity are good.
Sequence table
<110>Shanghai Xingyao Medical Technology Development Co., Ltd.
<120>A kind of human cytomegalovirus kit for detecting nucleic acid
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(20)
<223>Sense primer
<400> 1
agagagcgat tggtgttgcg 20
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(21)
<223>Downstream primer
<400> 2
ccgagttgga caacgagaag g 21
<210> 3
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(26)
<223>Fluorescence probe A; b=FAM; d=BHQ
<400> 3
batgcggctc acctcgtcaa tcttgd 26
<210> 4
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(26)
<223>Fluorescence probe B; b=FAM; d=BHQ
<400> 4
baagaacacc cccttctgca cacccd 26
Claims (6)
- A kind of 1. kit based on the highly sensitive detection human cytomegalovirus nucleic acid of Fluorescence PCR assay, which is characterized in that the examination Agent box has SEQ ID NO using a pair of of human cytomegalovirus amplimer and two fluorescence probes, pair of primers：1 and SEQ ID NO：2 sequence, two fluorescence probes have SEQ ID NO：3 and SEQ ID NO：4 sequences and the end of fluorescence probe 5 ' label Identical fluorescein or fluorescent dye reporter group, 3 ' end mark fluorescent quenching groups.
- 2. kit as described in claim 1, which is characterized in that the fluorescein or fluorescent dye reporter group are FAM Fluorophor or it is other it is any can as probe mark fluorescein or fluorescent dye, the fluorescent quenching group for BHQ, TAMRA or other is any can be as the compound of quenching group.
- 3. kit as described in claim 1, which is characterized in that the kit reaction system is respectively as follows into being grouped as： Tris-HCl, 50mM KCl, 0.2~0.5mM dNTPs, 1.5~5 mM MgCl of 10mM pH8.32, it is 0.1~0.5 μM each SEQ ID NO：1~4 primer and fluorescence probe, 1~5 U Taq archaeal dna polymerases and 0.01~1 U UNG enzymes, the template of extraction 10 μ l, 30 μ l of total reaction volume.
- 4. kit as described in claim 1, which is characterized in that each ingredient of kit reaction solution is final concentration of： Tris-HCl 10 mM, KCl 50 mM, dATP, dGTP, dCTP, dUTP each 0.2 mM, MgCl of pH8.323.5 mM, SEQ ID NO：1 and SEQ ID NO：Each 0.3 μM of 2 primers, SEQ ID NO：3 and SEQ ID NO：Each 0.2 μM of 4 fluorescence probes, Taq 2 U of archaeal dna polymerase, 0.5 U of UNG enzymes.
- 5. kit as described in claim 1, the amplification program used is as follows：According to 94 after 50 DEG C of 2min, 94 DEG C of 5min DEG C 10sec, 60 DEG C of 45sec cyclic amplifications 45 times acquire fluorescent reporter group launch wavelength fluorescence in 60 DEG C of each cycle Signal.
- 6. kit as described in claim 1, including negative control, positive control and viral nucleic acid quantitative calibration product.
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| CN111500779A (en) * | 2020-05-06 | 2020-08-07 | 深圳康美生物科技股份有限公司 | Kit for detecting human cytomegalovirus based on fluorescence PCR or mass spectrum |
| CN115044688A (en) * | 2022-06-22 | 2022-09-13 | 江苏康为世纪生物科技股份有限公司 | Freeze-dried PCR reagent and kit for helicobacter pylori nucleic acid detection |
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| CN109022620A (en) * | 2018-08-27 | 2018-12-18 | 郑州安图生物工程股份有限公司 | It is a kind of for detecting the kit of human cytomegalovirus |
| CN111500779A (en) * | 2020-05-06 | 2020-08-07 | 深圳康美生物科技股份有限公司 | Kit for detecting human cytomegalovirus based on fluorescence PCR or mass spectrum |
| CN115044688A (en) * | 2022-06-22 | 2022-09-13 | 江苏康为世纪生物科技股份有限公司 | Freeze-dried PCR reagent and kit for helicobacter pylori nucleic acid detection |
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