CN106191305A - Prenatal and postnatal care detection kit, detection oligonucleotide and method - Google Patents

Prenatal and postnatal care detection kit, detection oligonucleotide and method Download PDF

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CN106191305A
CN106191305A CN201610545337.XA CN201610545337A CN106191305A CN 106191305 A CN106191305 A CN 106191305A CN 201610545337 A CN201610545337 A CN 201610545337A CN 106191305 A CN106191305 A CN 106191305A
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probe
detection
hsv
primer
herpes simplex
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陈斐斐
张太松
毛露茜
刘学峰
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Acon Biotech Hangzhou Co Ltd
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Acon Biotech Hangzhou Co Ltd
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster

Abstract

The invention provides a kind of oligonucleotide, test kit and method for prenatal and postnatal care detection, including primer and probe, the primer of detection herpes simplex virus and probe, the primer of detection toxoplasma and probe, the primer of detection rubella virus and probe, the primer of detection herpes simplex virus I-type and probe, the primer of detection herpes simplex virus type II and the probe of detection cytomegalovirus.The present invention can diagnose rapidly and differentiate torch infection situation, has the detection cycle short, and specificity is good, and accuracy is high, highly sensitive, and condition relies on few, the advantages such as pollution risk is low.

Description

Prenatal and postnatal care detection kit, detection oligonucleotide and method
Technical field
The invention belongs to field of gene detection, be particularly used for prenatal and postnatal care TORCH (as toxoplasma, rubella virus, Cytomegalovirus, herpes simplex virus etc.) detection purpose test kit, detectable and method.
Background technology
TORCH is the abbreviation of one group of pathogenic microorganism English alphabet, and T refers to Toxoplasma gondii (toxoplasma, TOX), and O refers to Other microorganisms (Others, such as Coxsackie virus, prunus mume (sieb.) sieb.et zucc. green grass or young crops spirillum, piconavirus, hepatitis B virus etc.), R refers to rubella virus (rubella virus, RV), C refers to cytomegalovirus (cytomegalovirus, CMV), and H refers to herpes simplex virus (herpes Simplx virus, HSV) I, II type.This group infection caused by pathogen is referred to as torch infection.These several cause of disease body-sensing The scope of dye is wide, harm is big, and anemia of pregnant woman causes immunity of organisms to decline due to the change of hormonal system, is susceptible to primary sexuality Dye, the virus hidden in the anemia of pregnant woman's body infected in the past is the most easily activated and recurrent infection occurs.Trimester of pregnancy infection is not only endangered Evil parent, produces serious adverse consequences toward contact to fetus an d neonate, and miscarriage, premature labor, stillborn fetus or fetus can be caused raw Long slow and developmental disorder, can causing infection of newborn by birth canal and breast milk, if involving nervous system, can cause difference Dysnoesia and the various paralysis of degree, become deaf, the serious sequela such as blind, thus affect population quality.
Arch insect infection is unique infectious diseases common to human beings and animals in TORCH pathogen, all can become sense such as cat, dog, sheep, chicken Dye source, toxoplasma can enter human body, edible uncooked meat class or dirty goods, the water drinking pollution or suction by the wound of people Ill domestic animal excretion the spittle and infected.Most after infection is inapparent infection, and patient does not has obvious sings and symptoms, for pregnant The phase women that is pregnent infects for pregnant just 3 months, causes fetus and has a strong impact on, may result in miscarriage, premature labor, stillbirth or deformity.Rubella virus By respiratory tract infection, can directly pass through placental barrier after infecting anemia of pregnant woman, be broadcast to the infection rate progress along with the pregnancy period of fetus And reduce, therefore trimester of pregnancy determines that the time of rubella-infection is critically important.Cytomegalovirus belongs to herpesvirus, mainly by with row Poison person's close contact and infect, be one of maximum teratogenesis pathogen of intrauterine infection harm.After infection of pregnant women, virus may be caused Mass formed by blood stasis, infects fetus by blood, placental barrier, amniotic fluid etc., causes fetal development abnormal, causes miscarriage, stillborn fetus, premature labor, deformity Deng.Clinically, identify that the infection conditions of cytomegalovirus is damaged with or without organ dysfunction after identifying fetal infection further, and No sequel is had to play a very important role after birth.Herpes simplex virus has I, II type, wherein infects based on II type, propagates way Footpath is mainly birth canal and infects, and pregnant early stage mainly causes fetus stream to the infringement of fetus more than late trimester of pregnancy, the impact on pregnant early stage Product, congenital malformation, premature labor etc..
Torch infection has been subjected to world-wide medical circle especially department of obstetrics and gynecology and the great attention of pediatrician.American-European etc. The detections such as rubella, toxoplasma, cytomegalovirus are just classified as pregnancy period examination project as far back as the seventies by developed country.China with Deeply carrying out of prenatal and postnatal care work, pregnancy period TORCH detection is progressively carried out in medical treatment and family planning service.At present The methods for clinical diagnosis of torch infection includes the methods such as etiological examination, Serological testing, gene diagnosis.
Etiological examination includes body fluid or live tissue puncturing thing smear, tissue slice, microscopy and the separation of pathogen and training Support.Once microscopy finds or the existence of separation and Culture confirmation pathogen, can make a definite diagnosis.But smear, section verification and measurement ratio are low and required Time is long, costly, is not suitable as routine examination project.
It is elisa (ELISA) that Serological testing is applied most at present, utilizes antigen and antibody specific In conjunction with principle detection serum in antigen or the existence of antibody.TORCH is generally directed to lgM or lgG in clinical diagnosis.LgM exists Occur in early days, substituted by lgG later.China gets used to the IgM of TORCH 4 and carries out examination, but IgM antibody test affect because of Element is a lot, and domestic people TORCH projects IgM positive rate is the highest, and false positive easily occurs in simple use IgM examination.Additionally Along with the variation of life style, the ratio of HSV I reproductive tract infection is increasing year by year, as do not differentiated between HSV I and HSV II Screening method easily causes anemia of pregnant woman's perinatal stage to infect without the HSV differentiated, causes potentially serious harm to neonate.Therefore for obtaining Obtaining result more accurately and reliably, it is necessary for carrying out TORCH examination simultaneously.
Gene diagnosis includes that with nucleotide sequence be the molecule hybridization that carries out of probe and polymerase based on nucleic acid amplification Chain reactions (PCR) etc., this kind of detection sensitivity is high, can directly detect the hereditary material of pathogen, it was demonstrated that the existence of pathogen. Detection sample has drawn from amniotic fluid of pregnant woman cell, venous blood, fine hair specimen, umbilical cord blood, urine, milk etc. more.But nucleic acid speckle Dot blot, nucleic acid hybridization in situ method owe sensitive, and specificity is the highest, it is impossible to as clinical routine diagnostics, generally believe now PCR Method has reproducible, high specificity, highly sensitive feature, can make the target gene sample amplification million times of denier, have The highest sensitivity, overcomes missing inspection.Along with the use of PCR method, TORCH pathogen individual event PCR detectable occurs in succession, Due to symptom, the consequence all similar of torch infection, anemia of pregnant woman has to carry out multinomial detection simultaneously so that price is higher, still difficult pregnant Woman extensively carries out.When at present using PCR method examination TORCH, need independent four pipes detect respectively toxoplasma, rubella virus, Cytomegalovirus, the situation of herpes simplex virus.In order to determine which kind of type that sample is HSV, even to use five pipes respectively Detection toxoplasma, rubella virus, cytomegalovirus, herpes simplex virus I-type, the situation of herpes simplex virus type II.The most not The more sample of patient to be consumed, also greatly increases reagent and human cost simultaneously.
Summary of the invention
In order to overcome the deficiency in above-mentioned TORCH detection, the invention provides a kind of few core for prenatal and postnatal care detection Thuja acid includes:
The detection primer of cytomegalovirus and probe sequence be:
HCMV FP:AAGTGTTTATAATTCTGGTCGCA
HCMV RP:AGGGCCAGAAGCATCTGGTAA
HCMV P:ACCGGGACCACCGTCGTCTGA
The detection primer of herpes simplex virus and probe sequence be:
The general FP:GGAGAGGGACATCCAGGACT of HSV
The general RP:CATGAGCTTGTAATACACCGTCA of HSV
The general P:CTCACCGCCGAACTGAGCAGACA of HSV
The detection primer of toxoplasma and probe sequence be:
TOX FP:AGCCACAGAAGGGMCAGAAGT
TOX RP:CTCGYCGCYTCCCAACCA
TOX P:ATGCCGCTCCTCCAGCCGTC
The detection primer of rubella virus and probe sequence be:
RV FP:CGCATYTGGAAYGGYAC
RV RP:GYRAACACRCTCATCAC
RV P:AAGCARTACCAYCCYACCG
The detection primer of herpes simplex virus I-type and probe sequence be:
HSV-1FP:TGCAGAGCAACCCCATGAAG
HSV-1RP:GGCCTCGTCAAARTCGCC
HSV-1P:CTCAAGAACCCCACCARCCCGGA
The detection primer of herpes simplex virus type II and probe sequence be:
HSV-2FP:CTCCGGGTTTCACGTCGA
HSV-2RP:CGCAGGGAGAGCGTACTGA
HSV-2P:TGGTGGTGTTTGACTTTGCCAGCC
Wherein said M represents A or C, R and represents A or G, Y and represent C or T.
The fluorophor of described probe 5` end is labeled as FAM, VIC, Texas Red, Cy5 or HEX.
Further, the fluorophor of the 5` end labelling of probe HCMV P is FAM, the 5` end labelling of the general P of probe HSV Fluorophor is VIC, and the fluorophor of the 5` end labelling of probe TOX P is Texas Red, the 5` end labelling of probe RV P glimmering Light group is Cys5, and the fluorophor of the 5` end labelling of probe HSV-1P is FAM, the fluorescent base of the 5` end labelling of probe HSV-2P Group is VIC.
Present invention also offers a kind of TORCH detection kit for prenatal and postnatal care, try including DNA/RNA nucleic acid extraction Agent, fluorescent quantitative PCR reactant liquor, enzyme mixation, positive reference substance and negative controls, fluorescent quantitative PCR is reacted Liquid includes PCR reactant liquor 1 and PCR reactant liquor 2, and described PCR reactant liquor 1 includes primer and probe, the inspection detecting cytomegalovirus Survey primer and probe, the primer of detection toxoplasma and probe, the primer of detection rubella virus and the probe of herpes simplex virus, its In,
The detection primer of cytomegalovirus and probe sequence be:
HCMV FP:AAGTGTTTATAATTCTGGTCGCA
HCMV RP:AGGGCCAGAAGCATCTGGTAA
HCMV P:ACCGGGACCACCGTCGTCTGA
The detection primer of herpes simplex virus and probe sequence be:
The general FP:GGAGAGGGACATCCAGGACT of HSV
The general RP:CATGAGCTTGTAATACACCGTCA of HSV
The general P:CTCACCGCCGAACTGAGCAGACA of HSV
The detection primer of toxoplasma and probe sequence be:
TOX FP:AGCCACAGAAGGGMCAGAAGT
TOX RP:CTCGYCGCYTCCCAACCA
TOX P:ATGCCGCTCCTCCAGCCGTC
The detection primer of rubella virus and probe sequence be:
RV FP:CGCATYTGGAAYGGYAC
RV RP:GYRAACACRCTCATCAC
RV P:AAGCARTACCAYCCYACCG
Described PCR reactant liquor 2 includes primer and probe, the detection herpes simplex virus I I detecting herpes simplex virus I-type The primer of type and probe, wherein,
The detection primer of herpes simplex virus I-type and probe sequence be:
HSV-1FP:TGCAGAGCAACCCCATGAAG
HSV-1RP:GGCCTCGTCAAARTCGCC
HSV-1P:CTCAAGAACCCCACCARCCCGGA
The detection primer of herpes simplex virus type II and probe sequence be:
HSV-2FP:CTCCGGGTTTCACGTCGA
HSV-2RP:CGCAGGGAGAGCGTACTGA
HSV-2P:TGGTGGTGTTTGACTTTGCCAGCC
Wherein said M represents A or C, R and represents A or G, Y and represent C or T
The fluorophor of described probe 5` end is labeled as FAM, VIC, Texas Red, Cy5 or HEX.
Further, the fluorophor of probe HCMV P 5` end labelling is FAM, probe HSV general P 5` end labelling glimmering Light group is VIC, and the fluorophor of probe TOX P 5` end labelling is Texas Red, the fluorescent base of probe RV P 5` end labelling Group is Cys5, and the fluorophor of probe HSV-1P 5` end labelling is FAM, and the fluorophor of probe HSV-2P 5` end labelling is VIC。
Described positive reference substance include HCMV DNA clone plasmid, the general cloned plasmids of HSV, TOX cloned plasmids, containing RV The virus-like particle of genetic fragment, HSV-1 cloned plasmids and HSV-2 cloned plasmids
Described negative controls includes 0.9%NaCl.
The method that present invention also offers preparation prenatal and postnatal care TORCH detection kit, comprises the following steps:
(1) preparation DNA/RNA extracts reagent；
(2) preparation fluorescent quantitative PCR reactant liquor；
(3) preparation enzyme mixation；
(4) preparation positive reference substance；
(5) preparation negative controls；
HCMV FP, HCMV RP and HCMV P is included for preparing the oligonucleotide of fluorescent quantitative PCR reactant liquor； The general P of general RP and HSV of HSV general FP, HSV；TOX FP, TOX RP and TOX P；RV FP, RV RP and RV P；HSV-1FP、 HSV-1RP and HSV-1P；HSV-2FP, HSV-2RP and HSV-2P.Described fluorescent quantitative PCR reactant liquor includes that PCR reacts Liquid 1 and PCR reactant liquor 2, wherein PCR reactant liquor 1 includes primer and probe, the detection herpes simplex virus detecting cytomegalovirus Primer and probe, the primer of detection toxoplasma and probe, the primer of detection rubella virus and probe；PCR reactant liquor 2 includes inspection Survey primer and probe, the primer of detection herpes simplex virus type II and the probe of herpes simplex virus I-type.
The fluorophor of described probe 5` end is labeled as FAM, VIC, Texas Red, Cy5 or HEX.
Beneficial effect: the present invention uses the mode that primary dcreening operation and typing combine.Primary dcreening operation may determine that whether sample is bow Shape worm, rubella virus, cytomegalovirus, the herpes simplex virus positive.If primary dcreening operation result display sample herpes simplex virus is sun Property, then this sample is carried out herpes simplex virus detection.No matter when primary dcreening operation and typing detect, all employ and examine a pipe more Mode, this not only reduces the cost of examination, improves again the flux of detection.Test kit of the present invention uses DNA/RNA altogether Extraction agent extracts sample and DNA/RNA coamplification program detects, and this achieves in same reaction system the completeest Become RNA sample and the fluorescence quantitative PCR detection of DNA sample.In described test kit use the UNG antipollution reagent of enzyme+dUTP and Measure, can effectively get rid of false positive results, improves the accuracy of TORCH detection.
Accompanying drawing explanation
Fig. 1 is clinical sample HCMV testing result.
Fig. 2 is clinical sample HSV testing result.
Fig. 3 is TOX testing result.
Fig. 4 is clinical sample RV testing result.
Fig. 5 is clinical sample HSV-1 testing result.
Fig. 6 is clinical sample HSV-2 testing result.
Fig. 7 is the electrophoresis result of the dU-DNA experiment eliminating residual.
Fig. 8 be UNG enzyme be whether the electrophoresis result of 5 prime excision enzyme activity experiment.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.
Embodiment 1 detectable and test kit
Reagent for TORCH detection includes PCR reactant liquor 1 and PCR reactant liquor 2.Described PCR reactant liquor 1 includes detection The primer of cytomegalovirus and probe, the primer of detection herpes simplex virus and probe, the primer of detection toxoplasma and probe and The primer of detection rubella virus and probe.Wherein,
Primer (HCMV FP and HCMV RP) and probe (HCMV P) sequence of detection cytomegalovirus are as follows:
HCMV FP:AAGTGTTTATAATTCTGGTCGCA
HCMV RP:AGGGCCAGAAGCATCTGGTAA
HCMV P:ACCGGGACCACCGTCGTCTGA
Primer (the general RP of general FP and HSV of HSV) and probe (the general P of the HSV) sequence of detection herpes simplex virus are as follows:
The general FP:GGAGAGGGACATCCAGGACT of HSV
The general RP:CATGAGCTTGTAATACACCGTCA of HSV
The general P:CTCACCGCCGAACTGAGCAGACA of HSV
Primer (TOX FP and TOX RP) and probe (TOX P) sequence of detection toxoplasma are as follows:
TOX FP:AGCCACAGAAGGGMCAGAAGT
TOX RP:CTCGYCGCYTCCCAACCA
TOX P:ATGCCGCTCCTCCAGCCGTC
Primer (RV FP and RV RP) and probe (RV P) sequence of detection rubella virus are as follows:
RV FP:CGCATYTGGAAYGGYAC
RV RP:GYRAACACRCTCATCAC
RV P:AAGCARTACCAYCCYACCG
Described PCR reactant liquor 2 includes primer and probe, the detection herpes simplex virus I I detecting herpes simplex virus I-type The primer of type and probe, wherein,
Primer (HSV-1FP and HSV-1RP) and probe (HSV-1P) sequence of detection herpes simplex virus I-type are as follows:
HSV-1FP:TGCAGAGCAACCCCATGAAG
HSV-1RP:GGCCTCGTCAAARTCGCC
HSV-1P:CTCAAGAACCCCACCARCCCGGA
Primer (HSV-2FP and HSV-2RP) and probe (HSV-2P) sequence of detection herpes simplex virus type II are as follows:
HSV-2FP:CTCCGGGTTTCACGTCGA
HSV-2RP:CGCAGGGAGAGCGTACTGA
HSV-2P:TGGTGGTGTTTGACTTTGCCAGCC
Wherein said M represents A or C, R and represents A or G, Y and represent C or T.
The fluorophor of its middle probe 5` end is FAM, VIC, Texas Red, Cy5, HEX etc..
Test kit for TORCH detection includes DNA/RNA nucleic acid extracting reagent, fluorescent quantitative PCR reactant liquor, enzyme Mixed liquor, positive reference substance and negative controls.
DNA/RNA nucleic acid extracting reagent includes: lysate, cleaning mixture 1, cleaning mixture 2, eluent, Carrier RNA and core Acid purification column.
Fluorescent quantitative PCR reactant liquor includes PCR reactant liquor 1 and PCR reactant liquor 2.According to detection needs, probe 5` end Fluorophor be FAM, VIC, Texas Red, Cy5, HEX etc..
Positive reference substance includes: HCMV DNA clone plasmid, the general cloned plasmids of HSV, TOX cloned plasmids, containing RV gene The virus-like particle of fragment, HSV-1 cloned plasmids, HSV-2 cloned plasmids.
Negative controls includes: include 0.9%NaCl.
The preparation method of embodiment 2 detection kit and the Cleaning Principle of test kit
The preparation method of TORCH detection kit of the present invention, comprises the following steps:
(1) preparation DNA/RNA extracts reagent；
(2) preparation fluorescent quantitative PCR reactant liquor；
(3) preparation enzyme mixation；
(4) preparation positive reference substance；
(5) preparation negative controls；
The Cleaning Principle of test kit of the present invention: select toxoplasma, rubella virus, cytomegalovirus, simple bleb respectively Exanthema virus is universal, herpes simplex virus I and herpes simplex virus I I gene fragment design special primer and specific probe, this spy Pin can occur specific binding with the section of DNA of centre, primer amplified region or cDNA template, during PCR extension, 5 ' end fluorophors are cut down from probe by the 5 prime excision enzyme activity of Taq enzyme, are allowed to be free in reaction system, thus depart from The shielding of 3 ' end fluorescent quenching groups, can accept photostimulation and send the fluorescence that is available for instrument detection, thus realize entirely Aulomatizeted Detect to TORCH nucleic acid in capping system.Test kit uses the mode that primary dcreening operation and typing combine, and passes through Whether PCR reactant liquor 1 primary dcreening operation sample to be tested is toxoplasma, rubella virus, cytomegalovirus, the herpes simplex virus positive；If inspection The sample herpes simplex virus surveyed is positive, then this sample is carried out herpes simplex virus detection.In test kit the most also Employ UNG enzyme+dUTP anti-pollution measure, can effectively get rid of false positive results.
Embodiment 3 nucleic acid extraction step
1) taking out sample, concussion mixing, absorption 1mL sample is in 1.5mL centrifuge tube, and 12000rpm is centrifuged 1min, carefully Supernatant is abandoned in suction, retains 100 μ l samples.
2) often pipe (including negative control, positive control and sample to be tested) adds the 300 μ l cracking containing Carrier RNA Liquid, vortex vibration several seconds mix homogeneously, room temperature stands 10 minutes.
3) often pipe adds 320 μ l dehydrated alcohol, leniently upset 3-5 mix homogeneously of centrifuge tube.
4) take a nucleic acid purification post and put in 2ml centrifuge tube, aspiration step 3) in mixed liquor to join nucleic acid pure Changing in post, cover lid, 12000rpm is centrifuged 30 seconds.
5) abandon the filtrate in 2ml centrifuge tube, and 2ml centrifuge tube back-off on napkin is bounced once, by nucleic acid purification post Putting back in 2ml centrifuge tube, add 500 μ l cleaning mixture 1, cover lid in nucleic acid purification post, 12000rpm is centrifuged 30 seconds.
6) abandon the filtrate in 2ml centrifuge tube, and 2ml centrifuge tube back-off on napkin is bounced once, by nucleic acid purification post Putting back in 2ml centrifuge tube, add 600 μ l cleaning mixture 2, cover lid in nucleic acid purification post, 14000rpm is centrifuged 1 minute.
7) abandon the filtrate in 2ml centrifuge tube, and 2ml centrifuge tube back-off on napkin is bounced once, by nucleic acid purification post Putting back in 2ml centrifuge tube, 14000rpm is centrifuged 1 minute.
8) abandon 2ml centrifuge tube, nucleic acid purification post is placed in a clean 1.5ml centrifuge tube, in the film of purification column Centre adds 50 μ l eluents, covers lid, and room temperature stands 1 minute, and 12000rpm is centrifuged 30 seconds.
9) abandoning purification column, the nucleic acid of eluting is used for follow-up test；Or be stored in-20 DEG C standby.
Embodiment 4 Fluorescence PCR step
The PCR reactant liquor 1 of detection HCMV/HSV/RV/TOX is prepared, detection system 40 μ l.Formula is:
PCR reactant liquor 1:Tris-HCl (pH8.3) 10mmol/L, KCl 50mmol/L, MgCl24mmol/L, dNTPs are each 150umol/L、BSA 0.2μg/μl、HCMV FP 100nmol/L、HCMV RP 100nmol/L、HCMV P 75nmol/L、 HSV general FP 100nmol/L, HSV general RP 100nmol/L, HSV general P 75nmol/L, TOX FP 100nmol/L, TOX RP 100nmol/L, TOX P 75nmol/L, RV FP 200nmol/L, RV FP 250nmol/L, RV P 125nmol/ L, uses DEPC-H20 is adjusted to 0.6 μ l；
Enzyme mixation: Taq enzyme 3U, reverse transcriptase 5U, RNase inhibitor 5U, UNG enzyme 0.2U.
By above-mentioned PCR reactant liquor 1 18.6 μ l, after enzyme mixation 1.4 μ l fully mixes, it is dispensed into PCR reaction by 20 μ l amounts Guan Zhong, is transferred to sample process district.Corresponding reaction tube is separately added into the nucleic acid of gained in the embodiment 3 of 20 μ l, covers tightly anti- Ying Guan, is transferred to detection zone and carries out real-time fluorescent PCR amplification reaction, sample is carried out primary dcreening operation.
The PCR reactant liquor 2 of detection HSV-1/HSV-2 is prepared, detection system 40 μ l.Formula is as follows:
PCR reactant liquor 2:Tris-HCl (pH8.3) 10mmol/L, KCl 50mmol/L, MgCl24mmol/L, dNTPs are each 150umol/L、BSA 0.2μg/μl、HSV-1FP 100nmol/L、HSV-1RP 100nmol/L、HSV-1P 75nmol/L、 HSV-2FP 100nmol/L, HSV-2RP 100nmol/L, HSV-2P75nmol/L, be adjusted to 0.6 μ l with DEPC-H20；
Enzyme mixation: Taq enzyme 3U, reverse transcriptase 5U, RNase inhibitor 5U, UNG enzyme 0.2U.
By above-mentioned PCR reactant liquor 2 18.6 μ l, after enzyme mixation 1.4 μ l fully mixes, it is dispensed into PCR reaction by 20 μ l amounts Guan Zhong, is transferred to sample process district.Corresponding reaction tube is separately added into the nucleic acid of gained in the embodiment 3 of 20 μ l, covers tightly anti- Ying Guan, is transferred to detection zone and carries out real-time fluorescent PCR amplification reaction, sample is carried out HSV sample and carries out typing detection.
Real-time fluorescent PCR amplification response procedures such as table 1, wherein:
Table 1 real-time fluorescent PCR amplification response procedures
The most each passage result is explained as shown in table 2:
Table 2
Embodiment 5 clinical sample detects
By reagent and method detection clinical sample 135 example of embodiment 1 to 4.Business-like fluorescent PCR is selected in comparison respectively Detection kit carries out contrast test.Commercialization fluorescence PCR detection reagent kit is selected from human cytomegalic inclusion disease virus (HCMV) detection of nucleic acids Test kit (fluorescent PCR method) (matched group 1), herpes simplex virus nucleic acid (PCR) fluorescence detection reagent kit (matched group 2), arch Worm nucleic acid amplification (PCR) fluorescence detection reagent kit (matched group 3), rubella virus kit for detecting nucleic acid (PCR-fluorescence probe method) (matched group 4), herpes simplex virus I-type (HSV-I) kit for detecting nucleic acid (fluorescent PCR method) (matched group 5) and herpes simplex Virus Type II (HSV-II) kit for detecting nucleic acid (fluorescent PCR method) (matched group 6).Testing result is shown in Table 3 and Fig. 1 to 6.Fig. 1 It it is HCMV testing result.Fig. 2 is HSV testing result.Fig. 3 is TOX testing result.Fig. 4 is RV testing result.Fig. 5 is HSV-1 inspection Survey result.Fig. 6 is HSV-2 testing result.
Table 3 be comparing result (note: "+" represent positive findings, "-" represents less than reagent Monitoring lower-cut, " N/A " represent not Carry out this detection).
Experimental result shows:
In HCMV:135 example detection sample, use reagent of the present invention and method, detect that 81 examples HCMV are positive altogether, should It is positive that 81 example positive sample detect 80 examples through contrast agents, and 1 example is negative, this example matched group negative sample is confirmed through sequence measurement For positive findings.
In HSV:135 example detection sample, use reagent of the present invention and method, detect that 20 examples HSV are positive altogether, typing Detect 6 examples HSV-1,14 examples HSV-2；It is positive that this 20 example positive sample detects 20 examples HSV through contrast agents, and typing detects 5 examples HSV-1,14 examples HSV-2.Wherein the matched group 1 example HSV-1 positive does not detects.
In TOX:135 example detection sample, using reagent of the present invention and method, detect that 14 examples TOX are positive altogether, these are 14 years old It is positive that example positive sample detects 12 examples through contrast agents, and 2 examples are negative, this example matched group negative sample confirmed as through sequence measurement Positive findings.
In RV:135 example detection sample, use reagent of the present invention and method, detect that 5 examples RV are positive altogether, this 5 example positive sample It is positive that herbal classic contrast agents detects 4 examples, and 1 example is negative, and through sequence measurement, this example matched group negative sample is confirmed as positive findings.
Embodiment 6R&D test kit sensitivity technique result
HCMV, HSV, TOX, RV, HSV-1, HSV-2 positive sample is detected respectively according to reagent described in embodiment 1 to 4 and method This.Respectively positive sample being carried out gradient dilution, concentration respectively may be about 1 × 103copies/ml, 500copies/ml, 100copies/ml, each concentration is repeated 8 times.Test result is as shown in table 4, and the sensitivity of test kit of the present invention is 500copies/ml。
Table 4:(note: "+" represent positive findings, "-" represents less than reagent Monitoring lower-cut)
Embodiment 7UNG enzyme controls to pollute the result
(1) by UNG enzyme with dU-DNA as substrate, use the response procedures of the step 1 of embodiment 4, with UNG enzyme, substrate is entered Row digestion reaction, observing response result.And setting is not added with UNG enzyme as a control group.Testing result is as it is shown in fig. 7, result shows UNG enzyme can eliminate the dU-DNA of residual.UNG process can destroy residual product, stops to pollute, improves the specificity of detection.
(2) UNG enzyme and plasmid DNA are reacted 2 hours at 37 DEG C, observing response result.Testing result as shown in Figure 8, Result is shown as single amplified band.Single amplified band, illustrates to detect without 5 prime excision enzyme activity, if length difference band occurs, Illustrate that 5 prime excision enzyme activity detects.
Experimental result shows, the present invention uses UNG enzyme can destroy residual product, stops to pollute, prevents non-specific PCR Amplification and pollution, improve the accuracy of TORCH detection.

Claims (10)

1. for the oligonucleotide of prenatal and postnatal care detection, it is characterised in that include primer and probe, the inspection detecting cytomegalovirus Survey primer and probe, the primer of detection toxoplasma and probe, the primer of detection rubella virus and probe, the inspection of herpes simplex virus The primer of survey herpes simplex virus I-type and probe, the primer of detection herpes simplex virus type II and probe, wherein,
The detection primer of cytomegalovirus and probe sequence be:
HCMV FP:AAGTGTTTATAATTCTGGTCGCA
HCMV RP:AGGGCCAGAAGCATCTGGTAA
HCMV P:ACCGGGACCACCGTCGTCTGA
The detection primer of herpes simplex virus and probe sequence be:
The general FP:GGAGAGGGACATCCAGGACT of HSV
The general RP:CATGAGCTTGTAATACACCGTCA of HSV
The general P:CTCACCGCCGAACTGAGCAGACA of HSV
The detection primer of toxoplasma and probe sequence be:
TOX FP:AGCCACAGAAGGGMCAGAAGT
TOX RP:CTCGYCGCYTCCCAACCA
TOX P:ATGCCGCTCCTCCAGCCGTC
The detection primer of rubella virus and probe sequence be:
RV FP:CGCATYTGGAAYGGYAC
RV RP:GYRAACACRCTCATCAC
RV P:AAGCARTACCAYCCYACCG
The detection primer of herpes simplex virus I-type and probe sequence be:
HSV-1FP:TGCAGAGCAACCCCATGAAG
HSV-1RP:GGCCTCGTCAAARTCGCC
HSV-1P:CTCAAGAACCCCACCARCCCGGA
The detection primer of herpes simplex virus type II and probe sequence be:
HSV-2FP:CTCCGGGTTTCACGTCGA
HSV-2RP:CGCAGGGAGAGCGTACTGA
HSV-2P:TGGTGGTGTTTGACTTTGCCAGCC
Wherein said M represents A or C, R and represents A or G, Y and represent C or T.
Oligonucleotide the most according to claim 1, it is characterised in that the fluorophor of described probe 5` end be labeled as FAM, VIC, Texas Red, Cy5 or HEX.
Oligonucleotide the most according to claim 1, it is characterised in that the fluorophor of the 5` end labelling of probe HCMV P is The fluorophor of the 5` end labelling of the general P of FAM, probe HSV is VIC, and the fluorophor of the 5` end labelling of probe TOX P is The fluorophor of the 5` end labelling of Texas Red, probe RV P is Cys5, and the fluorophor of the 5` end labelling of probe HSV-1P is The fluorophor of the 5` end labelling of FAM, probe HSV-2P is VIC.
4. prenatal and postnatal care detection kit, including DNA/RNA nucleic acid extracting reagent, fluorescent quantitative PCR reactant liquor, enzyme mixing Liquid, positive reference substance and negative controls, it is characterised in that fluorescent quantitative PCR reactant liquor includes PCR reactant liquor 1 and PCR Reactant liquor 2, described PCR reactant liquor 1 include detecting the primer of cytomegalovirus and probe, the primer of detection herpes simplex virus and Probe, the primer of detection toxoplasma and probe, the primer of detection rubella virus and probe, wherein,
The detection primer of cytomegalovirus and probe sequence be:
HCMV FP:AAGTGTTTATAATTCTGGTCGCA
HCMV RP:AGGGCCAGAAGCATCTGGTAA
HCMV P:ACCGGGACCACCGTCGTCTGA
The detection primer of herpes simplex virus and probe sequence be:
The general FP:GGAGAGGGACATCCAGGACT of HSV
The general RP:CATGAGCTTGTAATACACCGTCA of HSV
The general P:CTCACCGCCGAACTGAGCAGACA of HSV
The detection primer of toxoplasma and probe sequence be:
TOX FP:AGCCACAGAAGGGMCAGAAGT
TOX RP:CTCGYCGCYTCCCAACCA
TOX P:ATGCCGCTCCTCCAGCCGTC
The detection primer of rubella virus and probe sequence be:
RV FP:CGCATYTGGAAYGGYAC
RV RP:GYRAACACRCTCATCAC
RV P:AAGCARTACCAYCCYACCG
Described PCR reactant liquor 2 includes detecting the primer of herpes simplex virus I-type and probe, detection herpes simplex virus type II Primer and probe, wherein,
The detection primer of herpes simplex virus I-type and probe sequence be:
HSV-1FP:TGCAGAGCAACCCCATGAAG
HSV-1RP:GGCCTCGTCAAARTCGCC
HSV-1P:CTCAAGAACCCCACCARCCCGGA
The detection primer of herpes simplex virus type II and probe sequence be:
HSV-2FP:CTCCGGGTTTCACGTCGA
HSV-2RP:CGCAGGGAGAGCGTACTGA
HSV-2P:TGGTGGTGTTTGACTTTGCCAGCC
Wherein said M represents A or C, R and represents A or G, Y and represent C or T.
Detection kit the most according to claim 4, it is characterised in that the fluorophor of described probe 5` end is labeled as FAM, VIC, Texas Red, Cy5 or HEX.
Detection kit the most according to claim 4, it is characterised in that the fluorophor of probe HCMV P 5` end labelling Fluorophor for FAM, probe HSV general P 5` end labelling is VIC, and the fluorophor of probe TOXP 5` end labelling is Texas Red, the fluorophor of probe RV P 5` end labelling is Cys5, and the fluorophor of probe HSV-1 P 5` end labelling is FAM, visits The fluorophor of pin HSV-2 P 5` end labelling is VIC.
Detection kit the most according to claim 4, it is characterised in that positive reference substance includes HCMV DNA clone matter Grain, the general cloned plasmids of HSV, TOX cloned plasmids, virus-like particle, HSV-1 cloned plasmids and HSV-2 containing RV genetic fragment Cloned plasmids.
Detection kit the most according to claim 4, it is characterised in that negative controls includes 0.9%NaCl.
9. the method preparing prenatal and postnatal care detection kit, comprises the following steps:
(1) preparation DNA/RNA extracts reagent；
(2) preparation fluorescent quantitative PCR reactant liquor；
(3) preparation enzyme mixation；
(4) preparation positive reference substance；
(5) preparation negative controls；
The oligonucleotide utilizing one of claim 1 to 3 described prepares described fluorescent quantitative PCR reactant liquor, described fluorescence Quantitative pcr amplification reaction liquid includes PCR reactant liquor 1 and PCR reactant liquor 2, and wherein PCR reactant liquor 1 includes detecting cytomegalovirus Primer and probe, the primer of detection herpes simplex virus and probe, the primer of detection toxoplasma and probe, detection rubella virus Primer and probe；PCR reactant liquor 2 includes primer and probe, the detection herpes simplex virus I I detecting herpes simplex virus I-type The primer of type and probe.
Method the most according to claim 9, it is characterised in that the fluorophor of described probe 5` end be labeled as FAM, VIC, Texas Red, Cy5 or HEX.
CN201610545337.XA 2016-07-08 2016-07-08 Prenatal and postnatal care detection kit, detection oligonucleotide and method Pending CN106191305A (en)

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