CN101748218A - Target sequence for detection of parainfluenza virus type I and kit - Google Patents
Target sequence for detection of parainfluenza virus type I and kit Download PDFInfo
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- CN101748218A CN101748218A CN200810204455A CN200810204455A CN101748218A CN 101748218 A CN101748218 A CN 101748218A CN 200810204455 A CN200810204455 A CN 200810204455A CN 200810204455 A CN200810204455 A CN 200810204455A CN 101748218 A CN101748218 A CN 101748218A
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Abstract
The invention provides a genetic marker and a method for detection of parainfluenza virus type I, in particular a nucleotide sequence for detection of the HN gene of the parainfluenza virus type I, oligonucleotide primers and a probe. The oligonucleotide primers and the probe are designed based on the nucleotide sequence. The invention also provides a method for rapid and specific detection of the parainfluenza virus type I with the primers. Moreover, the invention can detect the parainfluenza virus type I conveniently, rapidly, accurately, qualitatively and quantitatively.
Description
Technical field
The invention belongs to biological technical field, particularly relate to and a kind ofly utilize polymerase chain reaction (PCR) amplification I type parainfluenza virus HN gene, thus method of I type parainfluenza virus and the real-time fluorescent RT-PCR detection reagent box that utilizes this method to obtain in the special detection clinical sample in high sensibility ground.
Background technology
(parainfluenza virus PIV), belongs to myxovirus together with influenza virus to parainfluenza virus.1992 the 4th time into Paramyxoviridae (paramyxiviridae) incorporates it, paramyxovirus subfamily (paranyxivirinae) in ICNV.Its member's majority is the important pathogenic agent that causes zoonosis.Find again that in recent years some newly existing viruses are as megamyxoviruses (Hendra and Nipah viruses) and metapneumovirus.
Parainfluenza virus be child, children and adult showing the modal pathogenic agent of upper respiratory tract infection, according to statistics, infant's acute respiratory infection of about 30%～40% all is that (human parainfluenzavirus HPIV) causes by the human parainfluenza virus.To close spore virus the same with respiratory tract, and parainfluenza virus can superinfection.Parainfluenza virus is divided into one to four type, and every kind of hypotype all has different clinical and epidemiology characteristics.One type and two type parainfluenza viruses are major causes of children's croup disease, and symptom is the most serious in 2-4 year children.One type and two type parainfluenza viruses also can cause on other/the lower respiratory tract illness.The less discovery of two type parainfluenza viruses.Though parainfluenza type 3 virus also causes croup disease, its is more known as the pathogenic agent that is only second to child's bronchitis that respiratory tract closes spore virus, pneumonia.Parainfluenza type 3 virus symptom in children below 1 years old is the most serious.Four type parainfluenza viruses generally cause slight upper respiratory disease.
Eliminating I type parainfluenza virus need make great efforts in many ways, and as the development fast diagnosis method, develop new drug, carry out Study on Molecular Mechanism and vaccine research etc., it is the most urgent wherein to develop fast diagnosis method.I type parainfluenza virus direct or indirect immunofluorescence technique detection virus antigen method commonly used is diagnosed at present, does not reach the purpose of early diagnosis.In order to check out quickly the I type parainfluenza virus in the sample, augmentation detection is carried out to the nucleic acid of I type parainfluenza virus in needs development and use polymerase chain reactions (PCR).
The quantitative fluorescent PCR that development in recent years is got up (Fluorescence Quantitative PCR, FQ-PCR) technology is highly sensitive with it, specificity is good, advantages such as speed is fast are used widely at aspects such as the qualitative detection of gene expression dose analysis, pathogenic agent and detection by quantitative, and have become the quantitative main method of current viral nucleic acid.Domestic also existing at present test kit listing about hepatitis B, acquired immune deficiency syndrome (AIDS), tuberculosis detection by quantitative.
For polymerase chain reaction (PCR), select the base sequence of target DNA to be amplified the most important.At I type parainfluenza virus, this base sequence is necessary for that I type parainfluenza virus is special to be had and can not be present in for human or other biological DNA genome and may take place in other species of concurrent infection.
Parainfluenza virus is according to heredity and antigenicity, and HPIV can be divided into 4 kinds of serotype: HPIV1～4.Wherein HPIV4 can be divided into A again, two hypotypes of B.Virus has coating, and two kinds of furcellas are arranged on the coating, and a kind of is HN albumen, has the effect of HA and NA; Another kind is a F albumen, has the effect of cytogamy of making and lysed erythrocyte, and it is formed by connecting by 2 disulfide linkage by F1 and F2 subunit.In the peplos is nucleocapsid, also claims the RNP core, by viral RNA (vRNA), and N albumen, P albumen and L albumen are formed.The parainfluenza virus gene group is made up of the ssRNA of non-segmented negative, comprises about 15,500 Nucleotide, at least 6 kinds of common structural protein of encoding (3 '-N-P-C-M-F-HN-L-5 ').Discover that HN glycoprotein is present on the after birth of HPIV coating and infected cell with tetramer form probably, makes virus be adsorbed in host cell surface by the sialic acid acceptor, performance neuraminic acid enzymic activity.The experiment of Moscona and Paluso shows, the interaction of HN and cell receptor is special and complicated, and it relates to HN and these two kinds of surface glycoproteins of F, and changes because of the difference of HPIV type more, therefore, human parainfluenza virus's serotype can be carried out somatotype according to the HN gene.Based on this, this area presses for method and the test kit that exploitation newly detects I type parainfluenza virus with high specificity, so that can effectively detect I type parainfluenza virus.
Summary of the invention
Technical problem to be solved
Purpose of the present invention just provides method and the test kit of a kind of effective detection I type parainfluenza virus.
In a first aspect of the present invention, the genetic marker of a kind of I of detection type parainfluenza virus is provided, it has 75～1894 Nucleotide of successive among the SEQ IDNO:1.
In another preference, described genetic marker has the 1st～162 Nucleotide among the SEQ ID NO:2.
In a second aspect of the present invention, the test kit of a kind of I of detection type parainfluenza virus is provided, it is right that it contains the primer of specific amplification I type parainfluenza virus genetic marker, described primer length is 15～30 Nucleotide, and the sequence of a primer is identical with the sequence shown in the SEQ ID NO:1, and the sequence complementation shown in the sequence of another primer and the SEQ ID NO:1, and amplified production length is 75～1894 Nucleotide.
In another preference, described test kit also contains probe, and the length of described probe is 18～30 Nucleotide, and the sequence of this probe is identical or complementary with the sequence shown in the SEQ ID NO:1.
In another preference, a primer of described primer centering is selected from down group: SEQ ID NO:3 or 6; Another primer is selected from down group: SEQ ID NO:4 or 7; And described probe is the TaqMan probe, and its sequence is selected from SEQ ID NO:5 or 8.
In another preference, this test kit also comprises a) nucleic acid extraction liquid, b) RT-PCR damping fluid, c) quantitative calibration object, d) positive control, e) negative control.
In a third aspect of the present invention, 75～1894 method is provided in a kind of test sample, it comprises operation steps:
1. sample RNA extracts;
2. with the RAN that extracts as template, with the primer of specific amplification I type parainfluenza virus genetic marker to increasing, wherein said primer length is 15～30 Nucleotide, the sequence of a primer is identical with the sequence shown in the SEQ ID NO:1, and the sequence complementation shown in the sequence of another primer and the SEQ ID NO:1.
3. detect amplified production and whether exist, exist amplified production to represent that there is I type parainfluenza virus in sample.
In another preference, 3. described step is to come I type parainfluenza virus in the detection by quantitative sample by the power that detects fluorescent signal.
In a fourth aspect of the present invention, a kind of purposes of I type parainfluenza virus HN gene is provided, it is characterized in that it is used as the genetic marker whether vitro detection I type parainfluenza virus exists.
In another preference, I type parainfluenza virus HN gene has the nucleotide sequence shown in the SEQ ID NO:1.
Embodiment
The inventor is extensive studies through going deep into, gene-HN gene (the Kate E.Templeton of one section about about 1900 base pair from I type parainfluenza virus, finding, Sitha A.Scheltinga, Matthias F.C.Beersma, Aloys C.M.Kroes, and Eric C.J.Claas.Rapid and Sensitive Method Using Multiplex Real-Time PCR forDiagnosis of Infections by Influenza A and Influenza B Viruses, Respiratory SyncytialVirus, and Parainfluenza Viruses 1,2,3, and 4.Journal of Clinical Microbiology, April2004, p.1564-1569, Vol.42, No.4) the suitable extension increasing sequence of middle discovery.This gene order is an I type parainfluenza virus functioning gene, therefore, selects this ad hoc structure, functioning gene sequence as primer, has good characteristic.
As used herein, the nucleotide sequence of I type parainfluenza virus HN gene, its GenBank accession number HPU70936, concrete sequence is shown in SEQ ID NO:1.
Can in I type parainfluenza virus, expand according to the primer of I type parainfluenza virus HN gene design and the corresponding nucleic acids fragment, and, this primer does not amplify respective segments on other relevant bacterial strains, illustrate with I type parainfluenza virus HN gene order to be that the PCR reactive system of basic design has good specificity.
Though relatively by homology, a plurality of zones are arranged as suitable specificity marker zone in the full-length gene of I type parainfluenza virus HN gene, yet the nucleotide sequence shown in the SEQ ID NO:2 is a kind of sequence of the particularly preferred I of being suitable as type parainfluenza virus specificity genetic marker.
Therefore, based on SEQ ID NO:1 and 2, the invention provides a kind of method of utilizing polymerase chain reaction (PCR) amplification I type parainfluenza virus HN gene test I type parainfluenza virus; Also provide a kind of fast quantification to detect the fluorescence quantitative RT-PCR kit of I type parainfluenza virus.Key wherein has been to use the primer of specific amplification I type parainfluenza virus genetic marker right, described primer length is 15～30 Nucleotide, and a primer sequence is identical with the sequence shown in the SEQ ID NO:1, the sequence of another primer and SEQ IDNO:1 complementation, and the length of amplified production is 75～1894 Nucleotide.For example, SEQ ID NO:3 is identical with the sequence shown in the SEQ ID NO:1, and the sequence complementation shown in another primer SEQ ID NO:4 and the SEQID NO:1, and the length of amplified production is 162bp.SEQ ID NO:3 and 4 corresponds respectively to 531 and 673 positions of SEQID NO:1.
The probe of primer of specific amplification I type parainfluenza virus of the present invention and detection usefulness, can design according to the sequence (SEQ ID NO:1 or 2) of the genetic marker of inventing, and obtain (as can be synthetic) with business-like automatic dna synthesizer by conventional DNA synthetic method.
These primers of the present invention can carry out mark with radio isotope, vitamin H, enzyme, fluorescein or other chemiluminescent substance.
I type parainfluenza virus specificity nucleic acid molecule primer of the present invention has fabulous specific specificity.Carry out conventional PCR reaction with primer of the present invention, the result can amplify size and be 162bp specific PCR product from the RNA extract of the material that contains I type parainfluenza virus.Therefore, carry out conventional PCR reaction with primer of the present invention, and by judge having or not of corresponding big or small PCR product can be accurately, rapid detection I type parainfluenza virus, and required sample size is considerably less.
In order to reduce false positive, also can hybridize with I type parainfluenza virus specific probe amplified production.A kind of preferred probes is the TaqMan probe, it can be in PCR reaction directly whether and the height of quantity the existence by fluorescent signal reflection amplified production in real time.
Probe of the present invention can carry out mark with radio isotope, vitamin H, enzyme, fluorescein or other chemiluminescent substance.5 ' end report fluorophor such as Fluoresceincarboxylic acid (Carboxyfluorescein with this sequence, mark such as FAM), 3 ' end reacts with promptly can be used for the PCR that I type parainfluenza virus carries out qualitative and quantitative analysis behind the marks such as quenching group such as TAMRA or Dabcy1.
Though the complete complementation of primer and probe and template sequence is preferred, but those skilled in the art will know that, there be under the situation of certain not complementary (especially 5 ' of primer end) also can increase specifically (promptly only amplifying required fragment) at primer and template.The method that contains the test kit of these primers and use these primers is all within the scope of the invention, as long as the product that this primer amplification goes out contains fragment of the present invention.
Though the length of amplified production is not particularly limited, the length of amplified production is 70～1000bp usually, preferably is 75～500bp, more preferably is 100～300bp.
Primer of the present invention is combined with the TaqMan probe technique, just obtained of the present inventionly simultaneously I type parainfluenza virus being carried out qualitative detection and quantitative counting analytical procedure, this analytical procedure has not only overcome the error in the conventional quantifying PCR method, and it is few to analyze required sample size, detection limit is low, and is highly sensitive.
In a preference, a kind of fast quantification detects the fluorescence RT-PCR test kit of I type parainfluenza virus, and this test kit comprises a) RNA extracting solution, b) fluorescent quantitation reaction solution, and c) quantitative calibration object, d) positive reference substance, e) negative control product is characterized in that:
1) the fluorescent quantitation reaction solution contains PCR damping fluid, dNTPs solution, Taq archaeal dna polymerase, DEPC treating water, MgCl
2Solution, (as primer sequence is SEQ ID NO:3 and SEQ ID NO:4 for the primer of specific amplification I type parainfluenza virus HN gene genetic marker and fluorescent probe, the fluorescent probe sequence is SEQ ID NO:5), the fluorescence report group of fluorescent probe 5 ' end mark is FAM, and the fluorescent quenching group of 3 ' end mark is TAMRA.
2) quantitatively calibration object is to contain SEQ ID NO:2 totally 162 pGEM-T carriers that nucleotide fragments constitutes, and this carrier can be bred in intestinal bacteria.Positive reference substance is the positive culture of I type parainfluenza virus.
In a preferred version of the present invention, the fluorescent quantitation reaction solution comprises 50mM KCl, 10mM Tris-HClPH8.3 (25 ℃), 3mM MgCl
2, 3% methane amide, each 3pmol of forward primer and reverse primer, fluorescent probe 2pmol, 0.2mM dNTPs, DEPC treating water and mixed enzyme (each 1.5 unit of ThermoScript II and Taq enzyme).The nucleotide sequence shown in primer SEQID NO:3 and the SEQ ID NO:4 wherein, the nucleotide sequence shown in the fluorescent probe SEQ ID NO:5, the fluorescence report group of fluorescent probe 5 ' end mark is FAM, the fluorescent quenching group of 3 ' end mark is TAMRA.
In a preferred version of the present invention, quantitatively calibration object is to contain SEQ ID NO:2 to have the pGEM-T carrier that 162 nucleotide fragments constitute, and this carrier can be bred in intestinal bacteria.Storing concentration is 2 * 10
9Copies/ μ l uses preceding 10 times of gradient dilutions.After containing the segmental plasmid transformation escherichia coli propagation of purpose, use alkaline lysis method of extracting, through DNA purification kit purifying, with spectrophotometric instrumentation A
260Quantitatively and be diluted to 2 * 10
9Copies/ μ l ,-20 ℃ of preservations.
In a priority scheme of the present invention, the RNA extracting solution comprises 4M guanidine thiocyanate, 0.75M Trisodium Citrate (pH7.0) and 10% sodium N-lauroyl sarcosinate (Sarcosyl 1g/10mL), 14.3M beta-mercaptoethanol, 2M sodium-acetate (pH4.0) and water-saturated phenol.
In the invention provides the fluorescence quantitative RT-PCR kit that detects I type parainfluenza virus, there are two ends to be marked with the specific probe of fluorophor, when probe is complete, two groups distance on space structure is close, the fluorescence that 5 ' end reporter group produces is FRET (fluorescence resonance energy transfer) (FRET) and by the group cancellation of going out of 3 ' end quenching, thus in the system detection less than fluorescence.In PCR annealing and extension process, probe and template specificity combination, extension along with primer, the Taq archaeal dna polymerase utilizes it at 5 ' → 3 ' 5 prime excision enzyme activity fluorescent probe cutting to be discharged reporter group, destroyed the FRET between the group like this, the fluorescence that reporter group discharged can be built in the fluoroscopic examination in the quantitative PCR instrument, the proportional example relation of the accumulation volume of the increase of fluorescence volume and PCR product, to I type parainfluenza virus quantitatively can by with the cycle threshold (Ct of quantitative calibration object, Threshold Cycle) compares and draw, the Ct value is in the PCR process, and the accumulation of fluorescence volume surpasses substrate fluorescence volume cycle number.The Ct value is big more, and the starting template number is few more.Utilize the Ct value of the quantitative calibration object of gradient to make typical curve, again according to the initial copy number that can accurately measure this sample for the Ct value of test sample product.
The fluorescence quantitative RT-PCR kit of detection I type parainfluenza virus provided by the invention, process is to each component, as primer concentration, fluorescent probe concentration, Mg
2+Optimization such as concentration, annealing temperature (comprises round pcr and real-time fluorescence PCR system PE7000/7300/7500, LightCycler) combines.By prioritization scheme, experiment repeatedly, test kit can satisfy the actual requirement of quick special detection I type parainfluenza virus fully.
The present invention also provides a kind of method of utilizing test kit of the present invention to detect I type parainfluenza virus, and this method comprises following detection step:
A) from positive reference substance and sample to be tested, extract RNA with the RNA extracting solution;
B) getting the quantitative calibration object of RNA, gradient that the step extracts respectively adds in the fluorescent reaction liquid and carries out augmentation detection with the real-time fluorescence PCR instrument;
C) the initial copy number to testing sample carries out quantitatively by the cycle threshold that compares sample to be tested and standard substance.
The present invention compares with existing culture method commonly used has following major advantage and effect:
A) detection speed is fast, adds the extraction of RNA, finishes in 2～3 hours;
B) existence of fluorescent probe has guaranteed the specificity and the sensitivity that detect;
C) simple to operate;
D) quantitatively accurately;
E) can carry out the sample detection that height passes through simultaneously.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Hardor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1 separates and is used for the I type parainfluenza virus RNA that RT-PCR analyzes
To be used for the I type parainfluenza virus RNA that the polymerase chain reaction is detected in order from sample to be tested, separating, must use not influence the simple method of polymerase chain reaction.
1. get person under inspection's lung deep expectoration sputum 1～3ml with sterilization 5ml Glass tubing, airtight, or get throat swab sealing censorship.Sample can be used for immediately the test, also can be stored in-20 ℃ to be measured, preservation period is 6 months.Sample transports and should adopt 0 ℃ of curling stone.Sputum sample is handled: adds the 4%NaOH of 4 times of volumes in the sputum, shakes up, place about 30min under the room temperature and liquefy, get in 0.5ml to the 1.5ml centrifuge tube, add again after 0.5ml 4%NaOH room temperature places 10min, and 15, the centrifugal 5min of 000rpm.The precipitation add stroke-physiological saline solution 1ml beat even, 15, the centrifugal 5min of 000rpm; Repeated washing once removes most supernatant again.Throat swab is handled: add the 1ml stroke-physiological saline solution, fully concussion shakes up, and imbitition goes in the 1.5ml centrifuge tube, and 12, the centrifugal 5min of 000rpm removes most supernatant.
2. add 500 μ l RNA extracting solutions in above-mentioned precipitation, blow and beat mixing repeatedly with pipettor, room temperature was placed 5 minutes.
3. add 200 μ L trichloromethanes, with vibrate back and forth mixing 15 seconds of hand, room temperature was placed 5 minutes, and 4 ℃ 15, centrifugal 10 minutes of 000rpm moves into new centrifuge tube with supernatant.
4. add the equal-volume Virahol, vortex vibration 5 seconds, precipitation at room temperature 10 minutes, 4 ℃ 15, centrifugal 5 minutes of 000rpm abandons supernatant.
5. 75% washing with alcohol that adds 500 μ L precoolings turns upside down 10 times, and 4 ℃ 10, centrifugal 5 minutes of 000rpm blots supernatant, precipitation drying at room temperature 10-20 minute, and adding 20 μ L does not have RNA enzyme water dilution mixing, uses immediately or-70 ℃ of preservations.
The preparation of embodiment 2 detection kit
Prepare the fluorescence RT-PCR test kit that a kind of fast quantification detects I type parainfluenza virus with ordinary method, this test kit comprises a) RNA extracting solution, b) fluorescent quantitation reaction solution, c) quantitative calibration object, d) positive reference substance, e) negative control product.
Wherein:
1) the fluorescent quantitation reaction solution contains PCR damping fluid, dNTPs solution, Taq archaeal dna polymerase, DEPC treating water, MgCl
2Solution, (as primer sequence is SEQ ID NO:3 and SEQ ID NO:4 for the primer of specific amplification I type parainfluenza virus HN gene genetic marker and fluorescent probe, the fluorescent probe sequence is SEQ ID NO:5), the fluorescence report group of fluorescent probe 5 ' end mark is FAM, and the fluorescent quenching group of 3 ' end mark is TAMRA.(the amplified production size is 162bp)
Particularly, the fluorescent quantitation reaction solution comprises 50mM KCl, 10mM Tris-HCl PH8.3 (25 ℃), 3mM MgCl
2, 3% methane amide, each 3pmol of forward primer and reverse primer, fluorescent probe 2pmol, 0.2mM dNTPs, DEPC treating water and mixed enzyme (each 1.5 unit of ThermoScript II and Taq enzyme).
2) quantitatively calibration object is to contain SEQ ID NO:2 to have the pGEM-T carrier (this carrier is available from Promego company, and SEQ ID NO:2 inserts the β-Nei Xiananmei coding region) that 162 nucleotide fragments constitute, and this carrier can be bred in intestinal bacteria.
Particularly, quantitatively calibration object is 2 * 10 storing concentration
9Copies/ μ l uses preceding 10 times of gradient dilutions.After containing the segmental plasmid transformation escherichia coli propagation of purpose, use alkaline lysis method of extracting, through DNA purification kit purifying, with spectrophotometric instrumentation A
260Quantitatively and be diluted to 2 * 10
9Copies/ μ l ,-20 ℃ of preservations.
3) the RNA extracting solution comprises 4M guanidine thiocyanate, 0.75M Trisodium Citrate (pH7.0) and 10% sodium N-lauroyl sarcosinate (Sarcosyl 1g/10mL), 14.3M beta-mercaptoethanol, 2M sodium-acetate (pH4.0) and water-saturated phenol.
Embodiment 3 usefulness I type parainfluenza viruses carry out the polymerase chain reaction
Each the 26 μ l of fluorescent reaction liquid that get respectively among the embodiment 2 join in the different PCR reaction tubess, the RNA and the quantitative calibration object of gained among the embodiment 1 are added 4 μ l in the fluorescent quantitation reaction solution, cumulative volume 30 μ l begin augmentation detection on real-time fluorescence quantitative PCR instrument (ABI 7500).The amplification parameter be set to 37 ℃ 30 minutes, 94 ℃ 5 minutes, by 94 ℃ 10 seconds → 60 ℃ 45 seconds the circulation 40 times.At each round-robin annealing steps as a result the time, the detection wavelength is 530nm the programdesign of fluoroscopic examination.
After the loop ends, utilization instrument software kit reads sample copy number to be checked.The result is: quantitatively accurate product 2 * 10
6Copies/ μ l, 2 * 10
5Copies/ μ l, 2 * 10
422.87,26.05,29.58 of copies/ μ l; The Ct value scope of sample to be tested is 21～36, is 9.74 * 10 through the typical curve quantitative scope that converts
6Copies/ μ l～9copies/ μ l.Concrete outcome such as following table:
| Sample to be tested | The Ct value | Virus quantity (copies/ μ l) |
| 1 | 32.11 | 1.72×10 3 |
| 2 | 27.38 | 8.51×10 4 |
| 3 | 29.96 | 3.51×10 4 |
| 4 | 20.52 | 9.74×10 6 |
| Sample to be tested | The Ct value | Virus quantity (copies/ μ l) |
| 5 | 37.12 | 9.18×10 0 |
| 6 | 24.98 | 3.59×10 5 |
This shows that test kit of the present invention can be 9.74 * 10
6Extremely sensitivity amplify I type parainfluenza virus in the vast scope of copies/ μ l～9copies/ μ l.
The various pathogenic agent of embodiment 4 usefulness are carried out the polymerase chain reaction
Carry out augmentation detection with embodiment 1～3 identical method, difference is that the reaction template of present embodiment chooses the DNA of other close all kinds of pathogenic agent, as II type parainfluenza virus, III type parainfluenza virus, IV type parainfluenza virus, Pestivirus suis (CSFV), human leukocyte RNA, hepatitis C virus (HCV).
Detected result such as following table:
| The template source | The Ct value |
| II type parainfluenza virus | |
| III type parainfluenza virus | - |
| IV type parainfluenza virus | - |
| Pestivirus suis (CSFV) | - |
| Human leukocyte RNA | - |
| Hepatitis C virus (HCV) | - |
As mentioned above, present embodiment can amplify I type parainfluenza virus specifically to other close pathogen gene no cross reactions.
Therefore, the present invention has disclosed the advantage of amplification I type parainfluenza virus HN gene test I type parainfluenza virus aspect susceptibility, specificity for the first time, and the fluorescence quantitative RT-PCR kit of a kind of quick on this basis special detection I type parainfluenza virus is provided simultaneously.This test kit only needs 2～3 hours to sensitivity, high specificity, the good reproducibility of I type parainfluenza virus, has shortened detection time greatly.The entire operation process only needs one, once can detect the individual sample of 32～96 (decisions of real-time fluorescence PCR instrument model), has reduced waste of manpower resource.
The various pathogenic agent of embodiment 5 usefulness are carried out the polymerase chain reaction
Repeat the same procedure of embodiment 1～4, difference only is to replace the primer shown in the SEQID NO:3 and 4 with the primer shown in the SEQ ID NO:6 and 7, replaces the probe shown in the SEQ ID NO:5 with the probe shown in the SEQ ID NO:8.
Detected result shows that this primer is to amplifying I type parainfluenza virus specifically equally with probe.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.In addition, should be understood that those skilled in the art can make various changes or modifications the present invention after pronunciation has been read above-mentioned teachings of the present invention, these equivalent form of values fall within application appended claims restricted portion equally.
The nucleotides sequence tabulation
SEQUENCE?LISTING
＜110〉Shanghai Fosun Pharmaceutical (Group) Co., Ltd.
Fuxing Medical Science-Technology Development Co., Ltd., Shanghai
＜120〉a kind of target sequence and test kit that is used to detect I type parainfluenza virus
<130>6
<160>8
<170>PatentIn?version?3.3
<210>1
<211>1894
<212>DNA
<213>Human?parainfluenza?virus?1
<220>
<221>SEQ?ID?NO：1
<222>(1)..(1894)
<400>1
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agagtgtttg?caatgatgct?cttaagataa?cttggctaaa?gaaaagacaa?gttgtcaatg????1200
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actgcaactg?gttcaacaaa?tgtccgagag?aatgcatatc?aggtgtatac?actgatgcat????1500
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gtgttaatcc?taccataatg?tactcaaata?cctcaaaaat?catcaacatg?ctaagactca????1620
aaactggaca?attagaggca?gcatacacta?ctacatcatg?tatcactcat?ttcggaaagg????1680
gctactgctt?ccacattgtt?gaaatcaacc?aagccagcct?taatacctta?caacctatgt????1740
tgttcaagac?aagtatccct?aaaatatgta?aaatcacatc?ttgagcggat?caagacctaa????1800
cactatatca?attatgtgaa?aaccagatat?aatgtataaa?aatttaaaaa?taaagcataa????1860
atagacattt?atatgacaaa?tagaataaga?aaaa????????????????????????????????1894
<210>2
<211>162
<212>DNA
<213>Human?parainfluenza?virus??1
<220>
<221>SEQID?NO：2
<222>(1)..(162)
<400>2
tggagatgtc?ccgtaggaga?acccctactg?agcaacaacc?ctaatatctc?attattacct?????60
ggaccaagtc?tactttctgg?atccaccaca?atttcaggat?gtgttagatt?accttcatta?????120
tcaattggtg?atgcaatata?tgcgtattca?tcaaacttaa?tc????????????????????????162
<210>3
<211>20
<212>DNA
<213>Human?parainfluenza?virus?1
<220>
<221>SEQ?ID?NO：3
<222>(1)..(20)
<400>3
tggagatgt?c?ccgtaggaga????20
<210>4
<211>23
<212>DNA
<213>Human?parainfluenza?virus?1
<220>
<221>SEQ?ID?NO：4
<222>(1)..(23)
<400>4
gattaagttt?gatgaatacg?cat????23
<210>5
<211>26
<212>DNA
<213>Human?parainfluenza?virus??1
<220>
<221>SEQ?ID?NO：5
<222>(1)..(26)
<223>″n＝FAM″，″y＝TAMRA″
<220>
<221>misc_feature
<222>(1)..(1)
<400>5
nacctggacc?aagtctactt?tctggy????26
<210>6
<211>23
<212>DNA
<213>Human?parainfluenza?virus?1
<220>
<221>SEQ?ID?NO：6
<222>(1)..(23)
<400>6
gatttaaacc?cggtaatttc?tca????23
<210>7
<211>20
<212>DNA
<213>Human?parainfluenza?virus??1
<220>
<221>SEQ?ID?NO：7
<222>(1)..(20)
<400>7
gtgggcaagg?agcataactg????20
<210>8
<211>26
<212>DNA
<213>Human?parainfluenza?virus?1
<220>
<221>SEQ?ID?NO：8
<222>(1)..(26)
<223>″n＝FAM″，″y＝TAMRA″
<220>
<221>misc_feature
<222>(1)..(1)
<400>8
ncctatgaca?tcaacgacaa?caggay????26
Claims (10)
1. a genetic marker that detects I type parainfluenza virus is characterized in that, it has 75～1894 Nucleotide of successive among the SEQ ID NO:1.
2. genetic marker according to claim 1 is characterized in that, it has the 1st～162 Nucleotide among the SEQ ID NO:2.
3. test kit that detects I type parainfluenza virus, it is characterized in that, it is right that it contains the primer of specific amplification I type parainfluenza virus genetic marker, described primer length is 15～30 Nucleotide, and the sequence of a primer is identical with the sequence shown in the SEQ IDNO:1, and the sequence complementation shown in the sequence of another primer and the SEQ ID NO:1, and amplified production length is 75～1894 Nucleotide.
4. test kit according to claim 3 is characterized in that, also contains probe, and the length of described probe is 18～30 Nucleotide, and the sequence of this probe is identical or complementary with the sequence shown in the SEQ ID NO:1.
5. test kit according to claim 3 is characterized in that, a primer of described primer centering is selected from down group: SEQID NO:3 or 6; Another primer is selected from down group: SEQ ID NO:4 or 7; And described probe is the TaqMan probe, and its sequence is selected from SEQ ID NO:5 or 8.
6. test kit according to claim 3 is characterized in that, this test kit also comprises a) nucleic acid extraction liquid, b) RT-PCR damping fluid, c) quantitative calibration object, d) positive control, e) negative control.
7. the method for I type parainfluenza virus in the test sample is characterized in that it comprises operation steps:
1. sample RNA extracts;
2. with the RNA that extracts as template, with the primer of specific amplification I type parainfluenza virus genetic marker to increasing, wherein said primer length is 15～30 Nucleotide, the sequence of a primer is identical with the sequence shown in the SEQ ID NO:1, and the sequence complementation shown in the sequence of another primer and the SEQ ID NO:1.
3. detect amplified production and whether exist, exist amplified production to represent that there is I type parainfluenza virus in sample.
8. method according to claim 7 is characterized in that, 3. described step is to come I type parainfluenza virus in the detection by quantitative sample by the power that detects fluorescent signal.
9. the purposes of an I type parainfluenza virus HN gene is characterized in that, it is used as the genetic marker whether vitro detection I type parainfluenza virus exists.
10. purposes according to claim 9 is characterized in that, I type parainfluenza virus HN gene has the nucleotide sequence shown in the SEQ ID NO:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810204455A CN101748218A (en) | 2008-12-12 | 2008-12-12 | Target sequence for detection of parainfluenza virus type I and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810204455A CN101748218A (en) | 2008-12-12 | 2008-12-12 | Target sequence for detection of parainfluenza virus type I and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101748218A true CN101748218A (en) | 2010-06-23 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810204455A Pending CN101748218A (en) | 2008-12-12 | 2008-12-12 | Target sequence for detection of parainfluenza virus type I and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101748218A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111926114A (en) * | 2020-07-15 | 2020-11-13 | 四川大学华西医院 | Multiplex-time PCR (polymerase chain reaction) kit for detecting parainfluenza virus, method and application |
| CN115852046A (en) * | 2022-09-28 | 2023-03-28 | 圣湘生物科技股份有限公司 | Composition, kit, method and application for detecting and parting respiratory viruses |
-
2008
- 2008-12-12 CN CN200810204455A patent/CN101748218A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111926114A (en) * | 2020-07-15 | 2020-11-13 | 四川大学华西医院 | Multiplex-time PCR (polymerase chain reaction) kit for detecting parainfluenza virus, method and application |
| CN111926114B (en) * | 2020-07-15 | 2023-10-13 | 四川大学华西医院 | Multiplex real-time PCR kit, method and application for detecting parainfluenza virus |
| CN115852046A (en) * | 2022-09-28 | 2023-03-28 | 圣湘生物科技股份有限公司 | Composition, kit, method and application for detecting and parting respiratory viruses |
| CN115852046B (en) * | 2022-09-28 | 2024-01-30 | 圣湘生物科技股份有限公司 | Composition, kit, method and application for detecting and typing respiratory viruses |
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Open date: 20100623 |