CN101538618B - Fluorescence quantitative detection kit for human metapneumovirus - Google Patents
Fluorescence quantitative detection kit for human metapneumovirus Download PDFInfo
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Abstract
The invention relates to fluorescence quantitative detection kit for human metapneumovirus which is characterized by comprising a standard positive template (a), a negative control reference sample (b), a positive control reference sample (c), fluorescence quantitative reaction liquid (d), a PCR upstream primer (e), a PCR downstream primer (f) and a fluorescence probe (g) which are respectively packaged, and also comprising RNA extraction reagent and retrovirus reagent which are respectively packaged. The fluorescence quantitative detection kit has the following advantages: the quantization is accurate: the kit can accurately quantize and detect virus nucleic acid; the sensitivity and the specificity are high; the detection speed is high, with only 1 hour, and totally only 3-4 hours plus the time for extraction and retrovirus of the nucleic acid; the steps are simple; and high-flux sample detection can be conducted simultaneously. The fluorescence quantitative detection kit is applicable to the clinical or experimental quantitative and qualitative detection of the human metapneumovirus, early diagnosis of human metapneumovirus infection, the monitoring and prediction of the epidemicity of the human metapneumovirus and the monitoring and evaluation of curative effect.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of detection kit, be specifically related to the fluorescence quantitative detection kit of human metapneumovirus.
Background technology
Quantitative fluorescent PCR (Polymerase Chain Reaction; The polymerase chain reaction) technology has not only realized the leap of PCR from qualitative to quantitative; And compare with conventional PCR; It has specificity and more by force, effectively solves characteristics such as PCR pollution problem, level of automation height, is used widely at present.In scientific research and clinical diagnosis, be widely used at present.Wherein the principle of Taqman fluorescent quantitation technology is: be the basis with the Taqman fluorescent probe; The Taqman fluorescent probe is an oligonucleotide, and two ends are fluorescent emission group of mark and a fluorescent quenching group respectively.When probe was complete, emission group fluorescent signal emitted was absorbed by quenching group; During pcr amplification; 5 '-3 ' 5 prime excision enzyme activity of Taq enzyme is cut degraded with probe enzyme, the fluorescent emission group is separated with the fluorescent quenching group, thereby the fluorescence monitoring system can receive fluorescent signal; It is DNA chain of every amplification; Just there is a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form fully synchronously, thereby realized quantitatively.
The absolute quantitation method of Taqman fluorescent quantitation technology is the amount that the establishing criteria curve is directly measured unknown sample target gene.Being made as of typical curve is decided to be CT point [Maril Martell, et al.J Clin Microbiol, 1999:37 (2): 327-332] with 10 times of dilution cycle numbers of being carried out after the hMPV F gene clone.When the PCR condition was identical, the template amount was high more, and the CT value is low more, and the logarithmic value of CT value and template amount is linear, detected according to the DNA or the making curve of CT value with concentration known.C represents Cycle, and T represents threshold, and the implication of CT value is: the cycle number that the fluorescent signal in each reaction tubes is experienced when arriving the thresholding of setting.Utilize the standard substance of known initial copy number can make typical curve, wherein X-coordinate is represented the logarithm of initial copy number, and ordinate zou is represented the CT value.Therefore, as long as obtain the CT value of unknown sample, can obtain the initial copy number of this sample from typical curve.
(Human Metapneumovirus hMPV) separates a kind of new respiratory pathogen that obtains for Dutch scholar in calendar year 2001 to human metapneumovirus first in the childrens respiratory tract infection sample.This virus can cause people's last lower respiratory infection, and its Clinical symptoms is similar to RSV (respiratory syncytial virus), and cardinal symptom comprises cough, pharyngalgia, expiratory dyspnea, anoxic, pants etc.Since human metapneumovirus was found, all there was the epidemiology report of its infection in Asia, NA, Europe, South America, Africa, Oceania, present global fashion trend.The report that also infects in Chinese Chongqing and Beijing relevant for hMPV.Serological research shows that hMPV is popular more than 50 years the crowd, and most of people had all infected hMPV in the time of 5 years old.4～10.8% the respiratory tract infection infant of being in hospital is relevant with hMPV.HMPV still causes the common disease substance of old man and the serious respiratory infection diseases of immunodeficient patient, and can be with some other pathogenic agent concurrent infection, like RSV, and SARS-CoV (severe acute respiratory syndrome coronavirus).This shows that hMPV is a kind of common respiratory pathogen, set up that detection means is significant for its early diagnosis and therapy accurately.
Summary of the invention
The fluorescence quantitative detection kit that the purpose of this invention is to provide a kind of human metapneumovirus, it can be used to infect the detection of human metapneumovirus, and is easy and simple to handle, quick and precisely, and good reproducibility, versatility are good, economical and practical.
The fluorescence quantitative detection kit of human metapneumovirus of the present invention is characterized in that: comprise respectively packing: standard positive template (a), negative ginseng control article (b), positive ginseng control article (c), fluorescent quantitation reaction solution (d), PCR upstream primer (e), PCR downstream primer (f) and fluorescent probe (g).
The fluorescence quantitative detection kit of described human metapneumovirus, it comprises that also the RNA of packing extracts reagent and rt reagent respectively, handle the early stage that is used for sample to be tested.
The fluorescence quantitative detection kit of described human metapneumovirus, its described standard positive template (a) are to contain the pMD18-T vector plasmid that inserts 1620 Nucleotide distinguished sequences of human metapneumovirus F gene, and concentration is 1 * 10
10Copy/μ l, volume are 125 μ l to 500 μ l, and preservation condition is-20 ℃;
1620 Nucleotide of human metapneumovirus F gene that insert, its sequence is:
atgtcttgga?aagtgatgat?tatcatttcg?ttactcataa?cacctcagca?tggactaaaa 60
gaaagttatt?tagaagaatc?atgtagtact?ataactgaag?gatatctcag?tgttttaaga 120
acaggttggt?acaccaatgt?ctttacatta?gaagttggtg?atgttgaaaa?ccttacatgt 180
actgatggac?ctagcttaat?caaaacagaa?cttgacctaa?ccaaaagtgc?tctaagagaa 240
ctcaaaacag?tttctgctga?tcagttagcg?ggagaagaac?aaattgaaaa?tcccagacaa 300
tcaaggtttg?tcctaggtgc?aatagctctc?ggtgttgcca?cagcagcagc?agtcacagca 360
ggcattgcaa?tagccaaaac?cataaggctt?gagagtgaag?tgaatgcaat?caaaggtgct 420
ctcaaaacaa?ccagcgaggc?agtatccaca?ctaggaaatg?gagtgcgagt?cctagccacc 480
gcagtaagag?agctgaaaga?atttgtgagc?aaaaacctga?ctagtgcaat?taacaagaac 540
aaatgtgaca?ttgctgatct?gaagatggct?gtcagcttca?gtcaattcaa?cagaaggttc 600
ctaaatgttg?tgcggcagtt?ttcagacaat?gcagggataa?caccagcaat?atcattggac 660
ctaatgactg?atgctgagct?ggccagagct?gtatcataca?tgccaacatc?agcaggacag 720
ataaaactaa?tgttagagaa?ccgtgcaatg?gtgaggagaa?aaggatttgg?aatcttgata 780
ggggtctacg?gaagctccgt?gatttacatg?gtccagctgc?cgatctttgg?tgtcatagat 840
acaccttgtt?ggataatcaa?agcagctccc?tcttgttcag?aaaaagatgg?aaattatgct 900
tgcctcctaa?gagaagatca?aggatggtat?tgtaaaaatg?caggatccac?tgtttactac 960
ccaaatgaaa?aagactgcga?aacaagaggt?gatcatgttt?tttgtgacac?agcagcaggg 1020
atcaatgttg?ctgagcaatc?aagagaatgc?aacatcaaca?tatctacaac?caactaccca 1080
tgcaaagtca?gcacaggaag?acaccctatc?agcatggttg?cactatcacc?tctcggtgct 1140
ttggtagctt?gctacaaagg?ggttagctgt?tcgattggca?gtaatcgggt?tggaataatc 1200
aaacaactac?ctaaaggctg?ctcatacata?actaaccagg?acgcagacac?tgtaacaatt 1260
gacaacactg?tgtatcaact?aagcaaagtt?gagggtgaac?agcatgtaat?aaaagggaga 1320
ccagtttcaa?gcagtttcga?tccaatcaag?tttcctgagg?atcagttcaa?tgttgcgctt 1380
gatcaagtct?ttgaaagcat?tgaaaacagt?caagcactag?tggaccagtc?aaacaaaatt 1440
ctgaacagtg?cagaaaaagg?aaacactggc?ttcattattg?taataatttt?gattgctgtt 1500
cttgggttaa?ccatgatttc?agtgagcatc?atcatcataa?tcaaaaaaac?aaggaaaccc 1560
acaggggcac?ctccagagct?gaatggtgtt?accaacggcg?gttttatacc?gcatagttag 1620。
The fluorescence quantitative detection kit of described human metapneumovirus; Its described negative ginseng control article (b) be the Vero-E6 cell strain (African green monkey kidney cell strain) that never infects the human metapneumovirus type strain cDNA that extracts viral RNA and rt and obtain [cDNA (compl ementary DNA) be meant external through the reverse transcription synthetic, and RNA (being often referred to mRNA) complementary single stranded DNA]; Volume is 100 μ l to 400 μ l, and preservation condition is-20 ℃.
The fluorescence quantitative detection kit of described human metapneumovirus; Its described positive ginseng control article (c) are after the human metapneumovirus type strain infects the Vero-E6 cell strain; Extract the cDNA that viral RNA and rt obtain, volume is 100 μ l to 400 μ l, and preservation condition is-20 ℃.
The fluorescence quantitative detection kit of described human metapneumovirus, its described fluorescent quantitation reaction solution (d) is Premix Ex Taq
TmReagent (available from Japanese TaKaRa company), volume is 625 μ l to 2500 μ l, preservation condition is-20 ℃.
The fluorescence quantitative detection kit of described human metapneumovirus, its described PCR upstream primer (e) is according to human metapneumovirus F gene design; The concentration of upstream primer is that 10 μ M, volume are 25 μ l to 100 μ l; Preservation condition is-20 ℃;
PCR upstream primer sequence is: 5 '-CGTTTCTAACATGCCGACATCTG-3 '.
The fluorescence quantitative detection kit of described human metapneumovirus, its described PCR downstream primer (f) is according to human metapneumovirus F gene design; The concentration of downstream primer is that 10 μ M, volume are 25 μ l to 100 μ l; Preservation condition is-20 ℃;
PCR downstream primer sequence is: 5 '-GCTCCCGTAGACCCCTATCAG-3 '.
The fluorescence quantitative detection kit of described human metapneumovirus, its described fluorescent probe (g) is according to human metapneumovirus F gene design;
The fluorescent probe sequence is: 5 '-CCCTTTCTTCGCACCATCGCACGG-3 '
The fluorescence report gene of 5 ' end mark of fluorescent probe is FAM, and the fluorescent receptor gene of 3 ' end mark is the Eclipse quenching group; The volume of fluorescent probe is 50 μ l to 200 μ l; Preservation condition is-20 ℃.
It is that QIAamp Viral RNA Mini KitRNA extracts reagent (available from German QIAGEN company) that the fluorescence quantitative detection kit of described human metapneumovirus, its described RNA extract reagent (h), and preservation condition is a room temperature; Described rt reagent (i) is PrimeScript
TMRT Reagent Kit rt reagent (available from Japanese TaKaRa company), preservation condition is-20 ℃.
The present invention has the following advantages: quantitatively accurately, and accurate detection by quantitative viral nucleic acid; Sensitivity and specificity are high; Detection speed is fast, only needs 1 hour, adds the extraction and the rt of nucleic acid, only needs 3-4 hour altogether; Step is simple; Can carry out high-throughout pattern detection simultaneously.The present invention is applicable to that the clinical or laboratory of human metapneumovirus quantitatively reaches qualitative detection, and the early diagnosis that human metapneumovirus infects is kept watch on and predict human metapneumovirus popularity, and curative effect is monitored and assessed.
Description of drawings
Fig. 1 is the logarithmic typical curve synoptic diagram of CT value and copy number.
Embodiment
Embodiment one: the fluorescence quantitative detection kit of human metapneumovirus of the present invention, and comprise respectively packing: standard positive template (a), negative ginseng control article (b), positive ginseng control article (c), fluorescent quantitation reaction solution (d), PCR upstream primer (e), PCR downstream primer (f) and fluorescent probe (g);
This routine standard positive template (a) is to contain the pMD18-T vector plasmid that inserts 1620 Nucleotide distinguished sequences of human metapneumovirus F gene, and concentration is 1 * 10
10Copy/μ l, volume are 125 μ l; 1620 bases of human metapneumovirus F gene are cloned in this carrier, and after the transformed into escherichia coli DH5 a propagation, (concrete experiment condition and method are seen chief editors such as J. Sa nurse Brooker with alkali row solution; Science Press, 1992, the molecular cloning experiment guide; Second edition) extracts DNA; Use DNA purification kit (available from Clonetec company) purifying again, then, use spectrophotometer A
260Quantitatively and be diluted to 1 * 10
10Copy/μ l, preservation condition is-20 ℃; The pMD18-T carrier is formed through the reconstruction of pUC18 carrier by the said firm available from Japanese TaKaRa company, is a kind of dedicated carrier of high-efficient cloning PCR product, has with the identical function of pUC18 carrier, and can improve connection, the cloning efficiency of PCR product greatly.
This routine feminine gender ginseng control article (b) be the Vero-E6 cell strain that never infects the human metapneumovirus type strain (the African green monkey kidney cell strain get with fixed attention the cDNA that viral RNA and rt obtain [cDNA (complementaryDNA) be meant external through the reverse transcription synthetic, and RNA (being often referred to mRNA) complementary single stranded DNA]; Volume is 100 μ l, and preservation condition is-20 ℃.
These routine positive ginseng control article (c) are after the human metapneumovirus type strain infects the Vero-E6 cell strain, to extract the cDNA that viral RNA and rt obtain, and volume is 100 μ l, and preservation condition is-20 ℃.
This routine fluorescent quantitation reaction solution (d) is Premix Ex Taq
TmReagent (available from Japanese TaKaRa company), volume is 625 μ l, preservation condition is-20 ℃.
This routine PCR upstream primer (e) is according to human metapneumovirus F gene design; The concentration of upstream primer is that 10 μ M, volume are 25 μ l; Preserving bar CC is-20 ℃;
PCR upstream primer sequence is: 5 '-CGTTTCTAACATGCCGACATCTG-3 '.
This routine PCR downstream primer (f) is according to human metapneumovirus F gene design; The concentration of downstream primer is that 10 μ M, volume are 25 μ l; Preservation condition is-20 ℃;
PCR downstream primer sequence is: 5 '-GCTCCCGTAGACCCCTATCAG-3 '.
This routine fluorescent probe (g) is according to human metapneumovirus F gene design;
The fluorescent probe sequence is: 5 '-CCCTTTCTTCGCACCATCGCACGG-3 '
The fluorescence report gene of 5 ' end mark of fluorescent probe is FAM, and the fluorescent receptor gene of 3 ' end mark is the Eclipse quenching group; The volume of fluorescent probe is 50 μ l; Preservation condition is-20 ℃.
Embodiment two: the fluorescence quantitative detection kit of human metapneumovirus of the present invention, and comprise respectively packing: standard positive template (a), negative ginseng control article (b), positive ginseng control article (c), fluorescent quantitation reaction solution (d), PCR upstream primer (e), PCR downstream primer (f) and fluorescent probe (g); Comprise that also the RNA that handles the early stage that is used for sample to be tested of packing respectively extracts reagent and rt reagent.
This routine standard positive template (a) is to contain the pMD18-T vector plasmid that inserts 1620 Nucleotide distinguished sequences of human metapneumovirus F gene, and concentration is 1 * 10
10Copy/μ l, volume are 500 μ l; 1620 bases of human metapneumovirus F gene are cloned in this carrier, after the transformed into escherichia coli DH5 a propagation, extract DNA, use DNA purification kit (available from Clonetec company) purifying again, then, use spectrophotometer A with alkali row solution
260Quantitatively and be diluted to 1 * 10
10Copy/μ l, preservation condition is-20 ℃.
These routine feminine gender ginseng control article (b) are that the Vero-E6 cell strain (African green monkey kidney cell strain) that never infects the human metapneumovirus type strain extracts the cDNA of viral RNA and rt acquisition, and volume is 400 μ l, and preservation condition is-20 ℃.
These routine positive ginseng control article (c) are after the human metapneumovirus type strain infects the Vero-E6 cell strain, to extract the cDNA that viral RNA and rt obtain, and volume is 400 μ l, and preservation condition is-20 ℃.
This routine fluorescent quantitation reaction solution (d) is Premix Ex Taq
TmReagent (available from Japanese TaKaRa company), volume is 2500 μ l, preservation condition is-20 ℃.
This routine PCR upstream primer (e) is according to human metapneumovirus F gene design; The concentration of upstream primer is that 10 μ M, volume are 100 μ l; Preservation condition is-20 ℃;
PCR upstream primer sequence is: 5 '-CGTTTCTAACATGCCGACATCTG-3 '.
This routine PCR downstream primer (f) is according to human metapneumovirus F gene design; The concentration of downstream primer is that 10 μ M, volume are to 100 μ l; Preservation condition is-20 ℃;
PCR downstream primer sequence is: 5 '-GCTCCCGTAGACCCCTATCAG-3 '.
This routine fluorescent probe (g) is according to human metapneumovirus F gene design;
The fluorescent probe sequence is: 5 '-CCCTTTCTTCGCACCATCGCACGG-3 '
The fluorescence report gene of 5 ' end mark of fluorescent probe is FAM, and the fluorescent receptor gene of 3 ' end mark is the Eclipse quenching group; The volume of fluorescent probe is 200 μ l; Preservation condition is-20 ℃.
This routine RNA extracts test kit: be QIAamp Viral RNA Mini Kit test kit (available from German QIAGEN company), preservation condition is a room temperature.
The rt test kit that this is routine: be PrimeScript
TMRT Reagent Kit test kit (available from Japanese TaKaRa company), preservation condition is-20 ℃.
Embodiment three: the fluorescence quantitative detection kit of human metapneumovirus of the present invention, and comprise respectively packing: standard positive template (a), negative ginseng control article (b), positive ginseng control article (c), fluorescent quantitation reaction solution (d), PCR upstream primer (e), PCR downstream primer (f) and fluorescent probe (g); Comprise that also the RNA that handles the early stage that is used for sample to be tested of packing respectively extracts reagent and rt reagent.
This routine standard positive template (a) is to contain the pMD18-T vector plasmid that inserts 1620 Nucleotide distinguished sequences of human metapneumovirus F gene, and concentration is 1 * 10
10Copy/μ l, volume are 250 μ l; 1620 bases of human metapneumovirus F gene are cloned in this carrier, after the transformed into escherichia coli DH5a propagation, extract DNA, use DNA purification kit (available from Clonetec company) purifying again, then, use spectrophotometer A with alkali row solution
260Quantitatively and be diluted to 1 * 10
10Copy/μ l, preservation condition is-20 ℃.
These routine feminine gender ginseng control article (b) are that the Vero-E6 cell strain (African green monkey kidney cell strain) that never infects the human metapneumovirus type strain extracts the cDNA of viral RNA and rt acquisition, and volume is 200 μ l, and preservation condition is-20 ℃.
These routine positive ginseng control article (c) are after the human metapneumovirus type strain infects the Vero-E6 cell strain, to extract the cDNA that viral RNA and rt obtain, and volume is 200 μ l, and preservation condition is-20 ℃.
This routine fluorescent quantitation reaction solution (d) is Premix Ex Taq
TmReagent (available from Japanese TaKaRa company), volume is 1250 μ l, preservation condition is-20 ℃.
This routine PCR upstream primer (e) is according to human metapneumovirus F gene design; The concentration of upstream primer is that 10 μ M, volume are 50 μ l; Preservation condition is-20 ℃;
PCR upstream primer sequence is: 5 '-CGTTTCTAACATGCCGACATCTG-3 '.
This routine PCR downstream primer (f) is according to human metapneumovirus F gene design; The concentration of downstream primer is that 10 μ M, volume are to 50 μ l; Preservation condition is-20 ℃;
PCR downstream primer sequence is: 5 '-GCTCCCGTAGACCCCTATCAG-3 '.
This routine fluorescent probe (g) is according to human metapneumovirus F gene design;
The fluorescent probe sequence is: 5 '-CCCTTTCTTCGCACCATCGCACGG-3 '
The fluorescence report gene of 5 ' end mark of fluorescent probe is FAM, and the fluorescent receptor gene of 3 ' end mark is the Eclipse quenching group; The volume of fluorescent probe is 100 μ l; Preservation condition is-20 ℃.
This routine RNA extracts test kit: be QIAamp Viral RNA Mini Kit test kit (available from German QIAGEN company), preservation condition is a room temperature.
The rt test kit that this is routine: be PrimeScript
TMRT Reagent Kit test kit (available from Japanese TaKaRa company), preservation condition is-20 ℃.
Embodiment four: the fluorescence quantitative detection kit of using human metapneumovirus detects clinical respiratory tract sample (also available nasopharyngeal secretions sample or sputum specimen), uses this test kit to detect and may further comprise the steps:
The first step uses RNA to extract reagent (h), extracts the RNA of sample to be tested, be stored in-80 ℃ subsequent use; Sample to be tested is the human respiratory sample;
In second step, the RNA of the sample to be tested that the first step is obtained uses rt reagent (i) rt to be cDNA, and is subsequent use in-20 ℃ of preservations;
The 3rd step with dissolved in distilled water standard positive masterplate (a), and was 1 * 10 with its serial dilution
9Copy/μ l, 1 * 10
8Copy/μ l, 1 * 10
7Copy/μ l, 1 * 10
6Copy/μ l, 1 * 10
5Copy/μ l, 1 * 10
4Copy/μ l, 1 * 10
3Copy/μ l, 1 * 10
2Copy/μ l, 1 * 10
1Copy/μ l, totally 9 pipings row dilution article are subsequent use in-20 ℃ of preservations;
The 4th step; Pcr amplification; Get fluorescent quantitation reaction solution (d) 12.5 μ l, get PCR upstream primer (e) 0.5 μ l, get PCR downstream primer (f) 0.5 μ l, get fluorescent probe (g) 1 μ l, get zero(ppm) water 8.5 μ l and template 2 μ l, totally 25 μ l form a plurality of PCR reaction systems;
Described template is the cDNA that obtained in second step, the serial dilution article that the 3rd step obtained, negative ginseng control article (b), perhaps positive ginseng control article (c);
During pcr amplification, in each reaction system, need comprise serial dilution article, negative control article (b), positive ginseng control article (c) and at least 1 second cDNA that goes on foot the sample to be tested that obtains of joining that 5 the 3rd steps obtain at least; Wherein the serial dilution article are used for the production standard curve, and negative ginseng control article are as the negative control of PCR reaction, and whether positive ginseng control article are as the positive control of PCR reaction, normal to detect the PCR reactive system;
In the 5th step, the reaction system of using the fluorescent quantitation detector that the 4th step was obtained is carried out the PCR cycle detection, and the reaction conditions that PCR is set is: 95 ℃ of sex change of elder generation 10 seconds; 95 ℃ again, 10 seconds → 60 ℃, 30 seconds → 95 ℃, 10 seconds → 60 ℃, 30 seconds ... 40 circulations are set; Purpose is to detect round-robin CT value (that is: the cycle number that is experienced when the fluorescent signal in each reaction tubes arrives the thresholding of setting);
The 6th step; Through comparing the typical curve of sample to be tested and serial dilution standard positive template; Initial copy number to sample to be tested carries out quantitatively (seeing accompanying drawing 1); This step is accomplished by the CFX-96 fluorescent quantitative PCR detector of U.S. BIO-RAD company automatically, and quantitative result is calculated by this detector automatically;
The 7th step; Under the normal situation of PCR reactive system; Be that PCR reaction finishes back fluorescent quantitation detector and in feminine gender ginseng control QC, do not detect fluorescent signal, and in positive ginseng control QC, detect >=human metapneumovirus of 500 copies, obtain the human metapneumovirus copy number of sample to be tested through the reference standard curve; The human metapneumovirus that is judged to be of copy number >=500 infects, and all the other are negative.
Each respiratory tract sample is provided with 3 repetitions, to guarantee the particularity and the stability of experiment;
Embodiment five: the fluorescence quantitative detection kit of using human metapneumovirus detects the cell (also available supernatant sample) of Infection in Vitro experiment, uses this test kit to detect and may further comprise the steps:
The first step uses RNA to extract reagent (h), extracts the RNA of sample to be tested, be stored in-80 ℃ subsequent use; Sample to be tested is the cell of Infection in Vitro experiment;
In second step, the RNA of the sample to be tested that the first step is obtained uses rt reagent (i) rt to be cDNA, and is subsequent use in-20 ℃ of preservations;
The 3rd step with dissolved in distilled water standard positive masterplate (a), and was 1 * 10 with its serial dilution
9Copy/μ l, 1 * 10
8Copy/μ l, 1 * 10
7Copy/μ l, 1 * 10
6Copy/μ l, 1 * 10
5Copy/μ l, 1 * 10
4Copy/μ l, 1 * 10
3Copy/μ l, 1 * 10
2Copy/μ l, 1 * 10
1Copy/μ l, totally 9 pipings row dilution article are subsequent use in-20 ℃ of preservations;
The 4th step; Pcr amplification; Get fluorescent quantitation reaction solution (d) 12.5 μ l, get PCR upstream primer (e) 0.5 μ l, get PCR downstream primer (f) 0.5 μ l, get fluorescent probe (g) 1 μ l, get zero(ppm) water 8.5 μ l and template 2 μ l, totally 25 μ l form a plurality of PCR reaction systems;
Described template is the cDNA that obtained in second step, the serial dilution article that the 3rd step obtained, negative ginseng control article (b), perhaps positive ginseng control article (c);
During pcr amplification, in each reaction system, need comprise serial dilution article, negative control article (b), positive ginseng control article (c) and at least 1 second cDNA that goes on foot the sample to be tested that obtains of joining that 5 the 3rd steps obtain at least; Wherein the serial dilution article are used for the production standard curve, and negative ginseng control article are as the negative control of PCR reaction, and whether positive ginseng control article are as the positive control of PCR reaction, normal to detect the PCR reactive system;
In the 5th step, the reaction system of using the fluorescent quantitation detector that the 4th step was obtained is carried out the PCR cycle detection, and the reaction conditions that PCR is set is: 95 ℃ of sex change of elder generation 10 seconds; 95 ℃ again, 10 seconds → 60 ℃, 30 seconds → 95 ℃, 10 seconds → 60 ℃, 30 seconds ... 40 circulations are set; Purpose is to detect round-robin CT value (that is: the cycle number that is experienced when the fluorescent signal in each reaction tubes arrives the thresholding of setting);
The 6th step; Through comparing the typical curve of sample to be tested and serial dilution standard positive template; Initial copy number to sample to be tested carries out quantitatively (seeing accompanying drawing 1); This step is accomplished by the CFX-96 fluorescent quantitative PCR detector of U.S. BIO-RAD company automatically, and quantitative result is calculated by this detector automatically;
The 7th step; Under the normal situation of PCR reactive system; Be that PCR reaction finishes back fluorescent quantitation detector and in feminine gender ginseng control QC, do not detect fluorescent signal, and in positive ginseng control QC, detect >=human metapneumovirus of 500 copies, obtain the human metapneumovirus copy number of sample to be tested through the reference standard curve; The human metapneumovirus that is judged to be of copy number >=500 infects, and all the other are negative.
Sequence table
SEQUENCE?LISTING
< 110>Children's Hospital Attached to Chongqing Medical Univ.
< 120>fluorescence quantitative detection kit of human metapneumovirus
<160>4
<210>1
<211>1620
<212>DNA
< 213>metapneumovirus belongs to (Metapneumovirus genus.)
<220>
<221>Human?Metapneumovirus
<400>1
atgtcttgga?aagtgatgat?tatcatttcg?ttactcataa?cacctcagca?tggactaaaa 60
gaaagttatt?tagaagaatc?atgtagtact?ataactgaag?gatatctcag?tgttttaaga 120
acaggttggt?acaccaatgt?ctttacatta?gaagttggtg?atgttgaaaa?ccttacatgt 180
actgatggac?ctagcttaat?caaaacagaa?cttgacctaa?ccaaaagtgc?tctaagagaa 240
ctcaaaacag?tttctgctga?tcagttagcg?ggagaagaac?aaattgaaaa?tcccagacaa 300
tcaaggtttg?tcctaggtgc?aatagctctc?ggtgttgcca?cagcagcagc?agtcacagca 360
ggcattgcaa?tagccaaaac?cataaggctt?gagagtgaag?tgaatgcaat?caaaggtgct 420
ctcaaaacaa?ccagcgaggc?agtatccaca?ctaggaaatg?gagtgcgagt?cctagccacc 480
gcagtaagag?agctgaaaga?atttgtgagc?aaaaacctga?ctagtgcaat?taacaagaac 540
aaatgtgaca?ttgctgatct?gaagatggct?gtcagcttca?gtcaattcaa?cagaaggttc 600
ctaaatgttg?tgcggcagtt?ttcagacaat?gcagggataa?caccagcaat?atcattggac 660
ctaatgactg?atgctgagct?ggccagagct?gtatcataca?tgccaacatc?agcaggacag 720
ataaaactaa?tgttagagaa?ccgtgcaatg?gtgaggagaa?aaggatttgg?aatcttgata 780
ggggtctacg?gaagctccgt?gatttacatg?gtccagctgc?cgatctttgg?tgtcatagat 840
acaccttgtt?ggataatcaa?agcagctccc?tcttgttcag?aaaaagatgg?aaattatgct 900
tgcctcctaa?gagaagatca?aggatggtat?tgtaaaaatg?caggatccac?tgtttactac 960
ccaaatgaaa?aagactgcga?aacaagaggt?gatcatgttt?tttgtgacac?agcagcaggg 1020
atcaatgttg?ctgagcaatc?aagagaatgc?aacatcaaca?tatctacaac?caactaccca 1080
tgcaaagtca?gcacaggaag?acaccctatc?agcatggttg?cactatcacc?tctcggtgct 1140
ttggtagctt?gctacaaagg?ggttagctgt?tcgattggca?gtaatcgggt?tggaataatc 1200
aaacaactac?ctaaaggctg?ctcatacata?actaaccagg?acgcagacac?tgtaacaatt 1260
gacaacactg?tgtatcaact?aagcaaagtt?gagggtgaac?agcatgtaat?aaaagggaga 1320
ccagtttcaa?gcagtttcga?tccaatcaag?tttcctgagg?atcagttcaa?tgttgcgctt 1380
gatcaagtct?ttgaaagcat?tgaaaacagt?caagcactag?tggaccagtc?aaacaaaatt 1440
ctgaacagtg?cagaaaaagg?aaacactggc?ttcattattg?taataatttt?gattgctgtt 1500
cttgggttaa?ccatgatttc?agtgagcatc?atcatcataa?tcaaaaaaac?aaggaaaccc 1560
acaggggcac?ctccagagct?gaatggtgtt?accaacggcg?gttttatacc?gcatagttag 1620
<210>2
<211>23
<212>DNA
< 213>artificial sequence
<220>
< 221>PCR upstream primer
<400>2
cctttctaac?atgcccacat?ctc 23
<210>3
<211>21
<212>DNA
< 213>artificial sequence
<220>
< 221>PCR downstream primer
<400>3
gctcccgtag?acccctatca?g 21
<210>4
<211>24
<212>DNA
< 213>artificial sequence
<220>
< 221>fluorescent probe
<400>4
ccctttcttc?gcaccatccc?acgg 24
Claims (1)
1. the fluorescence quantitative detection kit of human metapneumovirus; It is characterized in that: comprise standard positive template (a), negative ginseng control article (b), positive ginseng control article (c), fluorescent quantitation reaction solution (d), PCR upstream primer (e), PCR downstream primer (f) and the fluorescent probe (g) of packing respectively, comprise that also the RNA of packing extracts reagent (h) and rt reagent (i) respectively;
Described standard positive template (a) is to contain the pMD18-T vector plasmid that inserts 1620 Nucleotide distinguished sequences of human metapneumovirus F gene, and concentration is 1 * 10
10Copy/μ l, volume are 125 μ l to 500 μ l, and preservation condition is-20 ℃;
1620 nucleotides sequences of human metapneumovirus F gene that insert are classified as:
atgtcttgga?aagtgatgat?tatcatttcg?ttactcataa?cacctcagca?tggactaaaa 60
gaaagttatt?tagaagaatc?atgtagtact?ataactgaag?gatatctcag?tgttttaaga 120
acaggttggt?acaccaatgt?ctttacatta?gaagttggtg?atgttgaaaa?ccttacatgt 180
actgatggac?ctagcttaat?caaaacagaa?cttgacctaa?ccaaaagtgc?tctaagagaa 240
ctcaaaacag?tttctgctga?tcagttagcg?ggagaagaac?aaattgaaaa?tcccagacaa 300
tcaaggtttg?tcctaggtgc?aatagctctc?ggtgttgcca?cagcagcagc?agtcacagca 360
ggcattgcaa?tagccaaaac?cataaggctt?gagagtgaag?tgaatgcaat?caaaggtgct 420
ctcaaaacaa?ccagcgaggc?agtatccaca?ctaggaaatg?gagtgcgagt?cctagccacc 480
gcagtaagag?agctgaaaga?atttgtgagc?aaaaacctga?ctagtgcaat?taacaagaac 540
aaatgtgaca?ttgctgatct?gaagatggct?gtcagcttca?gtcaattcaa?cagaaggttc 600
ctaaatgttg?tgcggcagtt?ttcagacaat?gcagggataa?caccagcaat?atcattggac 660
ctaatgactg?atgctgagct?ggccagagct?gtatcataca?tgccaacatc?agcaggacag 720
ataaaactaa?tgttagagaa?ccgtgcaatg?gtgaggagaa?aaggatttgg?aatcttgata 780
ggggtctacg?gaagctccgt?gatttacatg?gtccagctgc?cgatctttgg?tgtcatagat 840
acaccttgtt?ggataatcaa?agcagctccc?tcttgttcag?aaaaagatgg?aaattatgct 900
tgcctcctaa?gagaagatca?aggatggtat?tgtaaaaatg?caggatccac?tgtttactac 960
ccaaatgaaa?aagactgcga?aacaagaggt?gatcatgttt?tttgtgacac?agcagcaggg 1020
atcaatgttg?ctgagcaatc?aagagaatgc?aacatcaaca?tatctacaac?caactaccca 1080
tgcaaagtca?gcacaggaag?acaccctatc?agcatggttg?cactatcacc?tctcggtgct 1140
ttggtagctt?gctacaaagg?ggttagctgt?tcgattggca?gtaatcgggt?tggaataatc 1200
aaacaactac?ctaaaggctg?ctcatacata?actaaccagg?acgcagacac?tgtaacaatt 1260
gacaacactg?tgtatcaact?aagcaaagtt?gagggtgaac?agcatgtaat?aaaagggaga 1320
ccagtttcaa?gcagtttcga?tccaatcaag?tttcctgagg?atcagttcaa?tgttgcgctt 1380
gatcaagtct?ttgaaagcat?tgaaaacagt?caagcactag?tggaccagtc?aaacaaaatt 1440
ctgaacagtg?cagaaaaagg?aaacactggc?ttcattattg?taataatttt?gattgctgtt 1500
cttgggttaa?ccatgatttc?agtgagcatc?atcatcataa?tcaaaaaaac?aaggaaaccc 1560
acaggggcac?ctccagagct?gaatggtgtt?accaacggcg?gttttatacc?gcatagttag 1620
Described negative ginseng control article (b) are that the cDNA volume that the Vero-E6 cell strain that never infects the human metapneumovirus type strain extracts viral RNA and rt acquisition is 100 μ l to 400 μ l, and preservation condition is-20 ℃;
Described positive ginseng control article (c) are after the human metapneumovirus type strain infects the Vero-E6 cell strain, to extract the cDNA that viral RNA and rt obtain, and volume is 100 μ l to 400 μ l, and preservation condition is-20 ℃;
Described fluorescent quantitation reaction solution (d) is Premix Ex Taq
TmReagent, volume are 625 μ l to 2500 μ l, and preservation condition is-20 ℃;
Described PCR upstream primer (e) is according to human metapneumovirus F gene design; The concentration of PCR upstream primer (e) is that 10 μ M, volume are 25 μ l to 100 μ l; Preservation condition is-20 ℃;
PCR upstream primer (e) sequence is: 5 '-CGTTTCTAACATGCCGACATCTG-3 ';
Described PCR downstream primer (f) is according to human metapneumovirus F gene design; The concentration of PCR downstream primer (f) is that 10 μ M, volume are 25 μ l to 100 μ l; Preservation condition is-20 ℃;
PCR downstream primer (f) sequence is: 5 '-GCTCCCGTAGACCCCTATCAG-3 ';
Described fluorescent probe (g) is according to human metapneumovirus F gene design;
Fluorescent probe (g) sequence is: 5 '-CCCTTTCTTCGCACCATCGCACGG-3 '
The fluorescence report gene of 5 ' end mark of fluorescent probe (g) is FAM, and the fluorescent receptor gene of 3 ' end mark is the Eclipse quenching group; The volume of fluorescent probe is 50 μ l to 200 μ l; Preservation condition is-20 ℃;
It is that QIAamp Viral RNA Mini KitRNA extracts reagent that described RNA extracts reagent (h), and preservation condition is a room temperature;
Described rt reagent (i) is PrimeScript
TMRT Reagent Kit rt reagent, preservation condition is-20 ℃.
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| CN104450974B (en) * | 2014-12-30 | 2017-09-22 | 重庆医科大学附属儿童医院 | A kind of hMPV quantitative typings detection kit and its detection method |
| CN104878122A (en) * | 2015-03-31 | 2015-09-02 | 北京大学 | Method and composition for detecting human metapneumovirus based on real-time PCR |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544655A (en) * | 2003-11-14 | 2004-11-10 | 武汉大学 | Fluorescence quantitative reagent kit and detection method for human cytomegalovirus |
| CN101107366A (en) * | 2004-12-24 | 2008-01-16 | 卡洛斯Ⅲ世健康研究所 | Probes and methods for the simultaneous detection and identification of multiple viruses that cause respiratory infections in humans |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544655A (en) * | 2003-11-14 | 2004-11-10 | 武汉大学 | Fluorescence quantitative reagent kit and detection method for human cytomegalovirus |
| CN101107366A (en) * | 2004-12-24 | 2008-01-16 | 卡洛斯Ⅲ世健康研究所 | Probes and methods for the simultaneous detection and identification of multiple viruses that cause respiratory infections in humans |
Non-Patent Citations (5)
| Title |
|---|
| Liu WP et al.Human metapneumovirus isolate CS113 fusion protein (F) gene，complete cds，GenBank: EF081369，1620 bp cRNA linear.《NCBI》.2008,1. * |
| 朱汝南等.北京地区人偏肺病毒融合蛋白F编码基因的序列分析.《中华微生物学和免疫学杂志》.2005,第25卷(第10期),837-841. * |
| 毛华伟.人偏肺病毒分离、融合蛋白表达及主要蛋白的免疫信息学分析.《中国优秀硕士学位论文全文数据库，医药卫生科技辑，重庆医科大学硕士学位论文》.2007,53-55. * |
| 毛华伟等.人偏肺病毒在中国大陆的首次分离及其特征.《中华医学会第十四次全国儿科学术会议论文集》.2006,604. * |
| 陆柔剑等.偏肺病毒TaqMan-MGB探针实时定量RT-PCR检测方法的建立及初步应用.《生物技术通讯》.2008,第19卷(第2期),207-209. * |
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