CN104450974B - A kind of hMPV quantitative typings detection kit and its detection method - Google Patents
A kind of hMPV quantitative typings detection kit and its detection method Download PDFInfo
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Abstract
The present invention relates to biological technical field, more particularly to hMPV quantitative typings detection kit and its detection method.Primer sets include A hypotype hMPV primer sets and subtype B hMPV primer sets in the kit；A hypotype hMPV primer sets are by SEQ ID NO：Sense primer, SEQ ID NO shown in 1：Anti-sense primer and SEQ ID NO shown in 2：Probe composition shown in 3；Subtype B hMPV primer sets are by SEQ ID NO：Sense primer, SEQ ID NO shown in 4：Anti-sense primer and SEQ ID NO shown in 5：Probe composition shown in 6.Had shown that by research, the high specificity of primer sets of the invention, sensitivity are high, available for A hypotypes hMPV and subtype B hMPV effective amplification, so that hMPV accurate quantitative analysis and parting can be realized；And quantified simultaneously and genotyping result.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of hMPV quantitative typings detection kit and its detection side
Method.
Background technology
Human metapneumovirus (Human Metapneumovirus, hMPV) is for Dutch scholar in 2001 first in children
Isolated a kind of new respiratory pathogen in respiratory tract infection sample.The full length viral genome about 13550nt, according to
The difference of its sequence is segmented into two genotype of A, B.The virus can cause the upper ALRI of people, its Clinical symptoms class
It is similar to RSV (Respiratory Syncytial Virus(RSV)), cardinal symptom includes cough, pharyngalgia, expiratory dyspnea, anoxic, panted.From the inclined lung of the mankind
Since virus is found, there is the epidemiology report that it infects in Asia, North America, Europe, South America, Africa, Oceania, and present
Global prevalence trend.The report of hMPV infection is also related in Chongqing in China and Beijing.Serological research shows that hMPV exists
Crowd is popular more than 50 years, and most people infected hMPV at 5 years old.4~10.8% respiratory tract infection Hospitalized Children
It is relevant with hMPV.HMPV still causes the common causative of old man and immune deficient patients' serious respiratory tract infection disease, and can
With some other pathogen concurrent infection, such as RSV, SARS-CoV (SARS-CoV).As can be seen here,
HMPV is a kind of common respiratory pathogen, and set up accurate detection means has important meaning for its early diagnosis and therapy
Justice.
At present, detection hMPV method mainly includes Sanger methods, common fluorescent quantitative PCR method and Luminex.Its
In, Sanger methods refer to be directed to same sample, need to be by viral A, the specific primer of subtype B, and performing PCR is entered respectively can just enter
Row gene typing is identified, not only causes the waste of precious clinical resources, and take time and effort increase testing cost；It is common glimmering
Though Fluorescent Quantitative PCR method can carry out Viral Quantification detection, Viral typing identification can not be carried out simultaneously；Luminex detection is fast
It is fast, sensitive, but must could be detected after accumulation sample to sufficient amount, it is impossible to meet the need for clinical samples detect at any time,
And special instrument software etc. need to be configured, is detected expensive.
The order of severity of disease is different after being infected due to different genotype, and prognosis is also different, thus understand genotype for
Treatment has great importance.The deficiency existed for the above method, Viral typing mirror can be carried out simultaneously by being badly in need of exploitation one kind
Method that is fixed and quantitatively detecting, is that adjustment and prognosis evaluation of clinical treatment etc. provide foundation.
The content of the invention
In view of this, the invention provides a kind of hMPV quantitative typings detection kit and its detection method.The present invention is carried
The high specificity of the primer sets of confession, sensitivity are high, available for A hypotypes hMPV and subtype B hMPV effective amplification, so as to can realize
HMPV accurate quantitative analysis and parting；The hMPV quantitative typing detection kits that the present invention is provided are quantitatively accurate, can be with accurate quantitative analysis
Detect viral nucleic acid；Sensitivity and specificity are high；Detection speed is fast, only 1 hour, adds extraction and the reverse transcription of nucleic acid, altogether only
Need 3~4 hours；Step is simple；High-throughout pattern detection can be carried out simultaneously.The present invention is applied to the clinic of human metapneumovirus
Or laboratory is quantified and qualitative detection, the early diagnosis of human metapneumovirus infection, monitoring and prediction human metapneumovirus are popular
Property, and curative effect is monitored and assessed.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides the primer sets for expanding hMPV genetic fragments, including A hypotype hMPV primer sets and subtype B
HMPV primer sets；
A hypotype hMPV primer sets are by SEQ ID NO：Sense primer, SEQ ID NO shown in 1：Anti-sense primer shown in 2
With SEQ ID NO：Probe composition shown in 3；
Subtype B hMPV primer sets are by SEQ ID NO：Sense primer, SEQ ID NO shown in 4：Anti-sense primer shown in 5
With SEQ ID NO：Probe composition shown in 6.
In the present invention, A hypotypes hMPV primer sets are according to A hypotype type strain NL/1/00 (accession number:
AF371337) it is designed to come, subtype B hMPV primer sets are according to subtype B type strain NL/1/99 (accession number:
AF525843) it is designed to come.
The high specificity of the primer sets provided by research, the present invention, sensitivity are high, available for A hypotypes hMPV and subtype B
HMPV effective amplification, so as to realize hMPV accurate quantitative analysis and parting.
Application of the primer sets that the present invention is provided in hMPV quantitative typing detection kits are prepared；The primer sets include A
Hypotype hMPV primer sets and subtype B hMPV primer sets；A hypotype hMPV primer sets are by SEQ ID NO：Sense primer shown in 1,
SEQ ID NO：Anti-sense primer and SEQ ID NO shown in 2：Probe composition shown in 3；Subtype B hMPV primer sets are by SEQ ID
NO：Sense primer, SEQ ID NO shown in 4：Anti-sense primer and SEQ ID NO shown in 5：Probe composition shown in 6.
In some embodiments that the present invention is provided, hMPV is A hypotypes hMPV and/or subtype B hMPV.
Present invention also offers a kind of hMPV quantitative typings detection kit, including standard positive template, fluorescent quantitation are anti-
The primer sets for answering liquid and the present invention to provide；The primer sets include A hypotype hMPV primer sets and subtype B hMPV primer sets；A hypotypes
HMPV primer sets are by SEQ ID NO：Sense primer, SEQ ID NO shown in 1：Anti-sense primer and SEQ ID NO shown in 2：3
Shown probe composition；Subtype B hMPV primer sets are by SEQ ID NO：Sense primer, SEQ ID NO shown in 4：Under shown in 5
Swim primer and SEQ ID NO：Probe composition shown in 6.
In some embodiments that the present invention is provided, standard positive template is：Insert human metapneumovirus A hypotype F genes
The pMD18-T vector plasmids of 1620 nucleotide sequences, and insertion human metapneumovirus subtype B F 1620 nucleosides of gene
The pMD18-T vector plasmids of sour distinguished sequence.
In some embodiments that the present invention is provided, the concentration of standard positive template is 1 × 1010Copy/μ L, volume is
125 μ L to 500 μ L, preservation condition is -20 DEG C.
Human metapneumovirus 1620 nucleotide sequences of A hypotype F genes such as SEQ ID NO：Shown in 7.
1620 nucleotide sequences of human metapneumovirus subtype B F genes such as SEQ ID NO：Shown in 8.
Preferably, the kit that provides of the present invention also include RNA extracts reagents, reverse transcription reagents, positive ginseng control product or
One or more in feminine gender ginseng control product.
In some embodiments that the present invention is provided, positive ginseng control product are：Infect the Vero- of human metapneumovirus type strain
E6 cell lines extract the cDNA that viral RNA and reverse transcription are obtained.It includes：Human metapneumovirus A hypotypes type strain infects Vero-
After E6 cell lines, the cDNA that viral RNA and reverse transcription are obtained is extracted；Vero-E6 is infected with human metapneumovirus subtype B type strain
After cell line, the cDNA that viral RNA and reverse transcription are obtained is extracted.
The volume of positive ginseng control product is 100 μ L to 400 μ L, and preservation condition is -20 DEG C.
In some embodiments that the present invention is provided, feminine gender ginseng control product are water.
In some embodiments that the present invention is provided, it is poly- that fluorescent quantitation reaction solution includes buffer solution, dNTPs, dyestuff and DNA
Synthase.
In some embodiments that the present invention is provided, the volume of fluorescent quantitation reaction solution is 625 μ L to 2500 μ L, preserves bar
Part is -20 DEG C.
The concentration of primer is 10 μM in the present invention, and volume is 25 μ L to 100 μ L, and preservation condition is -20 DEG C.
5 ' end marks of fluorescence probe are respectively in the present invention：Genotype A (A hypotypes) fluorescent reporter group is FAM (carboxylics
Base fluorescein)-BHQ-1, genotype B (subtype B) fluorescent reporter group is HEX (chlorine fluorescein phosphoramidate)-BHQ-1.
The fluorescent receptor gene of 3 ' end marks of fluorescence probe is Eclipse quenching groups in the present invention.
The volume of fluorescence probe is 50 μ L to 200 μ L in the present invention, and preservation condition is -20 DEG C.
Present invention also offers a kind of method of non-diagnostic purpose hMPV quantitative typings detection, comprise the following steps：
Standard positive template is subjected to doubling dilution, standard dilution is obtained；Take fluorescent quantitation reaction solution, standard dilution
Quantitative fluorescent PCR is carried out with the primer sets that the present invention is provided, standard curve is drawn；
The RNA of testing sample is extracted, reverse transcription is cDNA, obtain testing sample cDNA；Take fluorescent quantitation reaction solution, it is to be measured
The primer sets that sample cDNA and the present invention are provided carry out quantitative fluorescent PCR, and according to standard curve, the hMPV for obtaining testing sample determines
Measure parting testing result.
Preferably, the response procedures of quantitative fluorescent PCR are：
(1) pre-degeneration：95 DEG C, 3min；
(2) expand：95 DEG C, 15s；62 DEG C, 10s；68 DEG C, 20s is a circulation, sets 40 circulations.
The invention provides a kind of hMPV quantitative typings detection kit and its detection method.The hMPV quantitative typings are detected
Primer sets in kit include A hypotype hMPV primer sets and subtype B hMPV primer sets；A hypotype hMPV primer sets are by SEQ ID
NO：Sense primer, SEQ ID NO shown in 1：Anti-sense primer and SEQ ID NO shown in 2：Probe composition shown in 3；Subtype B
HMPV primer sets are by SEQ ID NO：Sense primer, SEQ ID NO shown in 4：Anti-sense primer and SEQ ID NO shown in 5：6
Shown probe composition.The present invention at least has one of following advantage：
Had shown that by research, the high specificity for the primer sets that the present invention is provided, sensitivity are high, available for A hypotypes hMPV and B
Hypotype hMPV effective amplification, so that hMPV accurate quantitative analysis and parting can be realized；
A hypotypes hMPV is different with the fluorescence labeling of subtype B hMPV primed probe, can be detected in same test tube,
It is convenient rapid, reduce reagent and human cost；
The hMPV quantitative typing detection kits that the present invention is provided are quantitatively accurate, can detect viral nucleic acid with accurate quantitative analysis；
Sensitivity and specificity are high；Detection speed is fast, only 1 hour, adds extraction and the reverse transcription of nucleic acid, only needs 3~4 hours altogether；Step
It is rapid simple；High-throughout pattern detection can be carried out simultaneously；
The present invention can not only be quantified and parting to hMPV respectively, can also complete quantitative and parting with pipe simultaneously；
The present invention is quantified and qualitative detection suitable for the clinic of human metapneumovirus or laboratory, human metapneumovirus infection
Early diagnosis, monitoring and prediction human metapneumovirus are popular, and curative effect is monitored and assessed.
Brief description of the drawings
Fig. 1 shows the standard curve schematic diagram that A hypotypes hMPV is detected in embodiment 4；
Fig. 2 shows the standard curve schematic diagram that subtype B hMPV is detected in embodiment 4.
Embodiment
The invention discloses a kind of hMPV quantitative typings detection kit and its detection method, those skilled in the art can be with
Present disclosure is used for reference, technological parameter realization is suitably modified.In particular, all similar replacements and change are to ability
It is it will be apparent that they are considered as being included in the present invention for field technique personnel.The method of the present invention and application have been led to
Cross preferred embodiment to be described, related personnel substantially can be not departing from present invention, in spirit and scope to this paper institutes
The methods and applications stated are modified or suitably change is with combining, to realize and apply the technology of the present invention.
Agents useful for same or instrument can be by hMPV quantitative typings detection kit and its detection method that the present invention is provided
Market is bought.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1hMPV quantitative typing detection kits
The hMPV quantitative typings detection kit that the present invention is provided includes what is packed respectively：Standard positive template (a), feminine gender
Join control product (b), positive ginseng control product (c), fluorescent quantitation reaction solution (d), PCR sense primers (e), PCR anti-sense primers (f), fluorescence
Probe (g), PCR sense primers (h), PCR anti-sense primers (i) and fluorescence probe (j)；Also including packing respectively for treating test sample
The RNA extracts reagents of this early stage processing, reverse transcription reagents.
Standard positive template (a) is：Insert human metapneumovirus A hypotype F 1620 nucleotide sequences of gene
PMD18-T vector plasmids, and insert the pMD18-T carriers of human metapneumovirus subtype B F 1620 nucleotide sequences of gene
Plasmid.Concentration is 1 × 1010Copy/μ L, volume is 500 μ L；Respectively will insertion A hypotypes and subtype B human metapneumovirus F genes
1620 bases are cloned into the carrier, after conversion bacillus coli DH 5 а propagation, extract DNA with alkali row solution, then use DNA
Purification kit (being purchased from Clonetec companies) purifying, then, uses spectrophotometer A260Quantify and be diluted to 1 × 1010Copy/μ
L, preservation condition is -20 DEG C.
Feminine gender ginseng control product (b) are water.
The positive joins control product (c)：After human metapneumovirus A hypotypes type strain infection Vero-E6 cell lines, disease is extracted
The cDNA that malicious RNA and reverse transcription are obtained；After human metapneumovirus subtype B type strain infection Vero-E6 cell lines, virus is extracted
The cDNA that RNA and reverse transcription are obtained.Concentration is 1 × 1010Copy/μ L, volume is 400 μ L, and preservation condition is -20 DEG C.
Fluorescent quantitation reaction solution (d) is Premix Ex TaqTmReagent (is purchased from TaKaRa companies of Japan), and volume is 2500 μ
L, preservation condition is -20 DEG C.
For detecting A hypotypes hMPV primer sets by SEQ ID NO：Sense primer (e), SEQ ID NO shown in 1：2 institutes
The anti-sense primer (f) and SEQ ID NO shown：Probe (g) composition shown in 3：
PCR sense primers (e) are designed according to human metapneumovirus A hypotype F genes；The concentration of sense primer is 10 μ
M, volume are 100 μ L；Preservation condition is -20 DEG C；PCR upstream primer sequences such as SEQ ID NO：Shown in 1：5’-
GAGCAATAGCACTCGGTGTTG-3’；
PCR anti-sense primers (f) are designed according to human metapneumovirus A hypotype F genes；The concentration of anti-sense primer is 10 μ
M, volume are 100 μ L；Preservation condition is -20 DEG C；PCR downstream primer sequences such as SEQ ID NO：Shown in 2：5’-
TCACAAAATCTTTCAGCTCTCTCAC-3’；
Fluorescence probe (g) is designed according to human metapneumovirus A hypotype F genes；Fluorescence probe sequence such as SEQ ID
NO：Shown in 3：5’(FAM)-TTGCCAACACACGAACTCCATTCCC(Eclipse)-3’；The volume of fluorescence probe is 200 μ L；
Preservation condition is -20 DEG C.
For detecting subtype B hMPV primer sets by SEQ ID NO：Sense primer, SEQ ID NO shown in 4：Shown in 5
Anti-sense primer and SEQ ID NO：Probe composition shown in 6：
PCR sense primers (h) are designed according to human metapneumovirus subtype B F genes；The concentration of sense primer is 10 μ
M, volume are 100 μ L；Preservation condition is -20 DEG C；PCR upstream primer sequences such as SEQ ID NO：Shown in 4：5’-
GTGCGATAGCTCTCGGAGTTG-3’；
PCR anti-sense primers (i) are designed according to human metapneumovirus subtype B F genes；The concentration of anti-sense primer is 10 μ
M, volume are 100 μ L；Preservation condition is -20 DEG C；PCR downstream primer sequences such as SEQ ID NO：Shown in 5：5’-
TTCACCTCACTCTCAAGCCTTATG-3’；
Fluorescence probe (j) is designed according to human metapneumovirus subtype B F genes；Fluorescence probe sequence such as SEQ ID
NO：Shown in 6：5’(FAM)-CAGCAGCAGCAGTCACAGCAGGCAT(Eclipse)-3’；The volume of fluorescence probe is 200 μ L；
Preservation condition is -20 DEG C.
RNA extracts kits:It is QIAamp Viral RNA Mini Kit kits (being purchased from QIAGEN companies of Germany),
Preservation condition is room temperature.
Reverse Transcriptase kit:It is PrimeScriptTMRT Reagent Kit kits (are purchased from TaKaRa companies of Japan),
Preservation condition is -20 DEG C.
Embodiment 2hMPV quantitative typing detection kits
The hMPV quantitative typings detection kit that the present invention is provided includes what is packed respectively：Standard positive template (a), feminine gender
Join control product (b), positive ginseng control product (c), fluorescent quantitation reaction solution (d), PCR sense primers (e), PCR anti-sense primers (f), fluorescence
Probe (g), PCR sense primers (h), PCR anti-sense primers (i) and fluorescence probe (j).
Standard positive template (a) is：Insert human metapneumovirus A hypotype F 1620 nucleotide sequences of gene
PMD18-T vector plasmids, and insert the pMD18-T carriers of human metapneumovirus subtype B F 1620 nucleotide sequences of gene
Plasmid.Concentration is 1 × 1010Copy/μ L, volume is 500 μ L；Respectively will insertion A hypotypes and subtype B human metapneumovirus F genes
1620 bases are cloned into the carrier, after conversion bacillus coli DH 5 а propagation, with alkali row solution (specific experiment condition and method
See that J. Pehanorm Brookers etc. are edited, Science Press, 1992, Molecular Cloning:A Laboratory guide, the second edition) DNA is extracted, then use
DNA purification kits (being purchased from Clonetec companies) purifying, then, uses spectrophotometer A260Quantify and be diluted to 1 × 1010Copy
Shellfish/μ L, preservation condition is -20 DEG C；PMD18-T carriers are purchased from TaKaRa companies of Japan, by the said firm through the reconstruction of pUC18 carriers
Into being a kind of special carrier of high-efficient cloning PCR primer, with the same identical function of pUC18 carriers, and can greatly improve
The connection of PCR primer, cloning efficiency.
Feminine gender ginseng control product (b) are water.
Positive ginseng control product (c) are that human metapneumovirus type strain is infected after Vero-E6 cell lines, extract viral RNA simultaneously inverse
The cDNA obtained is transcribed, volume is 100 μ L, and preservation condition is -20 DEG C.
Fluorescent quantitation reaction solution (d) is Premix Ex TaqTmReagent (is purchased from TaKaRa companies of Japan), and volume is 625 μ
L, preservation condition is -20 DEG C.
For detecting A hypotypes hMPV primer sets by SEQ ID NO：Sense primer (e), SEQ ID NO shown in 1：2 institutes
The anti-sense primer (f) and SEQ ID NO shown：Probe (g) composition shown in 3：
PCR sense primers (e) are designed according to human metapneumovirus A hypotype F genes；The concentration of sense primer is 10 μ
M, volume are 25 μ L；Preservation condition is -20 DEG C；PCR upstream primer sequences such as SEQ ID NO：Shown in 1：5’-
GAGCAATAGCACTCGGTGTTG-3’；
PCR anti-sense primers (f) are designed according to human metapneumovirus A hypotype F genes；The concentration of anti-sense primer is 10 μ
M, volume are 25 μ L；Preservation condition is -20 DEG C；PCR downstream primer sequences such as SEQ ID NO：Shown in 2：5’-
TCACAAAATCTTTCAGCTCTCTCAC-3’；
Fluorescence probe (g) is designed according to human metapneumovirus A hypotype F genes；Fluorescence probe sequence such as SEQ ID
NO：Shown in 3：5’(FAM)-TTGCCAACACACGAACTCCATTCCC(Eclipse)-3’；The volume of fluorescence probe is 50 μ L；
Preservation condition is -20 DEG C.
For detecting subtype B hMPV primer sets by SEQ ID NO：Sense primer, SEQ ID NO shown in 4：Shown in 5
Anti-sense primer and SEQ ID NO：Probe composition shown in 6：
PCR sense primers (h) are designed according to human metapneumovirus subtype B F genes；The concentration of sense primer is 10 μ
M, volume are 25 μ L；Preservation condition is -20 DEG C；PCR upstream primer sequences such as SEQ ID NO：Shown in 4：5’-
GTGCGATAGCTCTCGGAGTTG-3’；
PCR anti-sense primers (i) are designed according to human metapneumovirus subtype B F genes；The concentration of anti-sense primer is 10 μ
M, volume are 25 μ L；Preservation condition is -20 DEG C；PCR downstream primer sequences such as SEQ ID NO：Shown in 5：5’-
TTCACCTCACTCTCAAGCCTTATG-3’；
Fluorescence probe (j) is designed according to human metapneumovirus subtype B F genes；Fluorescence probe sequence such as SEQ ID
NO：Shown in 6：5’(FAM)-CAGCAGCAGCAGTCACAGCAGGCAT(Eclipse)-3’；The volume of fluorescence probe is 50 μ L；
Preservation condition is -20 DEG C.
Embodiment 3hMPV quantitative typing detection kits
The hMPV quantitative typings detection kit that the present invention is provided includes what is packed respectively：Standard positive template (a), fluorescence
Under quantitative reaction liquid (d), PCR sense primers (e), PCR anti-sense primers (f), fluorescence probe (g), PCR sense primers (h), PCR
Swim primer (i) and fluorescence probe (j).
Standard positive template (a) is：Insert human metapneumovirus A hypotype F 1620 nucleotide sequences of gene
PMD18-T vector plasmids, and insert the pMD18-T carriers of human metapneumovirus subtype B F 1620 nucleotide sequences of gene
Plasmid.Concentration is 1 × 1010Copy/μ L, volume is 500 μ L；Respectively will insertion A hypotypes and subtype B human metapneumovirus F genes
1620 bases are cloned into the carrier, after conversion bacillus coli DH 5 а propagation, with alkali row solution (specific experiment condition and method
See that J. Pehanorm Brookers etc. are edited, Science Press, 1992, Molecular Cloning:A Laboratory guide, the second edition) DNA is extracted, then use
DNA purification kits (being purchased from Clonetec companies) purifying, then, uses spectrophotometer A260Quantify and be diluted to 1 × 1010Copy
Shellfish/μ L, preservation condition is -20 DEG C；PMD18-T carriers are purchased from TaKaRa companies of Japan, by the said firm through the reconstruction of pUC18 carriers
Into being a kind of special carrier of high-efficient cloning PCR primer, with the same identical function of pUC18 carriers, and can greatly improve
The connection of PCR primer, cloning efficiency.
Fluorescent quantitation reaction solution (d) is Premix Ex TaqTmReagent (is purchased from TaKaRa companies of Japan), and volume is 1250 μ
L, preservation condition is -20 DEG C.
For detecting A hypotypes hMPV primer sets by SEQ ID NO：Sense primer (e), SEQ ID NO shown in 1：2 institutes
The anti-sense primer (f) and SEQ ID NO shown：Probe (g) composition shown in 3：
PCR sense primers (e) are designed according to human metapneumovirus A hypotype F genes；The concentration of sense primer is 10 μ
M, volume are 50 μ L；Preservation condition is -20 DEG C；PCR upstream primer sequences such as SEQ ID NO：Shown in 1：5’-
GAGCAATAGCACTCGGTGTTG-3’；
PCR anti-sense primers (f) are designed according to human metapneumovirus A hypotype F genes；The concentration of anti-sense primer is 10 μ
M, volume are 50 μ L；Preservation condition is -20 DEG C；PCR downstream primer sequences such as SEQ ID NO：Shown in 2：5’-
TCACAAAATCTTTCAGCTCTCTCAC-3’；
Fluorescence probe (g) is designed according to human metapneumovirus A hypotype F genes；Fluorescence probe sequence such as SEQ ID
NO：Shown in 3：5’(FAM)-TTGCCAACACACGAACTCCATTCCC(Eclipse)-3’；The volume of fluorescence probe is 100 μ L；
Preservation condition is -20 DEG C.
For detecting subtype B hMPV primer sets by SEQ ID NO：Sense primer, SEQ ID NO shown in 4：Shown in 5
Anti-sense primer and SEQ ID NO：Probe composition shown in 6：
PCR sense primers (h) are designed according to human metapneumovirus subtype B F genes；The concentration of sense primer is 10 μ
M, volume are 50 μ L；Preservation condition is -20 DEG C；PCR upstream primer sequences such as SEQ ID NO：Shown in 4：5’-
GTGCGATAGCTCTCGGAGTTG-3’；
PCR anti-sense primers (i) are designed according to human metapneumovirus subtype B F genes；The concentration of anti-sense primer is 10 μ
M, volume are 50 μ L；Preservation condition is -20 DEG C；PCR downstream primer sequences such as SEQ ID NO：Shown in 5：5’-
TTCACCTCACTCTCAAGCCTTATG-3’；
Fluorescence probe (j) is designed according to human metapneumovirus subtype B F genes；Fluorescence probe sequence such as SEQ ID
NO：Shown in 6：5’(FAM)-CAGCAGCAGCAGTCACAGCAGGCAT(Eclipse)-3’；The volume of fluorescence probe is 100 μ L；
Preservation condition is -20 DEG C.
Embodiment 4hMPV quantitative typings are detected
The hMPV quantitative typing detection kits provided using embodiment 1, detect clinical respiratory sample (nasopharyngeal secretions
Sample be derived from Respiratory ward infant clinical sample 1,2,3), comprise the following steps：
Using RNA extracts reagents, extract the RNA of sample to be tested (clinical respiratory sample), be stored in -80 DEG C it is standby；
Use reverse transcription reagents reverse transcription to be cDNA the RNA of sample to be tested, saved backup in -20 DEG C；
Standard positive masterplate (a) is dissolved with distilled water, and is 1 × 10 by its doubling dilution9Copy/μ L, 1 × 108Copy/μ
L、1×107Copy/μ L, 1 × 106Copy/μ L, 1 × 105Copy/μ L, 1 × 104Copy/μ L, 1 × 103Copy/μ L, 1 × 102
Copy/μ L, 1 × 101Copy/μ L, totally 9 pipe series dilution product, are saved backup in -20 DEG C；
Take the μ L of 12.5 μ L, PCR sense primers (e) of fluorescent quantitation reaction solution (d), 0.5 μ L, PCR anti-sense primers (f) 0.5, it is glimmering
The μ L of 1 μ L, PCR sense primers (h) of light probe (g), 0.5 μ L, PCR anti-sense primers (i) 0.5, the μ L of fluorescence probe (j) 1, distilled water 3.5
The μ L and μ L of template 5, totally 25 μ L constitute PCR reaction systems；The template is the cDNA of one of above-mentioned sample to be tested, negative ginseng control product
(b), positive ginseng control product (c) or standard positive masterplate (a) are serially diluted one kind in product；Wherein, being serially diluted product is used to make
Standard curve, the negative control that feminine gender ginseng control product react as PCR, the positive control that positive ginseng control product react as PCR, to examine
Whether normal survey PCR reaction systems；Sample to be tested sets 3 repetitions, to ensure the accuracy and stability of experiment；
PCR cycle detection is carried out to each reaction system using fluorescent quantitative detector, the reaction condition for setting PCR is：
First 95 DEG C are denatured 3min；95 DEG C again, 15s → 62 DEG C, 10s → 68 DEG C, 20s, set 40 circulation；CT values to detect circulation
(i.e.：Fluorescence signal in each reaction tube reaches the period undergone during the thresholding of setting)；
By the standard curve for comparing sample to be tested He being serially diluted standard positive template, to the starting copies of sample to be tested
Number carries out quantitative (A hypotype standard curves are shown in accompanying drawing 1, and subtype B standard curve is shown in accompanying drawing 2), and the step is by BIO-RAD companies of the U.S.
CFX-96 fluorescent quantitative PCR detectors be automatically performed, quantitative result is calculated automatically by the detector；Sample 1,2,3
As a result 1,2,3 are shown in Table respectively.
The quantitative typing testing result of the sample to be tested of table 1
Terminate rear fluorescent quantitative detector by the results showed that PCR reactions of table 1 not detect in feminine gender ginseng control QC
To fluorescence signal, and the human metapneumovirus of >=500 copies is detected in positive ginseng control QC, show PCR reaction systems
Normally.Such a situation represents to infect human metapneumovirus A types.
The human metapneumovirus copy number of sample to be tested is obtained by reference standard curve, copy number >=500 are judged as people
Class metapneumovirus is positive, and is the A hypotypes of infection, specific numerical value (such as 5.6 × 106) represent to infect the tool of human metapneumovirus
Body copy number.
The quantitative typing testing result of the sample to be tested of table 2
Terminate rear fluorescent quantitative detector by the results showed that PCR reactions of table 2 not detect in feminine gender ginseng control QC
To fluorescence signal, and the human metapneumovirus of >=500 copies is detected in positive ginseng control QC, show PCR reaction systems
Normally.Such a situation represents to infect human metapneumovirus Type B.
The human metapneumovirus copy number of sample to be tested is obtained by reference standard curve, copy number >=500 are judged as people
Class metapneumovirus is positive, and is the subtype B of infection, specific numerical value (6.2 × 105) represent the specific of infection human metapneumovirus
Copy number.
The quantitative typing testing result of the sample to be tested of table 3
Terminate rear fluorescent quantitative detector by the results showed that PCR reactions of table 3 not detect in feminine gender ginseng control QC
To fluorescence signal, and the human metapneumovirus of >=500 copies is detected in positive ginseng control QC, show PCR reaction systems
Normally.Such a situation is represented while infecting human metapneumovirus A and subtype B.
The human metapneumovirus copy number of sample to be tested is obtained by reference standard curve, copy number >=500 are judged as people
Class metapneumovirus is positive, and is while the A and subtype B of infection, specific numerical value (A hypotypes 5.6 × 106, subtype B 6.2 × 105)
Represent the specific copy number of infection human metapneumovirus.
The hMPV quantitative typing detection kits provided using embodiment 2,3, detect above-mentioned sample to be tested 1,2,3, as a result
It is close with this.
Comparative example 1Sanger method detection examples
3 samples of embodiment 4 are detected using Sanger methods, comprised the following steps that：
(1) extraction of viral RNA：
1st, the ready buffer AVL of 560 μ L (containing carrier RNA) are drawn into 1.5mL centrifuge tubes.
2nd, by 140 μ L blood plasma, serum, urine, culture cell supernatant or acellular body fluid are added to equipped with buffer
In AVL-carrier RNA centrifuge tube.Mediate 15 seconds, mix.Room temperature places 10min.
3rd, 560 μ L absolute ethyl alcohols (96%~100%) are added in sample, mediation 15s is fully mixed.
4th, draw in the addition column (having been loaded into 2mL centrifuge tubes) of solution carefully in being walked on 630 μ L, note not
Encounter the edge of pillar.Close the lid, 6000 × g (8000rpm) centrifugations 1min.Column is put into new 2mL centrifuge tubes
In, discard old collecting pipe.If (solution can be centrifuged again not completely through film, until solution completely through).
5th, careful opening column lid, repeats the 6th step.If (sample size has exceeded 140 μ L, repeats this step
Suddenly, until all lysates all upper props).
6th, careful opening column lids, add 500 μ L buffer AW1.Close the lid, 8000rpm centrifugations,
1min.Column is put into new 2mL collecting pipes (Kit offers), old collecting pipe is discarded.Even if (sample size is more than 140 μ
L, without increase buffer AW1 amount).
7th, careful opening column lids, add 500 μ L buffer AW2.Close the lid, centrifuge at full speed
(14000rpm), 3min.Then the 11st step is carried out, or first carries out the 10th step, to avoid buffer AW2 from remaining, is then entered again
The step of row the 11st.(buffer AW2 residuals can influence downstream to test.Some centrifuges can vibrate when slowing down, and cause backflow,
So that buffer AW2 touch column.This may also can be caused to show when column and collecting pipe are taken out in centrifuge
As producing, therefore, the 10th step is preferably first carried out)
8th, column is put into new 2mL collecting pipes, discards old collecting pipe, at full speed centrifugation 1min.
9th, column is placed in 1.5mL centrifuge tubes.Discard old collecting pipe.Careful opening column, adds 60 μ L
The buffer AVE of room temperature.Close the lid, room temperature places 1min.8000rpm centrifuges 1min.Viral RNA can be with -80 DEG C
It is stable to preserve 1 year.
(2) RNA reverse transcriptions are cDNA
RT reaction solutions are prepared by following component (reaction solution, which is prepared, to be carried out on ice).
Reverse transcription reaction condition is as follows:
37 DEG C of 15min (reverse transcription reaction)
85 DEG C of 5sec (inactivation reaction of reverse transcriptase)
4℃
(3) PCR determines Viral typing：It is sick using being directed in embodiment 1 using cDNA products obtained in the previous step as template
The specific primer of malicious A/B hypotypes, is sequenced to determine Viral typing after entering performing PCR amplification.
PCR reaction solutions constitute (25 μ L systems)：
PCR reaction conditions are as follows：
(4) sequence verification
Amplified production to more than carries out sequence verification with Sanger methods, finally determines genotype.Result of the test
Consistent with the result of the test of embodiment 4, i.e., sample to be tested 1 infects human metapneumovirus A types, and sample to be tested 2 infects
Human metapneumovirus Type B, sample to be tested 3 infects human metapneumovirus A and subtype B simultaneously.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. the primer sets for expanding hMPV genetic fragments, it is characterised in that including A hypotype hMPV primer sets and subtype B hMPV
Primer sets；
The A hypotypes hMPV primer sets are by SEQ ID NO：Sense primer, SEQ ID NO shown in 1：Anti-sense primer shown in 2
With SEQ ID NO：Probe composition shown in 3；
The subtype B hMPV primer sets are by SEQ ID NO：Sense primer, SEQ ID NO shown in 4：Anti-sense primer shown in 5
With SEQ ID NO：Probe composition shown in 6；
It is described be used to expanding the response procedures for the quantitative fluorescent PCR that the primer sets of hMPV genetic fragments are used for：
(1) pre-degeneration：95 DEG C, 3min；
(2) expand：95 DEG C, 15s；62 DEG C, 10s；68 DEG C, 20s is a circulation, sets 40 circulations.
2. application of the primer sets as claimed in claim 1 in hMPV quantitative typing detection kits are prepared.
3. application according to claim 2, it is characterised in that the hMPV is A hypotypes hMPV and/or subtype B hMPV.
4. a kind of hMPV quantitative typings detection kit, it is characterised in that including standard positive template, fluorescent quantitation reaction solution and
Primer sets as claimed in claim 1.
5. kit according to claim 4, it is characterised in that the standard positive template is：Insert the A hypotype mankind inclined
The pMD18-T vector plasmids of 1620 nucleotide sequences of Pneumovirinae F genes, and insertion subtype B human metapneumovirus F genes
The pMD18-T vector plasmids of 1620 nucleotide sequences.
6. kit according to claim 4, it is characterised in that also including RNA extracts reagents, reverse transcription reagents, the positive
Join the one or more in control product or negative ginseng control product.
7. kit according to claim 6, it is characterised in that the positive joins control product and is：Infect human metapneumovirus
The Vero-E6 cell lines of type strain extract the cDNA that viral RNA and reverse transcription are obtained.
8. kit according to claim 6, it is characterised in that the negative ginseng control product are water.
9. a kind of method of non-diagnostic purpose hMPV quantitative typings detection, it is characterised in that comprise the following steps：
Standard positive template is subjected to doubling dilution, standard dilution is obtained；Take fluorescent quantitation reaction solution, the standard dilution
Quantitative fluorescent PCR is carried out with primer sets as claimed in claim 1, standard curve is drawn；
The RNA of testing sample is extracted, reverse transcription is cDNA, obtain testing sample cDNA；Take fluorescent quantitation reaction solution, it is described to be measured
Sample cDNA and primer sets as claimed in claim 1 carry out quantitative fluorescent PCR, according to the standard curve, are treated described in acquisition
The hMPV quantitative typing testing results of test sample product.
10. method according to claim 9, it is characterised in that the response procedures of the quantitative fluorescent PCR are：
(1) pre-degeneration：95 DEG C, 3min；
(2) expand：95 DEG C, 15s；62 DEG C, 10s；68 DEG C, 20s is a circulation, sets 40 circulations.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538618A (en) * | 2009-04-15 | 2009-09-23 | 重庆医科大学附属儿童医院 | Fluorescence quantitative detection kit for human metapneumovirus |
| CN104017901A (en) * | 2014-03-07 | 2014-09-03 | 邓瑛 | Method and kit for simultaneously detecting human type A and type B respiratory syncytial viruses and human metapneumoviruses |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538618A (en) * | 2009-04-15 | 2009-09-23 | 重庆医科大学附属儿童医院 | Fluorescence quantitative detection kit for human metapneumovirus |
| CN104017901A (en) * | 2014-03-07 | 2014-09-03 | 邓瑛 | Method and kit for simultaneously detecting human type A and type B respiratory syncytial viruses and human metapneumoviruses |
Non-Patent Citations (1)
| Title |
|---|
| Diagnosis and epidemiological studies of human metapneumovirus using real-time PCR;Kanti Pabbaraju，et al;《Journal of Clinical Virology》;20071231;第40卷(第3期);186-192页 * |
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