AU2021102785A4 - Primers, probe, kit and method for qpcr detection of phenacoccus manihoti - Google Patents
Primers, probe, kit and method for qpcr detection of phenacoccus manihoti Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 62
- 241000209267 Phenacoccus manihoti Species 0.000 title claims abstract description 42
- 238000001514 detection method Methods 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000011529 RT qPCR Methods 0.000 claims abstract description 39
- 239000013612 plasmid Substances 0.000 claims abstract description 27
- 230000003321 amplification Effects 0.000 claims abstract description 19
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 19
- 239000013642 negative control Substances 0.000 claims abstract description 6
- 239000013641 positive control Substances 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 17
- 238000003753 real-time PCR Methods 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 10
- 241000894007 species Species 0.000 claims description 10
- 241000238631 Hexapoda Species 0.000 claims description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000013461 design Methods 0.000 claims description 5
- 238000005516 engineering process Methods 0.000 claims description 5
- 238000007400 DNA extraction Methods 0.000 claims description 4
- 108020005196 Mitochondrial DNA Proteins 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 230000035772 mutation Effects 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000013492 plasmid preparation Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000011002 quantification Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 240000003183 Manihot esculenta Species 0.000 description 6
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 241001058119 Phenacoccus Species 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000607479 Yersinia pestis Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 2
- 241000549765 Phenacoccus aceris Species 0.000 description 2
- 241001058021 Phenacoccus solani Species 0.000 description 2
- 241001415279 Pseudococcidae Species 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 241001465977 Coccoidea Species 0.000 description 1
- 241001516600 Dysmicoccus Species 0.000 description 1
- 241001516601 Dysmicoccus neobrevipes Species 0.000 description 1
- 241000258937 Hemiptera Species 0.000 description 1
- 244000149260 Manihot esculenta subsp flabellifolia Species 0.000 description 1
- 241000209270 Phenacoccus solenopsis Species 0.000 description 1
- 241001492664 Solenopsis <angiosperm> Species 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention provides primers, probe, kit and method for qPCR detection of Phenacoccus manihoti
with high specificity and high sensitivity. The method for qPCR detection of Phenacoccus manihoti
comprises: Step (1): extracting DNA of a sample to be detected; Step (2): preparing a reaction system; Step
(3): constructing recombinant plasmids; step (4): conducting qPCR amplification on the sample to be
detected, plasmid sample, positive control sample and negative control sample using the primers and
probe; Step (5): plotting standard curves; and Step (6): calculating a result.
urawings o uescripion
PM-Fl-+
CTTTLACCTTTGATGATTTCTTCTTCTG TTTAATATTTCCACGAT
PM-Probel---+
TAAATA TTTTAGATTTTGATTACTACTTCCTTCTTTAATATTATTA
ATTTTAAATATAATATTAAACAATAATATTAATACAGGTTGGACTC
-PM-R1
TTTACCCCCCATTAATTAAC'AAAATTTCATTACTTTAAACTTC
Fig. 1
Standard Curve
128 -
2 2 ....- . ... .......... ... ..-. .....- .....- .....-. ....-.. ......-. .. .
-5 -4 -3 -2 -1 0
Log Starting Quantity
D Standard
X Unknown
-- FAM E=111-0% R^2=0.998 Slope=-3.OB4 y-int=12.14B
Fig.2
1
Description
urawings o uescripion
PM-Fl-+ CTTTLACCTTTGATGATTTCTTCTTCTG TTTAATATTTCCACGAT PM-Probel---+ TAAATA TTTTAGATTTTGATTACTACTTCCTTCTTTAATATTATTA
ATTTTAAATATAATATTAAACAATAATATTAATACAGGTTGGACTC -PM-R1
Fig. 1
Standard Curve
128 - 22 .. ..-. ... .......... .......- . . . .-. .- ...-....-.. ....-. .
. -5 -4 -3 -2 -1 0 Log Starting Quantity D Standard X Unknown -- FAM E=111-0% R^2=0.998 Slope=-3.OB4 y-int=12.14B
Fig.2
UeNZLp iuun
Technical Field
The present invention belongs to the technical field of real-time fluorescence quantitative
PCR (qPCR) detection of Phenacoccus manihoti, and particularly relates to primers, probe, kit
and method for qPCR detection of Phenacoccusmanihoti.
Background
Phenacoccus manihoti Matile-Ferrero, belonging to Phenacoccus, Phenacoccinae,
Pseudococcidae, Coccoidea, Hemiptera, is a pest to be internationally quarantined, and is also a
pest to be emphatically quarantined in entry-exit inspection and quarantine of Chinese ports. P.
manihoti is addictive to cassava, especially sweet cassava, and often outbreaks on cassava.
Existing studies have shown that host plants of P. manihoti cover more than 15 families, 27
species and 3 genera, including crops, landscape plants, weeds, fruit trees and the like. Native to
the central South America, P. manihoti is a primary pest on local cassava, is spread along with
host plants and products thereof, is discovered in West Africa, Central Africa and East Africa
one after another, and causes great loss to African cassava industry. P. manihoti is highly
suitable in most regions of Guangdong, Guangxi and Hainan as well as in partial regions of
Fujian, Jiangxi, Guizhou and Yunnan, and is moderately suitable in partial regions of Chongqing
and Sichuan. Therefore, it is a necessary prerequisite for the healthy development of the cassava,
vegetable and flower industry and the fruit and cotton industry in China to develop the rapid
detection technology for P. manihoti as soon as possible to strengthen the monitoring and
detection of P. manihoti. Therefore, the problem to be urgently solved by those skilled in the
field is to provide a qPCR detection method for specific detection of P. manihoti.
In this study, by means of the qPCR technology, based on mitochondrial COI genes of P.
manihoti, specific primers and TaqMan probe are designed, a kit and method for rapid and
UeNZLp iuun
accurate detection of P. manihoti are created, which can be popularized in Chinese ports, cassava
production bases, organic vegetable and organic fruit production bases, fresh cut flower
production bases, cotton planting areas, and allocation and transportation of vegetables,
ornamentals, fruit tree seedlings/plants in the form of kits.
Summary
In order to solve the problems proposed in the above background, the present invention
provides primers, probe, kit and method for qPCR detection of Phenacoccus manihoti, and has
the characteristics of high specificity and high sensitivity.
To achieve the above purpose, the present invention provides the following technical
solution: primers, probe and kit for qPCR detection of Phenacoccus manihoti, wherein the kit
comprises primers, probe, template, negative sample and premix.
A method for qPCR detection of Phenacoccus manihoti, wherein qPCR detection steps
comprise:
Step (1): extracting DNA of a sample to be detected;
Step (2): preparing a reaction system;
Step (3): constructing recombinant plasmids;
step (4): conducting qPCR amplification on the sample to be detected, plasmid sample,
positive control sample and negative control sample using the primers and probe;
Step (5): plotting standard curves; and
Step (6): calculating a result.
Preferably, step (1) comprises: extracting DNA of adult insects of more than six species,
putting adult insects in a 1.5ml centrifuge tube, and adding liquid nitrogen to grind into powder,
wherein DNA extraction is conducted using a DNeasy@ Tissue Kit from QIAgen, and the
specific steps are conducted in accordance with the kit instruction;
step (2) comprises target sequence selection and specific primers and probe design:
"UNZLp iuun
analyzing sequences of COI genes in mtDNA of P. manihoti, selecting a sequence fragment with
high mutation rate as a target fragment for amplification, designing multiple pairs of qPCR
primers and probe, conducting preliminary screening through test, and determining a pair of
qPCR primers and probe for detecting P. manihoti;
specific primers and probe design: for the determined pair, designing a forward primer as
PM-F1:5'-ACCTTTGATGATTTCTTCTTCTG-3', a reverse primer as
PM-R1:5'-TGAAGTTTAAAGTAATGAAATTTTG-3', the amplification product length of
PM-Fl/PM-Ri being 179bp; a probe as
PM-Probel:5'-ATTTTAGATTTTGATTACTACTTCCTTCTT-3', wherein the 5' end of the
probe is marked with an FAM reporter, and the 3'end is marked with a BHQ quencher.
Preferably, step (3) comprises: conducting conventional PCR amplification on the DNA of
P. manihoti by means of the primer pair PM-Fl/PM-R; connecting the obtained target fragment
with PUC57, converting the connected product, obtaining colonies of converted plasmids;
picking a monoclonal colony and inoculating in a culture medium for culture; extracting positive
recombinant plasmids by a plasmid preparation kit for PCR identification, and conducting
sequencing verification by Hangzhou Qingke Biotechnology Co., Ltd.; measuring the plasmid
concentration by a spectrophotometer after plasmid identification is correct, and taking same as a
gene standard product of the method of the present invention after quantification.
Preferably, in step (4), qPCR detection amplification system includes 10pL of 2xTaqMan
probe Real-time PCR Master Mixture (Takara Premix Ex Tag from Takara Biomedical
Technology (Dalian) Co., Ltd.), 0.4tL of forward and reverse primers (10M) respectively,
0.5tL of ROX Reference DyeII, 2L of template genomic DNA, 0.8tL of TaqMan probe (10M)
and ddH20 filled to 20tL, conducting qPCR amplification on a CFX Opus 96 Real-Time PCR
System quantitative PCR amplifier, starting "CFX Opus 96 Real-Time PCR System Software",
setting PCR reaction condition, two-step PCR procedure is: pre-denaturing at 95°C for 5min,
UeNZLp iuun
denaturing at 95°C for 10sec and annealing at 58°C for 30sec, repeating for 40 cycles, clicking
on Run to conduct PCR, ending the reaction in about 1h, saving a file, and starting analysis
software.
Compared with the prior detection methods, the present invention has the following
advantageous effects:
The present invention has the advantages that the identification of P. manihoti, and other
insects of Phenacoccus sp. and Dysmicoccus sp. is rapidly and accurately realized, the confusion
possibly caused by morphological identification is avoided; the time consumed for qPCR is
greatly shortened as compared with the conventional PCR, a detection result is obtained in only
1h; and has the advantages of high specificity and high sensitivity. The minimum DNA
concentration of detection can reach 10fg/uL, the result can be directly observed, the pollution
caused in the operation process can be effectively avoided. P. manihoti can be quickly and
accurately detected by means of the primers, probe and kit for qPCR detection of P. manihoti of
the present invention.
Description of Drawings
The drawings are used to provide further understanding for the present invention and
constitute part of the description. The drawings are used to explain the present invention together
with the embodiments of the present invention, and do not constitute a limitation to the present
invention. In the drawings:
Fig. 1 is a schematic diagram showing positions of the specific primer pair PM-F/PM-Ri
and probe PM-Probel on mtDNA of P. manihoti;
Fig. 2 shows a standard curve of qPCR detection based on the Taqman probe;
Fig. 3 shows species specificity detection of the fluorescent primers and probe; and
Fig. 4 shows sensitivity detection of the fluorescent primers and probe used by the
fluorescence quantitative detection system for P. manihoti.
"Uescptiuon
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and fully
described below in combination with the drawings in the embodiments of the present invention.
Apparently, the described embodiments are merely part of the embodiments of the present
invention, not all of the embodiments. Based on the embodiments in the present invention, all
other embodiments obtained by those ordinary skilled in the art without contributing creative
labor will belong to the protection scope of the present invention.
Embodiment 1
According to the research results at present, to ensure the specificity of the designed primers,
P. manihoti and related species such as Phenacoccus solani, Phenacoccus sp., Phenacoccus
solenopsis, Phenacoccus aceris as well as outer edge species such as Dysmicoccus neobrevipes
are selected, the primers PM-Fl/PM-Ri are designed using software primer5 on the basis of COI
gene sequences of mitochondrial genomes of P. manihoti, the probe PM-Probel is designed
using software Primer Exprss according to the amplified specific fragment, specific
amplification is conducted on P. manihoti, and both the primers and the probe are synthesized by
Hangzhou Qingke Biotechnology Co., Ltd.
Referring to Figs. 1-4, the present invention provides the following technical solution:
I. DNA extraction
Extracting DNA of adult insects of more than six species, putting adult insects in a 1.5ml
centrifuge tube, and adding liquid nitrogen to grind into powder, wherein DNA extraction is
conducted using a DNeasy@ Tissue Kit from QIAgen, and the specific steps are conducted in
accordance with the kit instruction.
II. Target sequence selection
Analyzing sequences of COI genes in mtDNA of P. manihoti, selecting a sequence
fragment with high mutation rate as a target fragment for amplification. Designing multiple pairs of qPCR primers and probe, conducting preliminary screening through test, and determining a pair of qPCR primers and probe for detecting P. manihoti.
III. Specific primers and probe design
As shown in Fig. 1: for the determined pair, designing a forward primer as
PM-F1:5'-ACCTTTGATGATTTCTTCTTCTG-3', a reverse primer as
PM-R1:5'-TGAAGTTTAAAGTAATGAAATTTTG-3', the amplification product length of
PM-Fl/PM-Ri being 179bp; a probe as
PM-Probel:5'-ATTTTAGATTTTGATTACTACTTCCTTCTT-3', wherein the 5' end of the
probe is marked with an FAM reporter, and the 3'end is marked with a BHQ quencher.
IV. Standard plasmid construction
Conducting conventional PCR amplification on the DNA of P. manihoti by means of the
primer pair PM-Fl/PM-Ri; connecting the obtained target fragment with PUC57, converting the
connected product, obtaining colonies of converted plasmids; picking a monoclonal colony and
inoculating in a culture medium for culture; extracting positive recombinant plasmids by a
plasmid preparation kit for PCR identification, and conducting sequencing verification by
Hangzhou Qingke Biotechnology Co., Ltd.; measuring the plasmid concentration by a
spectrophotometer after plasmid identification is correct, and taking same as a gene standard
product of the method of the present invention after quantification.
V. qPCR detection
As shown in Fig. 2, qPCR detection amplification system includes 10pL of 2xTaqMan
probe Real-time PCR Master Mixture (Takara Premix Ex Tag from Takara Biomedical
Technology (Dalian) Co., Ltd.), 0.4tL of forward and reverse primers (10M) respectively,
0.5tL of ROX Reference DyeII, 2L of template genomic DNA, 0.8tL of TaqMan probe (10M)
and ddH2 0 filled to 20jL, conducting real-time fluorescence PCR amplification on a CFX Opus
96 Real-Time PCR System quantitative PCR amplifier, starting "CFX Opus 96 Real-Time PCR
"UNZLp iuun
System Software", setting PCR reaction condition, two-step PCR procedure is: pre-denaturing at
°C for 5min, denaturing at 95°C for 10sec and annealing at 58°C for 30sec, repeating for 40
cycles, clicking on Run to conduct PCR, ending the reaction in about 1h, saving a file, and
starting an analysis software.
VI. Standard curve establishment
Taking the recombinant plasmids as a standard template and empty plasmids as a negative
control, conducting 10-fold decreasing gradient dilution on the recombinant plasmids, taking
24L of recombinant plasmid of each concentration as a template to conduct qPCR detection,
wherein the analysis result shows that the higher the concentration of template, the lower the
value of Ct, the parallel test result shows that the DNA concentration (x) has a correlation with
Ct(y) (the unit of x is in ng/ul), the relational expression is Y=-3.084x+12.148 (as shown in Fig.
2), the correlation coefficient is high, specifically, R2 =0.989 (greater than 0.98), to meet the
requirements of qPCR detection so as to quantitatively and quickly detect and analyze P.
manihoti.
VII. Species specificity detection of fluorescent primers and probe
As shown in Fig. 3, taking the DNA of the six species of adult insects as a template, the
recombinant plasmids as a positive control and the ddH2 0 as a negative control, conducting
qPCR detection, wherein the detection result shows that only P. manihoti and recombinant
plasmids gave out obvious fluorescence signals and have obvious amplification curves (as shown
in Fig. 3), while the other five species of Pseudococcidae and the negative control have no
amplification, indicating that the primers and probe designed by this patent have good specificity.
The detection system can be used for the detection, identification and analysis of P. manihoti.
In Fig. 3, numeral 1 represents plasmid DNA;
numeral 2 represents P. manihoti;
numeral 3-8 represent P. solani, Phenacoccus sp., P. solenopsis, P. aceris, D.
UeNZLp iuun
neobrevipes and negative control.
VIII. Sensitivity detection of fluorescent primers and probe
As shown in Fig. 4, conducting qRT-PCR detection on purified recombinant plasmids at
various dilutions, wherein the result shows that when the concentrations of DNA are1.ng/uL,
1OOpg/uL, 1pg/uL, 1.pg/uL, 100fg/uL and 10fg/uL, obvious positive amplification can be
detected; while the DNA with a concentration less than 10fg/uL shows negative amplification
and no increase of fluorescence signals. Therefore, the established qRT-PCR can detect 10fg/uL
at least.
In Fig. 4, a-g respectively represent1.ng/uL, OOpg/uL, Opg/uL, 1.pg/uL, 100fg/uL and
1Ofg/uL.
Finally, it should be noted that the above description is only a preferred embodiment of the
present invention, and is not intended to limit the present invention. Although the present
invention is described in detail with reference to the above embodiment, those skilled in the art
may still modify the technical solution recorded in the above embodiment, or equivalently
replace some of the technical features. Any modification, equivalent replacement, improvement,
etc. made within the spirit and the principle of the present invention shall be included within the
protection scope of the present invention.
Claims (5)
1. Primers, probe and kit for qPCR detection of Phenacoccus manihoti, wherein the kit
comprises primers, probe, template, negative sample and premix.
2. A method for qPCR detection of Phenacoccus manihoti, wherein qPCR detection steps
comprise:
Step (1): extracting DNA of a sample to be detected;
Step (2): preparing a reaction system;
Step (3): constructing recombinant plasmids;
step (4): conducting qPCR amplification on the sample to be detected, plasmid sample,
positive control sample and negative control sample using the primers and probe;
Step (5): plotting standard curves; and
Step (6): calculating a result.
3. The method for qPCR detection of Phenacoccus manihoti according to claim 2, wherein
step (1) comprises: extracting DNA of adult insects of more than six species, putting adult
insects in a 1.5ml centrifuge tube, and adding liquid nitrogen to grind into powder, wherein DNA
extraction is conducted using a DNeasy* Tissue Kit from QIAgen, and the specific steps are
conducted in accordance with the kit instruction;
step (2) comprises target sequence selection and specific primers and probe design:
analyzing sequences of COI genes in mtDNA of P. manihoti, selecting a sequence fragment with
high mutation rate as a target fragment for amplification, designing multiple pairs of qPCR
primers and probe, conducting preliminary screening through test, and determining a pair of
qPCR primers and probe for detecting P. manihoti;
specific primers and probe design: for the determined pair, designing a forward primer as
PM-F1:5'-ACCTTTGATGATTTCTTCTTCTG-3', a reverse primer as
PM-R1:5'-TGAAGTTTAAAGTAATGAAATTTTG-3', the amplification product length of
PM-Fl/PM-Ri being 179bp; a probe as
I-1u1ii1
PM-Probel:5'-ATTTTAGATTTTGATTACTACTTCCTTCTT-3', wherein the 5' end of the
probe is marked with an FAM reporter, and the 3'end is marked with a BHQ1 quencher.
4. The method for qPCR detection of Phenacoccus manihoti according to claim 2, wherein
step (3) comprises: conducting conventional PCR amplification on the DNA of P. manihoti by
means of the primer pair PM-Fl/PM-R; connecting the obtained target fragment with PUC57,
converting the connected product, obtaining colonies of converted plasmids; picking a
monoclonal colony and inoculating in a culture medium for culture; extracting positive
recombinant plasmids by a plasmid preparation kit for PCR identification, and conducting
sequencing verification by Hangzhou Qingke Biotechnology Co., Ltd.; measuring the plasmid
concentration by a spectrophotometer after plasmid identification is correct, and taking same as a
gene standard product of the method of the present invention after quantification.
5. The method for qPCR detection of Phenacoccus manihoti according to claim 2, wherein
in step (4), qPCR detection amplification system includes 10pL of 2xTaqMan probe Real-time
PCR Master Mixture (Takara Premix Ex Tag from Takara Biomedical Technology (Dalian) Co.,
Ltd.), 0.4tL of forward and reverse primers (10tM) respectively, 0.5pL of ROX Reference
DyeII, 2L of template genomic DNA, 0.8tL of TaqMan probe (10tM) and ddH 20 filled to
tL, conducting qPCR amplification on a CFX Opus 96 Real-Time PCR System quantitative
PCR amplifier, starting "CFX Opus 96 Real-Time PCR System Software", setting PCR reaction
condition, two-step PCR procedure is: pre-denaturing at 95°C for 5min, denaturing at 95°C for
sec and annealing at 58°C for 30sec, repeating for 40 cycles, clicking on Run to conduct PCR,
ending the reaction in about lh, saving a file, and starting analysis software.
2021102785 Drawings of Description
Fig. 1
Fig.2
2021102785 Drawings of Description
Fig.3
Fig. 4
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| AU2021102785A Ceased AU2021102785A4 (en) | 2021-05-24 | 2021-05-24 | Primers, probe, kit and method for qpcr detection of phenacoccus manihoti |
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Owner name: ZHEJIANG ACADEMY OF SCIENCE&TECHNOLOGY FOR INSPECTION&QUARANTINE Free format text: FORMER NAME(S): ZHEJIANG ACADEMY OF SCIENCE&TECHNOLOGY FOR INSPECTION&QUARANTINE; HANGZHU CUSTOMS DISTRICT P.R.CHINA ; ZHENGZHOU CUSTOMS TECHNICAL CENTER; GONGBEI CUSTOMS TECHNICAL CENTER Owner name: HANGZHOU CUSTOMS DISTRICT P.R.CHINA Free format text: FORMER NAME(S): ZHEJIANG ACADEMY OF SCIENCE&TECHNOLOGY FOR INSPECTION&QUARANTINE; HANGZHU CUSTOMS DISTRICT P.R.CHINA ; ZHENGZHOU CUSTOMS TECHNICAL CENTER; GONGBEI CUSTOMS TECHNICAL CENTER Owner name: ZHENGZHOU CUSTOMS TECHNICAL CENTER Free format text: FORMER NAME(S): ZHEJIANG ACADEMY OF SCIENCE&TECHNOLOGY FOR INSPECTION&QUARANTINE; HANGZHU CUSTOMS DISTRICT P.R.CHINA ; ZHENGZHOU CUSTOMS TECHNICAL CENTER; GONGBEI CUSTOMS TECHNICAL CENTER Owner name: GONGBEI CUSTOMS TECHNICAL CENTER Free format text: FORMER NAME(S): ZHEJIANG ACADEMY OF SCIENCE&TECHNOLOGY FOR INSPECTION&QUARANTINE; HANGZHU CUSTOMS DISTRICT P.R.CHINA ; ZHENGZHOU CUSTOMS TECHNICAL CENTER; GONGBEI CUSTOMS TECHNICAL CENTER |
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| MK22 | Patent ceased section 143a(d), or expired - non payment of renewal fee or expiry |