CN110396552A - Molecular labeling, primer and the method for based on PCR identification water droplet puppet health fibre worm - Google Patents

Molecular labeling, primer and the method for based on PCR identification water droplet puppet health fibre worm Download PDF

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CN110396552A
CN110396552A CN201910835831.3A CN201910835831A CN110396552A CN 110396552 A CN110396552 A CN 110396552A CN 201910835831 A CN201910835831 A CN 201910835831A CN 110396552 A CN110396552 A CN 110396552A
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puppet
water droplet
dna
seq
primer
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CN110396552B (en
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黎睿君
黄宇希
高延奇
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Dalian Ocean University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • 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
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa

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  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention discloses a kind of molecular labeling of based on PCR identification water droplet puppet health fibre worm, and DNA sequence dna is as shown in SEQ ID NO:1；The DNA sequence dna of upstream primer and downstream primer is respectively as shown in SEQ ID NO:2, SEQ ID NO:3；Identification method successively carries out in accordance with the following steps: separation fish body lesions position tissue extraction tissue DNA；Carry out PCR amplification；PCR product is examined with agarose gel electrophoresis, if there is the electrophoretic band of 498 bp, the shield infusorian of lesions position parasitism is water droplet puppet health fibre worm, is had many advantages, such as quick, simple and accurate.

Description

Molecular labeling, primer and the method for based on PCR identification water droplet puppet health fibre worm
Technical field
The invention belongs to field of biotechnology, especially a kind of molecular labeling of based on PCR identification water droplet puppet health fibre worm draws Object and method.
Background technique
Water droplet puppet health fibre worm (Pseudocohnilembus persalinus) it is the ciliophoran representative species of shield, it is under the jurisdiction of shield Fine mesh, Infusoria are the small facultative parasitism infusorians of individual, have been acknowledged as the main shield fibre class of marine fish Pathogen.Water droplet puppet health fibre worm adaptability is extremely strong, can free living in various breeding water bodies, once because of residual bait in breeding water body Excessively, environmental degradation etc. causes content of organics excessively high, and water droplet puppet health fibre worm is by mass propagation and parasitizes each culture-cycle fish The fish body of body, especially body-surface rauma.The helminth is food with the cell or tissue fragment of host, and illness fish body is secondary when serious Bacteriosis leads to cultured fishes mortality.Thus when insect pest occurs, need to parasitize the insect of host body surface into Row identification, to be accurately administered according to variety classes.
However, being difficult under optical microscopy and scanning electron microscope since shield fibre class helminth living body form is quite similar It distinguishes (attached drawing 1,2,3,4), ribosomes sequence is measured even with 18S rDNA technology, because the sequence similarity of shield fibre class is opposite Higher (reaching 90% or more), so as to determine kind of difficulty larger for shield infusorian.
Summary of the invention
The present invention is to provide a kind of based on PCR identification water droplet to solve above-mentioned technical problem present in the prior art Molecular labeling, primer and the method for pseudo- health fibre worm.
The technical solution of the invention is as follows: a kind of molecular labeling of based on PCR identification water droplet puppet health fibre worm, feature exist In DNA sequence dna as shown in SEQ ID NO:1.
A kind of primer of based on PCR identification water droplet puppet health fibre worm, it is characterised in that the DNA sequence of upstream primer and downstream primer Column are respectively as shown in SEQ ID NO:2, SEQ ID NO:3.
A kind of method of based on PCR identification water droplet puppet health fibre worm, it is characterised in that successively carry out in accordance with the following steps:
A. illness fish body table shield infusorian parasitism lesion site tissue is taken to extract DNA；
B. PCR amplification is carried out according to following condition:
PCR reaction system is 40 μ L systems: 20 8 μ L of μ L, 2 mM dNTPs of KOD buffer, upstream primer and downstream are drawn Each 2 μ L of object, 2 μ L, KOD enzyme 0.8 μ L, 2 μ L of shield infusorian DNA, 5.2 μ L of sterile purified water；The trip primer and under The DNA sequence dna of trip primer is respectively as shown in SEQ ID NO:2, SEQ ID NO:3；
PCR reaction condition are as follows: 94 DEG C of 3 min of initial denaturation；98 DEG C of 15 s of denaturation, 56 DEG C of 30 s of annealing, 68 DEG C extend 45 S, 35 circulations；68 DEG C of 5 min of extension；
C. PCR product is examined with agarose gel electrophoresis, if there is the electrophoretic band of 498 bp, separated obtained shield cilium Worm is water droplet puppet health fibre worm.
It, only need to be into present invention determine that the molecular labeling of based on PCR identification water droplet puppet health fibre worm and devise identification primer PCR amplification of row, can accurately identify that shield is ciliophoran to represent cause of disease-water droplet puppet health fibre worm, have quickly, it is easy And the advantages that accurate.
Detailed description of the invention
Fig. 1 is that water droplet puppet health fibre worm observes result under an optical microscope.
Fig. 2 is that Uronema marinum observes result under an optical microscope.
Fig. 3 is water droplet puppet health fibre worm scanning electron microscopic observation result.
Fig. 4 is Uronema marinum scanning electron microscopic observation result.
Fig. 5 is the embodiment of the present invention using tested Fish tissue DNA as template progress PCR amplification result electrophoretogram.
Fig. 6 carries out PCR amplification result electrophoretogram by template of Uronema marinum DNA and water droplet puppet health fibre worm DNA.
Specific embodiment
The technical solution of the invention is as follows: a kind of molecular labeling of based on PCR identification water droplet puppet health fibre worm, is to pass through survey Determine water droplet puppet health fibre worm (Pseudocohnilembus persalinus) mitochondria full-length genome, analyze water droplet puppet health fibre worm Opposite conservative gene in kind, shield infusorian interspecific difference gene and determination, DNA sequence dna is as shown in SEQ ID NO:1.
With the primer of the molecular labeling design of identified based on PCR identification water droplet puppet health fibre worm, upstream primer and downstream The DNA sequence dna of primer is respectively as shown in SEQ ID NO:2, SEQ ID NO:3.
The method that based on PCR identifies water droplet puppet health fibre worm successively carries out in accordance with the following steps:
A. it is not less than 20mg with aseptic inoculation ring scraping illness fish body table shield infusorian parasitism lesion site tissue, it is raw with ocean Object tissue DNA extracts kit extracts tissue DNA；
B. PCR amplification is carried out according to following condition:
PCR reaction system is 40 μ L systems: 20 8 μ L of μ L, 2 mM dNTPs of KOD buffer, upstream primer and downstream are drawn Each 2 μ L of object, 2 μ L, KOD enzyme 0.8 μ L, 2 μ L of shield infusorian DNA, 5.2 μ L of sterile purified water；The trip primer and under The DNA sequence dna of trip primer is respectively as shown in SEQ ID NO:2, SEQ ID NO:3；
PCR reaction condition are as follows: 94 DEG C of 3 min of initial denaturation；98 DEG C of 15 s of denaturation, 56 DEG C of 30 s of annealing, 68 DEG C extend 45 S, 35 circulations；68 DEG C of 5 min of extension；
C. PCR product is examined with agarose gel electrophoresis, as a result as shown in Figure 5.
M:Marker in Fig. 5；There is the electrophoretic band of 498 bp in swimming lane 1.Pcr amplification product after sequencing company is sequenced, DNA sequence dna is as shown in SEQ ID NO:1.
Comparative example 1:
1. the Uronema marinum DNA and water droplet puppet health fibre worm DNA to be determined by morphology and 18S rDNA sequencing analysis respectively PCR amplification, PCR reaction system and the same embodiment of the present invention of PCR reaction condition are carried out for standard form；
2. by gained PCR product agarose gel electrophoresis, as a result as shown in Figure 6.
M:Marker in Fig. 6；Swimming lane 1: Uronema marinum DNA is template；Swimming lane 2: water droplet puppet health fibre worm DNA is template.
From fig. 6, it can be seen that the PCR product electrophoretic band that Uronema marinum DNA is template is sky, and water droplet puppet health fibre worm DNA is the pcr amplification product of template, obtains the clear band of 498 bp.Pcr amplification product is after sequencing company is sequenced, DNA sequence dna As shown in SEQ ID NO:1.
By contrast verification as can be seen that based on PCR determined by the present invention identification water droplet puppet health fibre worm molecular labeling, Primer and method can quickly, simply and accurately identify water droplet puppet health fibre worm.
Sequence table
<110>Dalian Ocean University
<120>molecular labeling, primer and the method for based on PCR identification water droplet puppet health fibre worm
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 498
<212> DNA
<213>artificial sequence (Pseudocohnilembus persalinus)
<400> 1
tgactcttca ctaaaaatgt gaggtcaaac tgatttaaaa aagttagttg cttatggtac 60
aatccaagaa atgaatttaa tttttttagt tttttgttga ggtgatacaa atgctatagt 120
tggtggtatt ctttttaccg caactcactc ttttttatca gcattaatgt tttttttagt 180
cgattgtatt tacagaaggt accatacaag atctttaata gaggttaatg gtattttaca 240
tatgactcca aatttaggta tagctatttt agtaatgaca gttttctttt cagggttacc 300
tggaactata aaatatatta cagaatttta tatctttagt ggtttcttag aaacttctgt 360
tttttcttgc ttttttttaa tgtttgttgc taatgttcta ggtttaatag gatttagtaa 420
atgttgattt aatgcagttt ttggtatgat gagaaaacat gttggtacta taccacttga 480
tttaacaatt aaggaatt 498
<210> 2
<211> 26
<212> DNA
<213>artificial sequence (Pseudocohnilembus persalinus)
<220>
<221> misc_feature
<222> (1)..(26)
<400> 2
gttatggtta caatgtttgg tgttat 26
<210> 3
<211> 26
<212> DNA
<213>artificial sequence (Pseudocohnilembus persalinus)
<220>
<221> misc_feature
<222> (1)..(26)
<400> 3
tatagtacca acatgttttc tcatca 26

Claims (3)

1. a kind of molecular labeling of based on PCR identification water droplet puppet health fibre worm, it is characterised in that DNA sequence dna such as SEQ ID NO:1 institute Show.
2. a kind of primer of based on PCR identification water droplet puppet health fibre worm, it is characterised in that the DNA sequence of upstream primer and downstream primer Column are respectively as shown in SEQ ID NO:2, SEQ ID NO:3.
3. a kind of method of based on PCR identification water droplet puppet health fibre worm, it is characterised in that successively carry out in accordance with the following steps:
A. illness fish body table shield infusorian parasitism lesion site tissue is taken to extract DNA；
B. PCR amplification is carried out according to following condition:
PCR reaction system is 40 μ L systems: 20 8 μ L of μ L, 2 mM dNTPs of KOD buffer, upstream primer and downstream are drawn Each 2 μ L of object, 2 μ L, KOD enzyme 0.8 μ L, 2 μ L of shield infusorian DNA, 5.2 μ L of sterile purified water；The trip primer and under The DNA sequence dna of trip primer is respectively as shown in SEQ ID NO:2, SEQ ID NO:3；
PCR reaction condition are as follows: 94 DEG C of 3 min of initial denaturation；98 DEG C of 15 s of denaturation, 56 DEG C of 30 s of annealing, 68 DEG C extend 45 S, 35 circulations；68 DEG C of 5 min of extension；
C. PCR product is examined with agarose gel electrophoresis, if there is the electrophoretic band of 498 bp, separated obtained shield cilium Worm is water droplet puppet health fibre worm.
CN201910835831.3A 2019-09-05 2019-09-05 Molecular marker, primer and method for identifying pseudocercospora fusca based on PCR Active CN110396552B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117025810A (en) * 2023-07-04 2023-11-10 福建师范大学 Internal reference gene combination for expression analysis of pseudokanbrix drop genes and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140014042A (en) * 2013-12-23 2014-02-05 제주대학교 산학협력단 A primer for scuticociliate and a method for detecting scuticociliate using the same
CN104372069A (en) * 2013-08-12 2015-02-25 中国科学院海洋研究所 Specific probe capable of rapidly detecting pseudocohnilembus persalinus, detection method and applications thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104372069A (en) * 2013-08-12 2015-02-25 中国科学院海洋研究所 Specific probe capable of rapidly detecting pseudocohnilembus persalinus, detection method and applications thereof
KR20140014042A (en) * 2013-12-23 2014-02-05 제주대학교 산학협력단 A primer for scuticociliate and a method for detecting scuticociliate using the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ILSON WHANG等: "Identification of scuticociliates (Pseudocohnilembus persalinus, P. longisetus, Uronema marinum and Miamiensis avidus) based on the cox1 sequence", 《PARASITOL INT》 *
林能锋等: "水滴伪康纤虫全长cDNA文库的构建及EST分析", 《福建农业学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117025810A (en) * 2023-07-04 2023-11-10 福建师范大学 Internal reference gene combination for expression analysis of pseudokanbrix drop genes and application thereof
CN117025810B (en) * 2023-07-04 2024-03-26 福建师范大学 Internal reference gene combination for expression analysis of pseudokanbrix drop genes and application thereof

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