CN109161613A - A kind of MLPA detection kit detecting pig virus diarrhoea syndrome etiology nucleic acid - Google Patents
A kind of MLPA detection kit detecting pig virus diarrhoea syndrome etiology nucleic acid Download PDFInfo
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- 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
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- 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]
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- C12Q2600/16—Primer sets for multiplex assays
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Abstract
The present invention relates to inspection and quarantine fields, it is desirable to provide a kind of MLPA detection kit for detecting pig virus diarrhoea syndrome etiology nucleic acid.Include: (1) reverse transcription and pre- amplification mixed liquor in the kit: the sequence of reverse transcription and pre- amplimer is as shown in NO:1~12 SEQ ID；Wherein, reverse transcription primer is 12 base random primers；(2) multiple linking probe mixed liquor: where the sequence of probe is as shown in NO:13~24 SEQ ID, and the sequence of universal primer is as shown in NO:25~26 SEQ ID；(3) MLPA buffer；(4) buffer solution A and B are connected；(5) ligase Ligase-65；(6) PCR reaction mixture comprising universal primer shown in NO:25~26 sequence table SEQ ID；(7) SALSA polymerase；(8) negative control；(9) positive control, the positive control plasmid of the cause of disease comprising 6 kinds of pig virus diarrhoea syndromes.Compared with existing method, the present invention has the advantages that high throughput, saves money, specific good, high sensitivity.
Description
Technical field
It is used for the present invention provides one kind while detecting Porcine epidemic diarrhea virus (PEDV), transmissible gastro-enteritis virus
(TGEV), porcine rotavirus (RV), pig delta coronavirus (PDCoV), pig bocavirus (PBoV) and pig norovirus
(NoV) 6 multiple linking probe amplification identifies detection kit, it can be achieved that detecting 6 kinds simultaneously causes pig virus diarrhoea disease
The cause of disease of group, belongs to inspection and quarantine field.
Background technique
Pig virus diarrhoea is to endanger the most important disease of China's pig breeding industry at present, and pig virus diarrhoea can endanger any year
Age pig, lactation and the weanling pig especially mostly occurred at annual December to next year 1~2 month, morbidity and mortality
It is all higher, and fall ill and propagate rapidly, it is relatively difficult to control, greatly endangers the swinery safety of scale livestock farming, give
Pig breeding industry brings heavy losses [1].Once infecting the disease, piglet often shows watery diarrhea, dehydration until dead, disease incidence and
The death rate is very high, often brings crushing blow to farm.The encountered pathogenic of pig virus diarrhoea syndrome are as follows: pig is popular
Diarrhea virus (porcine epidemic diarrhea virus, PEDV), transmissible gastro-enteritis virus
(transmissible gastroenteritis virus of swine, TGEV), porcine rotavirus (porcine
Rotavirus, RV), pig delta coronavirus (porcine delta coronavirus, PDCoV), pig bocavirus
Six kinds of viruses such as (porcine bocavirus, PBoV), pig norovirus (swine norwalk viruses, NoV) cause
's.Rapidly due to syndrome morbidity, it needs to determine pathogenesis in the short time and prevent and treat, at present still without one kind
Method disposably can carry out antidiastole to a variety of disease pathogens for causing pig virus diarrhoea.
Therefore a kind of detection method that can precisely, quickly, efficiently distinguish Different Kinds of Pathogens is established, for pig pig virus diarrhoea
Prevention and occur it is most important.
Summary of the invention
The technical problem to be solved by the present invention is to overcome deficiency in the prior art, provide a kind of detection pig virus abdomen
Rush down the MLPA detection kit of syndrome etiology nucleic acid.The kit provides for clinic can detect Porcine Epidemic Diarrhea simultaneously
Malicious (PEDV), transmissible gastro-enteritis virus (TGEV), porcine rotavirus (RV), pig delta coronavirus (PDCoV), pig are rich
Block the detection means of virus (PBoV) and pig norovirus (NoV).
In order to solve the above technical problems, the invention adopts the following technical scheme:
A kind of MLPA detection kit detecting pig virus diarrhoea syndrome etiology nucleic acid is provided, is wrapped in the kit
It includes:
(1) reverse transcription and pre- amplification mixed liquor:
The mixed liquor includes reverse transcription and pre- amplimer, and sequence (is shown in Table 1) as shown in NO:1~12 SEQ ID, point
It Yong Yu not reverse transcription and pre- amplification；Wherein, reverse transcription primer is 12 base random primers；
(2) multiple linking probe mixed liquor:
The mixed liquor includes probe and universal primer；Wherein, the sequence of probe (is shown in Table as shown in NO:13~24 SEQ ID
2), the sequence of universal primer (is shown in Table 3) as shown in NO:25~26 SEQ ID；
(3) MLPA buffer；
(4) buffer solution A and B are connected；
(5) ligase Ligase-65；
(6) PCR reaction mixture comprising universal primer shown in NO:25~26 sequence table SEQ ID；
(7) SALSA polymerase；
(8) negative control；
(9) positive control includes Porcine epidemic diarrhea virus (PEDV), transmissible gastro-enteritis virus (TGEV), pig wheel
Shape virus (RV), pig delta coronavirus (PDCoV), pig bocavirus (PBocV) and pig norovirus (NV) positive control matter
Grain.
In the present invention, the sequence SEQ ID NO:1-2 is the pre- amplimer of pig bocavirus；The sequence SEQ ID
NO:3-4 is the pre- amplimer of pig norovirus；The sequence SEQ ID NO:5-6 is that pig delta coronavirus expands draw in advance
Object；The sequence SEQ ID NO:7-8 is the pre- amplimer of Porcine epidemic diarrhea virus respectively；The sequence SEQ ID NO:9-
10 be the pre- amplimer of porcine rotavirus；The sequence SEQ ID NO:11-12 is that transmissible gastro-enteritis virus expands draw in advance
Object；The sequence SEQ ID NO:13-14 is the left side probe and right side probe for detecting pig bocavirus；The sequence SEQ ID
NO:15-16 is respectively the left side probe and right side probe for detecting pig norovirus；The sequence SEQ ID NO:17-18 difference
For the left side probe and right side probe for detecting pig delta coronavirus；The sequence SEQ ID NO:19-20 is respectively to detect pig
The left side probe and right side probe of epidemic diarrhea virus；The sequence SEQ ID NO:21-22 is respectively to detect pig colyliform disease
The left side probe and right side probe of poison；The sequence SEQ ID NO:23-24 is respectively to detect transmissible gastro-enteritis virus
Left side probe and right side probe；Wherein, 5 ' ends of SEQ ID NO:14,16,18,20,22,24 carry out phosphatizing treatment；
Sequence SEQ ID NO:25,26 be respectively general PCR amplification forward and reverse primer.
In the present invention, positive control in kit from viral nucleic acid RNA after reverse transcription, gene-specific fragments with
Plasmid connects resulting recombinant plasmid mixture；Wherein specific gene be respectively Porcine epidemic diarrhea virus (PEDV) S gene,
The N gene of transmissible gastro-enteritis virus (TGEV), the NSP1 gene of porcine rotavirus (RV), pig delta coronavirus
(PDCoV) the NS1 gene of N gene, pig bocavirus (PBoV) and the RdRp gene of pig norovirus (NV).
Invention further provides detect 6 kinds of pig virus diarrhoea syndrome etiology nucleic acids simultaneously using aforementioned agents box
MLPA detect method for testing, comprising the following steps:
(1) paramagnetic particle method extracts sample DNA/RNA；
Be total to extracts kit and Full automatic instrument for extracting nucleic acid using magnetic bead DNA/RNA, at the same extract the DNA in sample and
RNA obtains 200 μ L samples；
(2) RNA reverse transcription is at cDNA and pre- amplification
Carry out one-step method reverse transcription RT-PCR reaction；
Prepare 25 μ L reaction systems: 10 μ L OneStep Ahead RT-PCR Master Mix, 1 μ L OneStep
It is (every final concentration of that Ahead RT-Mix, 5 μ L DNA or RNA, the random reverse transcription primer of 1 μ L and 5 μ L expand primer mixed liquor in advance
0.5 μM of primer), 3 μ L H2O is supplied；
Reaction condition: 50 DEG C of 10min, 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 20s, 72 DEG C of 20s, 40 circulations；72
℃ 2min；
(3) MLPA amplification and detection
A) DNA is denaturalized
0.2mL PCR reaction tube is taken, 0.5 μ L DNA solution is added in every pipe and 4.5 μ L TE, 98 DEG C of denaturation 5min are down to room
25 DEG C of temperature；
B) probe and sample DNA hybridize
Prepare the probe mixed liquor of 3 μ L mixing :+1.5 μ L probe mixed liquor of 1.5 μ L MLPA buffer；
Probe mixture is added in above-mentioned PCR pipe, 95 DEG C of incubation 1min, 60 DEG C of hybridization 1-16h, 54 DEG C incubate；
C) connection of hybridization probe
Prepare 32 μ L connection enzymatic mixtures :+1 μ L connection of 25+3 μ L connection buffer solution B of μ L dH2O+3 μ L connection buffer solution A
Enzyme Ligase-65；
PCR instrument temperature is down to 54 DEG C, opens pipe lid, 32 μ L connection enzymatic mixtures is added, 54 DEG C of incubation 15min, 98 DEG C add
Hot 5min inactivates ligase, 20 DEG C of incubations；
D) PCR amplification of linking probe
Prepare 10 μ L PCR mixed liquors: 7.5 μ L dH2+ 0.5 μ L SALSA polymerase of O+2 μ L PCR reaction mixture；
PCR pipe is taken out, 10 μ L PCR mixtures are added at room temperature；Start PCR reaction, reaction condition are as follows: 95 DEG C of 30s, 60
DEG C 30s, 72 DEG C of 60s, 35 circulations；72 DEG C of incubation 20min, are down to 15 DEG C；
E) full-automatic nucleic acids instrument analysis:
Pcr amplification product takes 20 μ L, is analyzed with full-automatic nucleic acids instrument；
(4) result description and judgement
A) quality control standard:
Positive control has specific amplification band at 102bp, 110bp, 117bp, 124bp, 131bp, 138bp；
Negative control is without specific amplification band；
If negative control and positive condition are unsatisfactory for conditions above, this time test is considered as invalid；
B) result judges:
It is positive: to have specific amplification band at 102bp, indicate that there are pig bocavirus in sample；There is spy at 110bp
Specific amplification band indicates that there are pig norovirus in sample；There is specific amplification band at 117bp, indicates to deposit in sample
In pig delta coronavirus；There is specific amplification band at 124bp, indicates that there are Porcine epidemic diarrhea virus in sample；?
There is specific amplification band at 131bp, indicates that there are porcine rotavirus in sample；There are specific amplification band, table at 138bp
There are transmissible gastro-enteritis virus in sample sheet；MLPA amplified production can be sequenced, be further confirmed that simultaneously；
It is negative: without specific amplification band, to show in sample without Porcine epidemic diarrhea virus, transmissible gastroenteritis of swine disease
Poison, porcine rotavirus, pig delta coronavirus, pig bocavirus, pig norovirus.
Compared with existing method, the present invention has the advantage that and effect:
1) high-throughput.One reaction can detect 6 kinds of cause of diseases simultaneously, using full-automatic nucleic acids instrument, can examine simultaneously every time
96 samples are surveyed, provide high-throughput detection technique for the antidiastole of pathogen and emergency diagnosis.
2) it saves money.A usual sample detects 6 kinds of disease pathogen nucleic acid using fluorescent quantitation method, and expense is usually
200 × 6=1200 member, and this kit is in a pipe due to being reacted, about 400 yuan of expense or so.
3) specificity is good.It is analyzed by sequence alignment and blast, reinforces probe design, it is ensured that probe and target gene
It combines.
4) high sensitivity.Common MLPA method at least needs the target dna of 6000 copies.The present invention passes through increase
The step for RT-PCR, is enriched with target gene, the minimum target gene for detecting 10 copies or so.
Detailed description of the invention
Fig. 1 is the positive control construction of recombinant plasmid PCR verifying of 6 kinds of diseases；
In figure, M is DL2000DNA Marker；1 is PDCoV-N, and 2 be TGEV-N, and 3 be RV-NSP1, and 4 be PEDV, and 5 are
PBoV, 6 be NoV.
Fig. 2 .1 to 2.6 is the peak figure and simulation electrophoretogram of the specificity verification of pig virus diarrhoea syndrome MLPA method
(single template)；
TGEV, RV, PEDV, PDCoV, NoV, PBoV the gene masculine plasmid built is shown as in figure, it is single respectively to add
Enter in the system with 6 pairs of pre- amplimers and 6 pairs of probes, the result detected according to MLPA response procedures.
The peak figure and simulation electrophoretogram (hybrid guided mode of the specificity verification of Fig. 3 pig virus diarrhoea syndrome MLPA method
Plate).
TGEV-N, RV-Nsp1, PEDV-S, PDCoV-N, NoV-RdRp, PBoV-NS1 gene built is shown as in figure
Positive plasmid is added in the system with 6 pairs of pre- amplimers and 6 pairs of probes together, is detected according to MLPA response procedures
Result.Pig bocavirus has specific amplification band in 102bp；Pig norovirus has specific amplification band in 110bp；Pig
Delta coronavirus has specific amplification band in 117bp；Porcine epidemic diarrhea virus has specific amplification band in 124bp；
Porcine rotavirus has specific amplification band in 131bp；Transmissible gastro-enteritis virus has specific amplification band in 138bp.
Specific embodiment
Multiplex ligation-dependent probe amplification (multiplex ligation-dependent probe
Amplification, MLPA) it is a kind of new skill high-throughput, that qualitative and quantitative analysis is carried out for target sequence in determined nucleic acid
Art.The technology combines the hybridization check of nucleic acid and PCR chain amplification, thus realize the efficient specificity analysis of target molecule,
Detection and quantitative analysis are carried out to 45 different target genes in same reaction tube.The basic principle of MLPA includes probe and target
Sequence DNA is hybridized, and later by connection, PCR amplification, product passes through capillary electrophoresis separation and data collection, DNA analysis
Software analyze to the data of collection and finally be drawn a conclusion.Each MLPA probe include two oligonucleotide fragments, one by
Chemical synthesis, one is prepared by the phage-derived method of M13；Each probe includes one section of primer sequence and one section of specific sequence
Column.In MLPA reaction, two oligonucleotide fragments are all hybridized with target sequence, connect two parts spy using ligase later
Needle.Connection reaction height is special, and only when two probes hybridize completely with target sequence, i.e. target sequence and probe-specific sequence is complete
Complete complementary, two sections of probes could be connected into a complete single nucleic acid strands by ligase；, whereas if target sequence and probe sequence
It is not fully complementary, it even if the difference of only one base, will lead to hybridization not exclusively, carry out connection reaction can not.Connection
After the reaction was completed, expand the probe that connects with a pair of of universal primer, the length of the amplified production of each pair of probe be all it is unique,
Range is in 90~480bp.Finally, by capillary electrophoresis separation amplified production, software analysis, it was therefore concluded that.
While the present invention is constructed based on MLPA technology detect 6 kinds of pig virus diarrhoea syndromes detection method, be
The disease of such symptom provides the high-throughput diagnostic technology of high specificity, high sensitivity.
The present invention passes through to improve the detection sensitivity of MLPA according to the distinguished sequence on the GenBank of 6 kinds of diseases
Blast analysis, each disease devise a pair of pre- amplimer (sequence is shown in Table 1), these sequences include the sequence of MLPA probe
Column, by synthesis and pre- amplification based on one-step method cDNA, then operating process according to the invention carries out MLPA, probe
(sequence is shown in Table 2) carries out PCR amplification after denaturation, hybridization, connection, using universal primer (sequence is shown in Table 3), then carries out capillary
Electrophoresis tube detection.
16 kinds of pig virus diarrhoea multiplex ligation-dependent probe amplifications (MLPA) of table expand primer sequence (5 ' -3 ') in advance
26 kinds of pig virus diarrhoea multiplex ligation-dependent probe amplification (MLPA) probe sequences (5 ' -3 ') of table
Wherein, above-mentioned SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID
5 ' the ends of NO:22 and SEQ ID NO:24 carry out phosphatizing treatment；
3 universal primer sequence of table
The present invention establishes while detecting Porcine epidemic diarrhea virus, pig transmissible using multiplex ligation-dependent probe amplification
The multiple linking probe expansion of marcy agent, porcine rotavirus, pig delta coronavirus, pig bocavirus and pig norovirus 6
Increase detection method and assemble kit, this reagent constituents composition is as follows:
(1) reverse transcription and pre- amplification primer mixed liquor, including Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus,
Porcine rotavirus, pig delta coronavirus, pig bocavirus and pig norovirus pre- amplimer, the primer sequence is shown in
Table 1, the concentration of every primer is 2.5 μM in mixed liquor；
(2) probe mixed liquor comprising detection Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pig colyliform disease
Poison, pig delta coronavirus, pig bocavirus and pig norovirus left and right side probe, the sequence of the probe is shown in Table
2, wherein 5 ' ends of SEQ ID NO:14,16,18,20,22,24 carry out phosphatizing treatment；Every kind of probe in mixed liquor
Concentration is 1.33nM；
(3) MLPA buffer；
(4) buffer solution A is connected；
(5) buffer solution B is connected；
(6) ligase Ligase-65；
(7) PCR reaction mixture comprising sequence table SEQ ID NO:25, universal primer shown in 26；
(8) SALSA polymerase；
(9) negative control: TE；
(10) positive control: for TGEV-N, RV-Nsp1, PEDV-S, PDCoV-N, NoV-RdRp, PBoV-NS1 gene sun
The mixture of property Plasmid DNA.
The kit 6 kinds of pig virus diarrhoea diseases of detection simultaneously are invented to using one's duty combined with specific embodiments below
The MLPA detection method of group's etiology nucleic acid is described in detail.
One, the operation instruction of kit
The composition of 1 kit
Wherein, MLPA buffer, connection buffer solution A, connection buffer solution B, ligase Ligase-65 and SALSA polymerase
Purchased from MRC-Holland company.
Condition of storage:
In addition to positive control, other components are stored in -15 DEG C to -25 DEG C.Positive control is stored in -80 DEG C.
In order to guarantee experiment effect, kit should be within 1 year using finishing.
2 detection (use) methods
2.1 paramagnetic particle methods extract sample DNA/RNA；
It is total to extracts kit (article No.: DP422) with the magnetic bead DNA/RNA of TIANGEN company, it is complete certainly using the grand NP968 in day
Dynamic instrument for extracting nucleic acid extracts DNA and RNA in sample simultaneously, obtains 200 μ L samples.
2.2RNA reverse transcription is at cDNA and pre- amplification
One-step method reverse transcription RT-PCR reaction is carried out according to QIAGEN OneStep Ahead RT-PCR Kit specification.
Prepare 25 μ L reaction systems: 10 μ L OneStep Ahead RT-PCR Master Mix, 1 μ L OneStep Ahead RT-
Mix, 5 μ L DNA or RNA, the random reverse transcription primer of 1 μ L and 5 μ L expand primer mixed liquor (final concentration of 0.5 μ of every primer in advance
M), 3 μ L H2O are supplied.Reaction condition: 50 DEG C of 10min, 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 20s, 72 DEG C of 20s, 40 circulations；
72℃2min。
2.3MLPA amplification and detection
2.3.1DNA denaturation
0.2mL PCR reaction tube is taken, 0.5 μ L DNA solution is added in every pipe and 4.5 μ L TE, 98 DEG C of denaturation 5min are down to room
25 DEG C of temperature.
2.3.2 probe and sample DNA hybridize
Prepare the probe mixed liquor of 3 μ L mixing :+1.5 μ L probe mixed liquor of 1.5 μ L MLPA buffer.By probe mixture
It is added in above-mentioned PCR pipe, 95 DEG C of incubation 1min, 60 DEG C of hybridization 1-16h, 54 DEG C incubate.
2.3.3 the connection of hybridization probe
Prepare 32 μ L connection enzymatic mixtures :+1 μ L connection of 25+3 μ L connection buffer solution B of μ L dH2O+3 μ L connection buffer solution A
Enzyme Ligase-65.PCR instrument temperature is down to 54 DEG C, opens pipe lid, is added 32 μ L connection enzymatic mixtures, 54 DEG C of incubation 15min, and 98
DEG C heating 5min inactivate ligase, 20 DEG C incubation.
2.3.4 the PCR amplification of linking probe
Prepare 10 μ L PCR mixed liquors :+0.5 μ L SALSA polymerase of 7.5 μ L dH2O+2 μ L PCR reaction mixture.It takes
10 μ L PCR mixtures are added in PCR pipe out at room temperature.Start PCR reaction, reaction condition are as follows: 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C
60s, 35 circulations；72 DEG C of incubation 20min, are down to 15 DEG C.
2.3.5 full-automatic nucleic acids instrument analysis:
Pcr amplification product takes 20 μ L, is analyzed with full-automatic nucleic acids instrument (Qsep100DNA Analyzer).
The description of 2.4 results and judgement
2.4.1 quality control standard:
Positive control has specific amplification band at 102bp, 110bp, 117bp, 124bp, 131bp, 138bp.
Negative control is without specific amplification band.
If negative control and positive condition are unsatisfactory for conditions above, this time test is considered as invalid.
2.4.2 result judges:
It is positive: to have specific amplification band at 102bp, indicate that there are pig bocavirus in sample；There is spy at 110bp
Specific amplification band indicates that there are pig norovirus in sample.There is specific amplification band at 117bp, indicates to deposit in sample
In pig delta coronavirus；There is specific amplification band at 124bp, indicates that there are Porcine epidemic diarrhea virus in sample；?
There is specific amplification band at 131bp, indicates that there are porcine rotavirus in sample；There are specific amplification band, table at 138bp
There are transmissible gastro-enteritis virus in sample sheet.MLPA amplified production can be sequenced, be further confirmed that simultaneously.
It is negative: without specific amplification band, to show in sample without Porcine epidemic diarrhea virus, transmissible gastroenteritis of swine disease
Poison, porcine rotavirus, pig delta coronavirus, pig bocavirus, pig norovirus.
Points for attention:
1) DNA sample:
A. the content of salt ion, alcohols etc. in DNA solution is reduced as far as possible.
B. with TE rather than water dissolution or dilution DNA, depurination when avoiding DNA high temperature.
C. by all DNA sample concentration dilutions to roughly equal (it is recommended that 20-40ng/ul) before experiment.
2) probe, ligase Ligase-65, connection buffer solution A should be dispensed before use, avoid multigelation；
3) mixing centrifugation should be shaken before buffer and reagent use, when mixing gently blows and beats mixing with pipette tips, pays attention to avoiding
It generates bubble or will get on tube wall, all steps containing enzyme can not be centrifuged；
4) when plus connecting enzyme reaction, PCR pipe will be placed in PCR instrument, be sure not to take out；
5) evaporate Quality Control: tube bottom at least 5 μ L of residue are completed in the connection of 8 μ L TE/ water blank, connection.
6) full-automatic nucleic acids instrument setting: optimization injection voltage and time, PCR product can be on after diluteds
Sample, so that signal is in optimized analysis range.
Two, the specificity of kit
1 material
The used virus of this test is shown in Table 4 with bacterium.
4, table tests used virus and bacterium
Note: virus described in table 4 or bacterial strain, are existing well-known technique, and those skilled in the art can pass through commercially available mode
Voluntarily obtain.The applicant promises to undertake from the application submits in 20 years, provides above-mentioned virus or bacterial strain sample to the public
This.
2 methods
2.1 use Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, porcine rotavirus, the coronal disease of pig delta respectively
Poison, pig bocavirus, pig norovirus six kinds of single probes of cause of disease be directed to common swine disease cause of disease such as pig circular ring virus respectively
2b type, 2 porcine circovirus d type, pig japanese encephalitis virus, porcine pseudorabies virus, pig parvoviral, pig breeding and breathing trace integration
It levies virus, swine fever virus, C.perfringens and actinobacillus pleuropneumoniae and carries out MLPA detection, verify the special of probe
Property.
2.2 carry out MLPA detection to all viral and bacteriums in table 4 respectively with the mixed probe of six kinds of cause of diseases.
3 results
3.1 are detected with any one group of probe of design, can only be amplified from corresponding viral template correspondingly sized
Band, and to 2 porcine circovirus b type, 2 porcine circovirus d type, pig japanese encephalitis virus, porcine pseudorabies virus, the tiny disease of pig
Poison, pig breeding are with respiratory syndrome virus, swine fever virus, C.perfringens and actinobacillus pleuropneumoniae without amplification
Signal.Illustrate that probe specificity is good.
3.2 probes mixed with six kinds of cause of diseases carry out MLPA detection, Zhi Nengcong to all viral and bacteriums in table 4 respectively
Band of corresponding size is amplified in corresponding viral template, and to 2 porcine circovirus b type, 2 porcine circovirus d type, pig Japan
Encephalitis viruses, porcine pseudorabies virus, pig parvoviral, pig breeding and respiratory syndrome virus, swine fever virus, perfringens shuttle
Bacterium and actinobacillus pleuropneumoniae are without amplified signal.Show that established method specificity is good.
Three, the sensitivity of kit
1 material
It is shown in Table 4.
2 methods
The building of 2.1 plasmids
By TGEV-N, RV-Nsp1, PEDV-S, PDCoV-N, NoV-RdRp, PBoV-NS1 genetic fragment of amplification with
The connection of pZERO-blunt carrier, Transformed E scherichia coli DH5a extract recombinant plasmid, and carry out PCR verifying and survey
Sequence verifying.
2.2 sensitivity verifying
With the light absorption value A260 and A280 of ultraviolet specrophotometer measurement recombinant plasmid, the copy of recombinant plasmid is calculated
Number, is diluted to 10 for positive recombinant plasmid with EASY Dilution reagent10Copy μ L- 1, then 10 times of gradient dilutions are carried out, with
108, 107, 106, 105, 104, 103, 102, 101, 100Copy μ L- 19 copy number gradients carry out MLPA as standard form
Reaction.Plasmid mass concentration (gL- 1)=λ (A260) × 50 (gL- 1)/1 000 × quasi-mode plate releases extension rate.Copy number
Calculation formula: copy number (copy μ L- 1)=plasmid concentration (g μ L- 1) × Avgadro constant/recombinant plasmid molecular weight.
3 results
The minimum Porcine epidemic diarrhea virus that 12 copies can be detected of this method, 8 copy transmissible gastroenteritis of swine diseases
Poison, 24 copy porcine rotavirus, 5 copy pig delta coronavirus, 18 copy pig bocavirus, 4 copy pig promises are such as
Virus.
Sequence table
<110>Zhejiang A & F University
<120>a kind of MLPA detection kit for detecting pig virus diarrhoea syndrome etiology nucleic acid
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
gtctcggtgc agctgcg 17
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
gtagctgtgt tctgtctagc cagc 24
<210> 3
<211> 16
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
gccgtgttgg cagcag 16
<210> 4
<211> 28
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
tcatcatcac catagaagga gaagaggg 28
<210> 5
<211> 29
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 5
ctcaaaacaa aaaggctact catcctcag 29
<210> 6
<211> 21
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 6
gattgttggg gttgcgtttg g 21
<210> 7
<211> 34
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 7
ttgcaatctg ttaatgatta cctgtctttt agca 34
<210> 8
<211> 32
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 8
tgtccagaat cagatgtata ataaacacct gc 32
<210> 9
<211> 24
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 9
atgggaaata ggcaatccag cttg 24
<210> 10
<211> 50
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 10
gaaatggatt attgtattta ataagttcca attttagagg ttttctagac 50
<210> 11
<211> 19
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 11
atggccaacc agggacaac 19
<210> 12
<211> 27
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 12
tctttaaatt tggcatctgc atgaggt 27
<210> 13
<211> 49
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 13
gggttcccta agggttggac ctggctgggt ccagatatct gcactccga 49
<210> 14
<211> 53
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 14
gcgtagctca gtttgagtcc aacaagacct tctagattgg atcttgctgg cac 53
<210> 15
<211> 53
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 15
gggttcccta agggttggat caatgagggt cttccctctg gggtgccctg cgc 53
<210> 16
<211> 57
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 16
ctcccaatgg aactccatcg cccactggct cctctctaga ttggatcttg ctggcac 57
<210> 17
<211> 57
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 17
gggttcccta agggttggac ttaactccgc catcaaaccc gttgaaaacc atggcta 57
<210> 18
<211> 60
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 18
ctggctgcgt tacaccagac aaaagccagg tggcacttct agattggatc ttgctggcac 60
<210> 19
<211> 61
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 19
gggttcccta agggttggat ttcaattcac aaagggtgag ttgattactg gcacgcctaa 60
a 61
<210> 20
<211> 63
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 20
ccacttgaag gtgttacgga cgtttctttt atgactctgg tctagattgg atcttgctgg 60
cac 63
<210> 21
<211> 63
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 21
gggttcccta agggttggac ttccttcaaa gtgcatcttc tgctgaattc aaaaactacc 60
atc 63
<210> 22
<211> 68
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 22
taataattac tatcggcgct gccttgattg ctttgctatt cgctttctag attggatctt 60
gctggcac 68
<210> 23
<211> 83
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 23
gggttcccta agggttggat ggccaaccag ggacaacgtg tcagttgggg agatgaatct 60
accaaaacac gtggtcgttc caa 83
<210> 24
<211> 55
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 24
ttcccgtggt cggaagaata ataacatacc tctctagatt ggatcttgct ggcac 55
<210> 25
<211> 19
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 25
gggttcccta agggttgga 19
<210> 26
<211> 23
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 26
gtgccagcaa gatccaatct aga 23
Claims (4)
1. a kind of MLPA detection kit for detecting pig virus diarrhoea syndrome etiology nucleic acid, which is characterized in that the kit
In include:
(1) reverse transcription and pre- amplification mixed liquor:
The mixed liquor includes reverse transcription and pre- amplimer, and sequence is respectively used to reverse transcription as shown in NO:1~12 SEQ ID
With pre- amplification；Wherein, reverse transcription primer is 12 base random primers；
(2) multiple linking probe mixed liquor:
The mixed liquor includes probe and universal primer；Wherein, the sequence of probe is as shown in NO:13~24 SEQ ID, universal primer
Sequence as shown in NO:25~26 SEQ ID；
(3) MLPA buffer；
(4) buffer solution A and B are connected；
(5) ligase Ligase-65；
(6) PCR reaction mixture comprising universal primer shown in NO:25~26 sequence table SEQ ID；
(7) SALSA polymerase；
(8) negative control；
(9) positive control includes Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, porcine rotavirus, pig delta hat
Shape virus, pig bocavirus and pig norovirus positive control plasmid.
2. kit according to claim 1, which is characterized in that the sequence SEQ ID NO:1-2 is pig bocavirus
Pre- amplimer；The sequence SEQ ID NO:3-4 is the pre- amplimer of pig norovirus；The sequence SEQ ID NO:5-6
It is the pre- amplimer of pig delta coronavirus；The sequence SEQ ID NO:7-8 is that Porcine epidemic diarrhea virus expands in advance respectively
Primer；The sequence SEQ ID NO:9-10 is the pre- amplimer of porcine rotavirus；The sequence SEQ ID NO:11-12 is pig
The pre- amplimer of infectious gastroenteritis virus；The sequence SEQ ID NO:13-14 is the left side probe for detecting pig bocavirus
With right side probe；The sequence SEQ ID NO:15-16 is respectively the left side probe and right side probe for detecting pig norovirus；Institute
Stating sequence SEQ ID NO:17-18 is respectively the left side probe and right side probe for detecting pig delta coronavirus；The sequence
SEQ ID NO:19-20 is respectively the left side probe and right side probe for detecting Porcine epidemic diarrhea virus；The sequence SEQ ID
NO:21-22 is respectively the left side probe and right side probe for detecting porcine rotavirus；The sequence SEQ ID NO:23-24 difference
For the left side probe and right side probe for detecting transmissible gastro-enteritis virus；Wherein, SEQ ID NO:14,16,18,20,
22,24 5 ' ends carry out phosphatizing treatment；Sequence SEQ ID NO:25,26 are the forward direction of general PCR amplification respectively and anti-
To primer.
3. kit according to claim 1, which is characterized in that the positive control in kit comes from viral nucleic acid RNA
After reverse transcription, gene-specific fragments connect resulting recombinant plasmid mixture with plasmid；Wherein specific gene is respectively pig
The S gene of epidemic diarrhea virus, the N gene of transmissible gastro-enteritis virus, porcine rotavirus NSP1 gene, pig delta
The RdRp gene of the N gene of coronavirus, the NS1 gene of pig bocavirus and pig norovirus.
4. the MLPA for being detected 6 kinds of pig virus diarrhoea syndrome etiology nucleic acids simultaneously using kit described in claim 1 is expanded
Detect method for testing, which comprises the following steps:
(1) paramagnetic particle method extracts sample DNA/RNA；
It is total to extracts kit and Full automatic instrument for extracting nucleic acid using magnetic bead DNA/RNA, while extracting the DNA in sample and RNA, is obtained
To 200 μ L samples；
(2) RNA reverse transcription is at cDNA and pre- amplification
Carry out one-step method reverse transcription RT-PCR reaction；
Prepare 25 μ L reaction systems: 10 μ L OneStep Ahead RT-PCR Master Mix, 1 μ L OneStep Ahead
RT-Mix, 5 μ L DNA or RNA, the random reverse transcription primer of 1 μ L and 5 μ L expand primer mixed liquor (final concentration of every primer in advance
0.5 μM), 3 μ L H2O is supplied；
Reaction condition: 50 DEG C of 10min, 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 20s, 72 DEG C of 20s, 40 circulations；72℃2min；
(3) MLPA amplification and detection
A) DNA is denaturalized
0.2mL PCR reaction tube is taken, 0.5 μ L DNA solution is added in every pipe and 4.5 μ L TE, 98 DEG C of denaturation 5min are down to room temperature 25
℃；
B) probe and sample DNA hybridize
Prepare the probe mixed liquor of 3 μ L mixing :+1.5 μ L probe mixed liquor of 1.5 μ L MLPA buffer；
Probe mixture is added in above-mentioned PCR pipe, 95 DEG C of incubation 1min, 60 DEG C of hybridization 1-16h, 54 DEG C incubate；
C) connection of hybridization probe
Prepare 32 μ L connection enzymatic mixtures :+1 μ L ligase of 25+3 μ L connection buffer solution B of μ L dH2O+3 μ L connection buffer solution A
Ligase-65；
PCR instrument temperature is down to 54 DEG C, opens pipe lid, and 32 μ L connection enzymatic mixtures, 54 DEG C of incubation 15min, 98 DEG C of heating are added
5min inactivates ligase, 20 DEG C of incubations；
D) PCR amplification of linking probe
Prepare 10 μ L PCR mixed liquors: 7.5 μ L dH2+ 0.5 μ L SALSA polymerase of O+2 μ L PCR reaction mixture；
PCR pipe is taken out, 10 μ L PCR mixtures are added at room temperature；Start PCR reaction, reaction condition are as follows: 95 DEG C of 30s, 60 DEG C
30s, 72 DEG C of 60s, 35 circulations；72 DEG C of incubation 20min, are down to 15 DEG C；
E) full-automatic nucleic acids instrument analysis:
Pcr amplification product takes 20 μ L, is analyzed with full-automatic nucleic acids instrument；
(4) result description and judgement
A) quality control standard:
Positive control has specific amplification band at 102bp, 110bp, 117bp, 124bp, 131bp, 138bp；
Negative control is without specific amplification band；
If negative control and positive condition are unsatisfactory for conditions above, this time test is considered as invalid；
B) result judges:
It is positive: to have specific amplification band at 102bp, indicate that there are pig bocavirus in sample；There is specificity at 110bp
Amplified band indicates that there are pig norovirus in sample；There is specific amplification band at 117bp, indicates that there are pigs in sample
Delta coronavirus；There is specific amplification band at 124bp, indicates that there are Porcine epidemic diarrhea virus in sample；?
There is specific amplification band at 131bp, indicates that there are porcine rotavirus in sample；There are specific amplification band, table at 138bp
There are transmissible gastro-enteritis virus in sample sheet；MLPA amplified production can be sequenced, be further confirmed that simultaneously；
It is negative: without specific amplification band, to show in sample without Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pig
Rotavirus, pig delta coronavirus, pig bocavirus, pig norovirus.
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| CN201811091224.2A CN109161613B (en) | 2018-09-19 | 2018-09-19 | MLPA detection kit for detecting pathogenic nucleic acid of porcine viral diarrhea syndrome |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706269A (en) * | 2019-02-06 | 2019-05-03 | 浙江农林大学 | The multiple linking probe that a variety of fowl respiratory pathogens can be detected expands identification reagent box |
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