CN109207635A - Detect the MLPA detection kit of pig virus breeding difficulty syndrome etiology nucleic acid - Google Patents
Detect the MLPA detection kit of pig virus breeding difficulty syndrome etiology nucleic acid Download PDFInfo
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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 breeding difficulty syndrome etiology nucleic acid.Include: 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；Multiple linking probe mixed liquor: the sequence of its middle 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；MLPA buffer；Connect buffer solution A and B；Ligase Ligase-65；PCR reaction mixture comprising universal primer shown in NO:25~26 sequence SEQ ID；SALSA polymerase；Negative control；Positive control, the positive control plasmid of viral CSFV, PRV, JEV, PRRSV, PCV2, PPV comprising six kinds of pig virus breeding difficulty 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 reproductive and respiratory syndrome virus (PRRSV), pig Japanese B
Encephalitis viruses (JEV), swine fever virus (CSFV), porcine circovirus 2 type (PCV2), porcine pseudorabies virus (PRV) and the tiny disease of pig
The multiple linking probe amplification of poison (PPV) 6 identifies detection kit, it can be achieved that detecting 6 kinds simultaneously causes pig virus breeding barrier
The cause of disease for hindering syndrome, belongs to inspection and quarantine field.
Background technique
Pig virus breeding difficulty is to endanger one of most important disease of China's pig breeding industry at present, disease main harm pregnancy
Sow group leads to period of pregnancy sow premature labor, miscarriage, produces the mummification of fetus, and disease incidence and the piglet death rate are all higher, and normal companion
Endometrial inflammation and immunosupress are sent out, morbidity and propagation rapidly, are relatively difficult to control, greatly endanger scale livestock farming
Swinery safety, bring heavy losses to pig breeding industry.Once infecting the disease, often band is malicious for sow, easily brings destructiveness to farm
Strike.The encountered pathogenic of pig virus breeding difficulty syndrome are as follows: porcine reproductive and respiratory syndrome virus (Porcine
Reproductive and respiratory syndrome virus, PRRSV), pig Japanese B encephalitis virus
(Japanese encephalitis virus, JEV), swine fever virus (Classic swine fever virus, CSFV), pig
Circovurus type 2 (Porcine circovirus 2types, PCV2), porcine pseudorabies virus (Pseudorabies virus,
PRV) and caused by six kinds of viruses such as pig parvoviral (Porcine parvovirus, PPV).Since syndrome morbidity is fast
Speed, needs to determine in the short time and pathogenesis and prevents and treats, at present there is no one way to disposably to causing
A variety of disease pathogens of pig virus diarrhoea carry out antidiastole.
Therefore a kind of detection method that can precisely, quickly, efficiently distinguish Different Kinds of Pathogens is established, pig pig virus is bred
The prevention and control of obstacle symptom grouping disease are most important.
Summary of the invention
The technical problem to be solved by the present invention is to overcome deficiency in the prior art, it is numerous to provide a kind of detection pig virus
Grow the MLPA detection kit of obstacle syndrome etiology nucleic acid.The kit can detect porcine reproductive and respiratory syndrome virus simultaneously
(PRRSV), pig Japanese B encephalitis virus (JEV), swine fever virus (CSFV), porcine circovirus 2 type (PCV2), porcine pseudorabies
Malicious (PRV) and pig parvoviral (PPV).
In order to solve the above technical problems, the invention adopts the following technical scheme:
A kind of MLPA detection kit for detecting pig virus breeding difficulty syndrome etiology nucleic acid is provided, in the kit
Include:
(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 O:25~26 sequence SEQ ID N；
(7) SALSA polymerase；
(8) negative control；
(9) positive control includes porcine reproductive and respiratory syndrome virus (PRRSV), pig Japanese B encephalitis virus
(JEV), swine fever virus (CSFV), porcine circovirus 2 type (PCV2), porcine pseudorabies virus (PRV) and pig parvoviral (PPV)
Positive control plasmid.
In the present invention, the sequence SEQ ID NO:1-2 is the pre- amplimer of porcine reproductive and respiratory syndrome virus；It is described
Sequence SEQ ID NO:3-4 is the pre- amplimer of pig Japanese B encephalitis virus；The sequence SEQ ID NO:5-6 is hog cholera
The pre- amplimer of poison；The sequence SEQ ID NO:7-8 is the pre- amplimer of porcine circovirus 2 type respectively；The sequence SEQ
ID NO:9-10 is the pre- amplimer of porcine pseudorabies virus；The sequence SEQ ID NO:11-12 is that pig parvoviral expands in advance
Primer；The sequence SEQ ID NO:13-14 is the left side probe and right side probe for detecting porcine reproductive and respiratory syndrome virus；
The sequence SEQ ID NO:15-16 is respectively that the left side probe for detecting pig norovirus Japanese B encephalitis virus and right side are visited
Needle；The sequence SEQ ID NO:17-18 is respectively the left side probe and right side probe for detecting swine fever virus；The sequence SEQ
ID NO:19-20 is respectively the left side probe and right side probe for detecting porcine circovirus 2 type；The sequence SEQ ID NO:21-
22 be respectively the left side probe and right side probe for detecting porcine pseudorabies virus；The sequence SEQ ID NO:23-24 is respectively to examine
Survey the left side probe and right side probe of pig parvoviral；Wherein, the 5 ' of SEQ ID NO:14,16,18,20,22,24 hold into
Row 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 is respectively porcine reproductive and respiratory syndrome virus (PRRSV)
M gene, the E gene of pig Japanese B encephalitis virus (JEV), swine fever virus (CSFV) raq gene, porcine circovirus 2 type
(PCV2) the gE gene of ORF2 gene, porcine pseudorabies virus (PRV) and the VP2 gene of pig parvoviral (PPV).
Invention further provides realized using the kit while detecting 6 kinds of pig virus breeding difficulty syndromes
The method of the multiple linking probe augmentation detection of etiology nucleic acid, 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
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；It is every final concentration of
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: 25 μ L dH2+ 1 μ L connection of+3 μ L connection buffer solution B of O+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 94bp, 100bp, 106bp, 116bp, 122bp, 127bp；
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 in 94bp, indicate that there are the cores of porcine reproductive and respiratory syndrome virus in sample
Acid has specific amplification band at 100bp, indicates that there are the nucleic acid of pig Japanese B encephalitis virus in sample；At 106bp
There is specific amplification band, indicates that there are the nucleic acid of swine fever virus in sample；There is specific amplification band at 116bp, indicates
There are the nucleic acid of porcine circovirus 2 type in sample；There is specific amplification band at 122bp, indicates that there are pig puppet is mad in sample
The nucleic acid of dog disease poison；There is specific amplification band at 127bp, indicates that there are the nucleic acid of pig parvoviral in sample；It is right simultaneously
MLPA amplified production is sequenced, and is further confirmed that；
It is negative: without specific amplification band, to show in sample without porcine reproductive and respiratory syndrome virus, pig Japanese B brain
Scorching virus, swine fever virus, porcine circovirus 2 type, porcine pseudorabies virus and pig parvoviral.
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 viral breeding difficulty syndrome disease pathogens；
In figure, M is DL2000DNA Marker；1 is PCV2；2 be PRRSV；3 be CSFV；4 be PPV；5 be PRV；6 are
JEV。
Fig. 2 .1 to 2.6 is peak figure (the single mould of the specificity verification of pig virus breeding difficulty syndrome MLPA method
Plate)；
CSFV-E2, PRV-gE, PRRSV-M, PCV2-Cap, JEV-E, PPV-VP2 positive plasmid built, it is single respectively
It is a to be added in the system with 6 pairs of pre- amplimers and 6 pairs of probes, it is detected according to MLPA response procedures, the results showed that pig
Porcine reproductive and respiratory syndrome virus has specific peak figure in 94bp；Pig pig Japanese B encephalitis virus has specificity in 100bp
Peak figure；Swine fever virus has specific peak figure in 106bp；Porcine circovirus 2 type has specific peak figure in 116bp；Porcine pseudorabies
Poison has specific peak figure in 122bp；Pig pig parvoviral has specific peak figure in 127bp.
Fig. 3 is the simulation electrophoretogram of the specificity verification of pig virus breeding difficulty syndrome MLPA method；
The result shows that: there is specific amplification band in 94bp, indicates that there are porcine reproductive and respiratory syndrome virus in sample
Nucleic acid, have specific amplification band at 100bp, indicate that there are the nucleic acid of pig Japanese B encephalitis virus in sample；?
There is specific amplification band at 106bp, indicates that there are the nucleic acid of swine fever virus in sample.There is specific amplification item at 116bp
Band indicates that there are the nucleic acid of porcine circovirus 2 type in sample；There is specific amplification band at 122bp, indicates exist in sample
The nucleic acid of porcine pseudorabies virus；There is specific amplification band at 127bp, indicates that there are the nucleic acid of pig parvoviral in sample.
Fig. 4 .1 to 4.6 is specificity verification peak figure (the single template disease of pig virus breeding difficulty syndrome MLPA method
Venom is template)；
It is respectively template with the cDNA of CSFV, PRV, JEV, PRRSV, PCV2, PPV Viral extraction or DNA, band is individually added
In the system for having 6 pairs of pre- amplimers and 6 pairs of probes, detected according to MLPA response procedures, the results showed that pig pig breeding with
Breath syndrome virus has specific peak figure in 94bp；Pig pig Japanese B encephalitis virus has specific peak figure in 100bp；Swine fever
Virus has specific peak figure in 106bp；Porcine circovirus 2 type has specific peak figure in 116bp；Porcine pseudorabies virus is in 122bp
There is specific peak figure；Pig pig parvoviral has specific peak figure in 127bp.
Fig. 5 is the peak figure (hybrid template) of the specificity verification of pig virus breeding difficulty syndrome MLPA method.
CSFV, PRV, PRRSV, PCV2, JEV, PPV the gene masculine plasmid built is added together with 6 pairs of pre- amplifications
In the system of primer and 6 pairs of probes, the result that is detected according to MLPA response procedures.Pig porcine reproductive and respiratory syndrome virus
There is specific peak figure in 94bp；Pig pig Japanese B encephalitis virus has specific peak figure in 100bp；Swine fever virus has in 106bp
Specific peak figure；Porcine circovirus 2 type has specific peak figure in 116bp；Porcine pseudorabies virus has specific peak figure in 122bp；
Pig pig parvoviral has specific peak figure in 127bp.
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 breeding difficulty symptom grouping multiplex ligation-dependent probe amplifications (MLPA) of table expand primer sequence in advance
(5’-3’)
26 kinds of pig virus breeding difficulty symptom grouping multiplex ligation-dependent probe amplification (MLPA) probe sequences (5 '-of table
3’)
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 reproductive and respiratory syndrome virus, pig using multiplex ligation-dependent probe amplification
The multiple connection of Japanese B encephalitis virus, swine fever virus, porcine circovirus 2 type, porcine pseudorabies virus and pig parvoviral 6 is visited
Needle amplification detection method simultaneously assembles kit, this reagent constituents composition is as follows:
(1) reverse transcription and pre- amplification primer mixed liquor, including porcine reproductive and respiratory syndrome virus, pig Japanese B encephalitis
Virus, swine fever virus, porcine circovirus 2 type, porcine pseudorabies virus and pig parvoviral pre- amplimer, the primer sequence
It is shown in Table 1, the concentration of every primer is 2.5 μM in mixed liquor；
(2) probe mixed liquor comprising detection porcine reproductive and respiratory syndrome virus, pig Japanese B encephalitis virus, pig
Pestivirus, porcine circovirus 2 type, porcine pseudorabies virus and pig parvoviral left and right side probe, the sequence of the probe
It 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 spy in mixed liquor
The concentration of needle 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 CSFV-E2, PRV-gE, PRRSV-M, PCV2-Cap, JEV-E, PPV-VP2 gene masculine
The mixture of Plasmid DNA.
The present invention also further establishes using aforementioned agents box while detecting porcine reproductive and respiratory syndrome virus, pig day
This japanese encephalitis virus, swine fever virus, porcine circovirus 2 type, porcine pseudorabies virus and pig parvoviral 6 kinds of virus diarrheas
The identification detection method of disease.
The present invention will be described in detail combined with specific embodiments below.
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 application 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 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。
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 94bp, 100bp, 106bp, 116bp, 122bp, 127bp.
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 in 94bp, indicate that there are the cores of porcine reproductive and respiratory syndrome virus in sample
Acid has specific amplification band at 100bp, indicates that there are the nucleic acid of pig Japanese B encephalitis virus in sample；At 106bp
There is specific amplification band, indicates that there are the nucleic acid of swine fever virus in sample.There is specific amplification band at 116bp, indicates
There are the nucleic acid of porcine circovirus 2 type in sample；There is specific amplification band at 122bp, indicates that there are pig puppet is mad in sample
The nucleic acid of dog disease poison；There is specific amplification band at 127bp, indicates that there are the nucleic acid of pig parvoviral in sample.It simultaneously can
MLPA amplified production is sequenced, is further confirmed that.
It is negative: without specific amplification band, to show in sample without porcine reproductive and respiratory syndrome virus, pig Japanese B brain
Scorching virus, swine fever virus, porcine circovirus 2 type, porcine pseudorabies virus and pig parvoviral.
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 reproductive and respiratory syndrome virus, pig Japanese B encephalitis virus, swine fever virus, pig circular ring virus 2 respectively
Six kinds of single probes of cause of disease of malicious 2 types, porcine pseudorabies virus and pig parvoviral are directed to common swine disease cause of disease such as pig respectively and flow
Row diarrhea virus, transmissible gastro-enteritis virus, porcine rotavirus, pig delta coronavirus, pig bocavirus, pig promise are such as
Virus, C.perfringens and actinobacillus pleuropneumoniae carry out MLPA detection, verify the specificity of probe.
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 Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, porcine rotavirus, pig delta coronavirus, pig
Bocavirus, pig norovirus, C.perfringens and actinobacillus pleuropneumoniae are without amplified signal.Illustrate that probe is special
Property is good.
3.2 use porcine reproductive and respiratory syndrome virus, pig Japanese B encephalitis virus, swine fever virus, 2 porcine circovirus
The probe that six kinds of type, porcine pseudorabies virus and pig parvoviral cause of diseases mix is respectively to all viruses and bacteriums progress in table 4
MLPA detection can only amplify band of corresponding size from corresponding viral template, and pass to Porcine epidemic diarrhea virus, pig
Metachromia marcy agent, porcine rotavirus, pig delta coronavirus, pig bocavirus, pig norovirus, C.perfringens
With actinobacillus pleuropneumoniae 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 CSFV-E2, PRV-gE, PRRSV-M, PCV2-Cap, JEV-E, PPV-VP2 genetic fragment and pZERO- of amplification
The connection of blunt carrier, Transformed E scherichia coli DH5a extract recombinant plasmid, and carry out PCR verifying and sequence verification.
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 nucleic acid of the minimum porcine reproductive and respiratory syndrome virus that 7 copies can be detected of this method, 8 copy pig Japan
The nucleic acid of japanese encephalitis virus, the nucleic acid of 8 copy swine fever virus, 3 the copy nucleic acid of porcine circovirus 2 type, 7 copy pigs
The nucleic acid of Pseudorabies virus, 9 copy pig parvoviral nucleic acid.
With porcine reproductive and respiratory syndrome virus, pig Japanese B encephalitis virus, swine fever virus, porcine circovirus 2 type, pig
After 6 kinds of viruses of Pseudorabies virus and pig parvoviral carry out doubling dilution, RNA or DNA is extracted, RNA is synthesized cDNA, made with this
For template according to the program of MLPA carry out reaction and fluorescence quantitative RT-RCR react, detect this method minimum detection limit (see
Table 5), the results showed that MLPA method is than about poor 5 times or so of fluorescent quantitation method.
56 kinds of table viral breeding difficulty syndrome MLPA methods and fluorescent quantitative RT-PCR method remolding sensitivity compared with
A is indicated with the minimum extension rate of 5 times of progress doubling dilutions of virus.
Four, the repeatability of kit
Doubling dilution is carried out by template of 6 kinds of viruses, takes three concentration gradients 5-2、5-3、5-4, 3 weights of each gradient setting
It is multiple, MLPA reaction is carried out according to the program of 4.2.2-4.2.5 in this, as template, while carrying out 3 repetitions according to the method described above
Test, obtain between the group of this method and organize in the coefficient of variation, institute's value is respectively less than 10%, and the coefficient of variation is small, illustrates this method
It is reproducible to be shown in Table 6.
66 kinds of table viral breeding difficulty syndrome MLPA detection method repeatability
Sequence table
<110>Zhejiang A & F University
<120>the MLPA detection kit of pig virus breeding difficulty syndrome etiology nucleic acid is detected
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
ggtacatgac attcgcgcac 20
<210> 2
<211> 21
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
cggatgaaag cccgcggcac t 21
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
agaagcgagc tgatagtagc t 21
<210> 4
<211> 23
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
gccgcctggg acgccccaac ttg 23
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 5
tggacatgtg tgaaaggcga 20
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 6
catctgatgc atgcaccttg ac 22
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 7
atctcatcat gtccaccgcc 20
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 8
gacgtatcca aggaggcgtt 20
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 9
tgatgaccgt gacgtactcg 20
<210> 10
<211> 17
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 10
cgtgcagctt cacctcg 17
<210> 11
<211> 24
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 11
ctgtgaatgt tagtgttcct ttcc 24
<210> 12
<211> 32
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 12
gaacttgata cagatctaaa acctagacta ca 32
<210> 13
<211> 45
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 13
gggttcccta agggttggag aaacctggaa attcatcacc tccag 45
<210> 14
<211> 49
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 14
atgccgtttg tgcttgctag gccgcatcta gattggatct tgctggcac 49
<210> 15
<211> 49
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 15
gggttcccta agggttggag tggacttttc gggaagggaa gcattgaca 49
<210> 16
<211> 51
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 16
catgtgcaaa attctcctgc accagtaatc tagattggat cttgctggca c 51
<210> 17
<211> 47
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 17
gggttcccta agggttggag gtaagtgcat tttggcaaat gagacag 47
<210> 18
<211> 59
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 18
gttacagaat agtagattca acggactgta acagagtcta gattggatct tgctggcac 59
<210> 19
<211> 57
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 19
gggttcccta agggttggag gtgcgggaga ggcgggtgtt gaagatgcca tttttcc 57
<210> 20
<211> 59
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 20
ttctccagcg gtaacggtgg cgggggtgga cgagcctcta gattggatct tgctggcac 59
<210> 21
<211> 59
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 21
gggttcccta agggttggat cggggaccgg tggctgaccg cctgcccctt cgacgcctt 59
<210> 22
<211> 63
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 22
cggcgaggag gtgcacacga acgccaccgc ggacgagtcg tctagattgg atcttgctgg 60
cac 63
<210> 23
<211> 58
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 23
gggttcccta agggttggag caccaaacct aacagatgat ttcaatgctg actctcct 58
<210> 24
<211> 69
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 24
caacaaccta gaataataac ttattcaaac ttttggtgga aaggaatcta gattggatct 60
tgctggcac 69
<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 breeding difficulty syndrome etiology nucleic acid, which is characterized in that the examination
Include: in agent box
(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 O:25~26 sequence SEQ ID N；
(7) SALSA polymerase；
(8) negative control；
(9) positive control includes porcine reproductive and respiratory syndrome virus, pig Japanese B encephalitis virus, swine fever virus, pig annulus
The positive control plasmid of viral 2 types, porcine pseudorabies virus and pig parvoviral.
2. kit according to claim 1, which is characterized in that the sequence SEQ ID NO:1-2 is that pig breeds and exhales
Inhale the pre- amplimer of syndrome virus；The sequence SEQ ID NO:3-4 is the pre- amplimer of pig Japanese B encephalitis virus；Institute
Stating sequence SEQ ID NO:5-6 is the pre- amplimer of swine fever virus；The sequence SEQ ID NO:7-8 is pig circular ring virus respectively
The pre- amplimer of 2 types；The sequence SEQ ID NO:9-10 is the pre- amplimer of porcine pseudorabies virus；The sequence SEQ ID
NO:11-12 is the pre- amplimer of pig parvoviral；The sequence SEQ ID NO:13-14 is that the breeding of detection pig is integrated with breathing
Levy the left side probe and right side probe of virus；The sequence SEQ ID NO:15-16 is respectively to detect pig norovirus Japan second
The left side probe and right side probe of type encephalitis viruses；The sequence SEQ ID NO:17-18 is respectively the left side for detecting swine fever virus
Side probe and right side probe；The sequence SEQ ID NO:19-20 is respectively left side probe and the right side for detecting porcine circovirus 2 type
Side probe；The sequence SEQ ID NO:21-22 is respectively the left side probe and right side probe for detecting porcine pseudorabies virus；It is described
Sequence SEQ ID NO:23-24 is respectively the left side probe and right side probe for detecting pig parvoviral；Wherein, the SEQ ID
5 ' ends of NO:14,16,18,20,22,24 carry out phosphatizing treatment；Sequence SEQ ID NO:25,26 are general PCR respectively
The forward and reverse primer of amplification.
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
M gene, the E gene of pig Japanese B encephalitis virus, the raq gene of swine fever virus, the pig annulus of Reproductive and respiratory syndrome virus
ORF2 gene, the gE gene of porcine pseudorabies virus and the VP2 gene of pig parvoviral of viral 2 types.
4. realized using kit described in claim 1 while detecting 6 kinds of pig virus breeding difficulty syndrome etiology nucleic acids
The method of MLPA detection, 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 in advance；Final concentration of every primer
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: 25 μ L dH2+ 1 μ L ligase of+3 μ L connection buffer solution B of O+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 94bp, 100bp, 106bp, 116bp, 122bp, 127bp；
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: have specific amplification band in 94bp, indicate in sample there are the nucleic acid of porcine reproductive and respiratory syndrome virus,
There is specific amplification band at 100bp, indicates that there are the nucleic acid of pig Japanese B encephalitis virus in sample；There is spy at 106bp
Specific amplification band indicates that there are the nucleic acid of swine fever virus in sample；There is specific amplification band at 116bp, indicates sample
It is middle that there are the nucleic acid of porcine circovirus 2 type；There is specific amplification band at 122bp, indicates that there are porcine pseudorabies in sample
The nucleic acid of poison；There is specific amplification band at 127bp, indicates that there are the nucleic acid of pig parvoviral in sample；Simultaneously to MLPA
Amplified production is sequenced, and is further confirmed that；
It is negative: without specific amplification band, to show in sample without porcine reproductive and respiratory syndrome virus, pig Japanese B encephalitis disease
Poison, swine fever virus, porcine circovirus 2 type, porcine pseudorabies virus and pig parvoviral.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102943129A (en) * | 2012-11-30 | 2013-02-27 | 中华人民共和国北京出入境检验检疫局 | Multiplex ligation-dependent probe amplification detection kit for simultaneously detecting five swine disease viruses, primers and probes |
| CN103555861A (en) * | 2013-11-11 | 2014-02-05 | 辽宁医学院 | Triplex ligation-dependent probe amplification detection method for swine disease diagnosis |
| CN103981286A (en) * | 2014-05-19 | 2014-08-13 | 广西壮族自治区兽医研究所 | GeXP rapid detection kit for identifying eight types of porcine viral diseases and primer group thereof |
| CN105734172A (en) * | 2016-03-10 | 2016-07-06 | 中山大学达安基因股份有限公司 | Kit capable of quickly detecting classical swine fever virus/porcine reproductive and respiratory syndrome virus/pseudorabies virus/porcine parvovirus |
| US9434997B2 (en) * | 2007-08-24 | 2016-09-06 | Lawrence Livermore National Security, Llc | Methods, compounds and systems for detecting a microorganism in a sample |
| CN108220479A (en) * | 2018-01-05 | 2018-06-29 | 浙江农林大学 | The multiple linking probe amplification identification reagent box of a variety of pig Sudden Death Syndrome cause of diseases can be detected |
-
2018
- 2018-09-19 CN CN201811091220.4A patent/CN109207635B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9434997B2 (en) * | 2007-08-24 | 2016-09-06 | Lawrence Livermore National Security, Llc | Methods, compounds and systems for detecting a microorganism in a sample |
| CN102943129A (en) * | 2012-11-30 | 2013-02-27 | 中华人民共和国北京出入境检验检疫局 | Multiplex ligation-dependent probe amplification detection kit for simultaneously detecting five swine disease viruses, primers and probes |
| CN103555861A (en) * | 2013-11-11 | 2014-02-05 | 辽宁医学院 | Triplex ligation-dependent probe amplification detection method for swine disease diagnosis |
| CN103981286A (en) * | 2014-05-19 | 2014-08-13 | 广西壮族自治区兽医研究所 | GeXP rapid detection kit for identifying eight types of porcine viral diseases and primer group thereof |
| CN105734172A (en) * | 2016-03-10 | 2016-07-06 | 中山大学达安基因股份有限公司 | Kit capable of quickly detecting classical swine fever virus/porcine reproductive and respiratory syndrome virus/pseudorabies virus/porcine parvovirus |
| CN108220479A (en) * | 2018-01-05 | 2018-06-29 | 浙江农林大学 | The multiple linking probe amplification identification reagent box of a variety of pig Sudden Death Syndrome cause of diseases can be detected |
Non-Patent Citations (2)
| Title |
|---|
| NAN CHENG等: "Rapid and low-cost strategy for detecting genome-editing induced deletion: A single-copy case", 《ANALYTICA CHIMICA ACTA》 * |
| 史喜菊等: "多重连接探针扩增(MLPA)技术同时检测五种病毒的研究", 《农业生物技术学报》 * |
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