CN110438259B - LAMP primer combination, detection method and kit capable of distinguishing 1-7 porcine parvovirus types for typing detection - Google Patents
LAMP primer combination, detection method and kit capable of distinguishing 1-7 porcine parvovirus types for typing detection Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a primer combination, a detection method and a kit of LAMP (loop-mediated isothermal amplification) capable of distinguishing 1-7 porcine parvovirus types for typing detection. According to the invention, multiple sets of LAMP primers are designed by respectively utilizing conserved segments of the porcine parvovirus NS1 gene and the NP2 gene, and a primer combination which has good specificity and high sensitivity and can distinguish the 1-7 porcine parvovirus types and be used for typing detection is screened out. The primer combination can be used for respectively detecting porcine parvovirus types 1-7, and has no cross reaction with other porcine viruses. According to the invention, the fluorescent dye is added into the LAMP reaction system, the porcine parvovirus 1-7 can be detected by one-step method, the whole-process real-time monitoring is realized by combining fluorescence quantification, the result can be obtained within 1 hour, and the detection sensitivity can reach 100 copies/mu L. The detection method disclosed by the invention has the advantages of simplicity, convenience, rapidness, sensitivity and specificity, and also avoids errors caused by artificial judgment and environmental pollution caused by uncovering.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a LAMP primer combination, a detection method and a kit for parting detection capable of distinguishing porcine parvovirus types 1-7, which are used for detecting and distinguishing porcine parvovirus types 1-7.
Background
Porcine parvovirus (porcine parovirus) is one of the major pathogens causing reproductive failure in pregnant sows, and is characterized by infected sows, particularly primiparous sows, exhibiting abortion, stillbirth, mummy and malformed fetuses, or having a low litter size, and sometimes causing infertility in boars and sows. Porcine parvovirus has high infectivity, and once the virus is introduced into susceptible healthy swinery, the infection of the swinery can almost reach 100 percent within 3 months; the pigs in the infected herd remained serologically positive for a longer period. Porcine parvovirus diseases mostly occur in spring and summer sections or in farrowing and mating seasons of sows. When the sow is infected in the early stage of pregnancy, the death rate of the embryo and the fetal pig can reach 80-100 percent. The sows are most susceptible to infection in the first 30-40 days of the gestation period, and the infection at different time of the gestation period can cause different symptoms such as stillbirth, abortion, mummy, weak piglets and long-term infertility of the sows. Therefore, the rapid and accurate identification of porcine parvovirus in the early stage is of great significance for controlling virus spread.
Mary and Mahnel discovered porcine parvovirus for the first time in 1966, and then the virus developed in several countries and regions of the world. In the 80 s of the 20 th century, porcine parvovirus diseases were found separately in various places of China. Classical Porcine parvovirus disease is caused by infection with Porcine parvovirus type 1 (PPV 1). Porcine parvovirus type 2 (PPV2) was discovered from porcine serum when Burma performed a hepatitis E survey in 2001 by tiijikata et al. In 2008, a new porcine parvovirus, hokovirus (phov), was identified in hong kong, and its genome sequence was closely related to human parvovirus 4(PARV4) and bovine hokovirus (bhop), i.e., porcine parvovirus type 3 (PPV 3). PPV4 was discovered in 2005 from a swine herd infected with porcine circovirus associated disease (PCVAD), North Carolina, USA, and was subsequently reported in China. In 2013, Xiao et al found a novel PPV in the U.S. herd, tentatively designated PPV5, which has a typical parvovirus genome structure. In 12 months 2014, Chinese scholars adopt a sequence-independent single-primer amplification method to obtain a parvovirus which is different from the conventional parvovirus in aborted pig fetuses and is temporarily named as PPV 6. PPV7 was first identified in the U.S. swinery in 2016 by metagenomic sequencing, PPV7 is a highly differentiated novel parvovirus, and the presence of PPV7 virus was also found in China in 2017. Sequence alignment found PPV7 to have no more than 17% sequence homology with NS1 of other parvoviruses. Due to the continuous discovery of novel strains, the typing detection of the porcine parvovirus has important significance for the genetic evolution of the virus, the investigation and monitoring of molecular epidemiology, the pathological research and the prevention and control of epidemic situations, but the typing detection research of the porcine parvovirus 1-7 is not reported at present.
Common methods for detecting porcine parvovirus include: enzyme-linked immunosorbent assay (ELISA), qualitative PCR technology, real-time fluorescence quantitative PCR technology and loop-mediated isothermal amplification (LAMP). ELISA sensitivity was high, but the detection results may include false positives. The qualitative PCR technology has low cost and easy operation, but has lower sensitivity, and because the amplification product needs to be analyzed once through gel electrophoresis, the method is very easy to generate pollution. Although the real-time fluorescence quantitative PCR technology has the characteristics of high sensitivity, high specificity and the like, the experiment has high requirements on detection personnel, the experiment operation steps need frequent heating and cooling processes, and the time consumed by light detection is at least more than 1 h. Loop-mediated isothermal amplification (LAMP) is a strand displacement nucleic acid amplification reaction in which a Bst DNA polymerase having a strand displacement activity and a waterfall nucleic acid amplification function is used to perform denaturation and automatic cycle of nucleic acid under isothermal conditions. LAMP designs 4-6 specific primers for 6 regions of a target sequence, and uses DNA polymerase with a strand displacement function to continuously replicate and amplify DNA at a constant temperature. In order to improve the reaction efficiency, two loop primers can be added into the reaction system, so that the two loop primers are respectively combined with the stem-loop structure to start strand displacement synthesis and cycle replication. Therefore, one of the cores of the LAMP technology is primer design, primers are key factors determining the sensitivity and specificity of detection results, and efficient, specific and sensitive LAMP primer combinations adapted to a reaction system can be found only through multiple primer screening and verification.
Moreover, most of the established LAMP reaction methods at present need agarose gel electrophoresis or a color developing agent is added after uncovering, so that laboratory pollution or false positive results are easily caused. In addition, most of the color development methods for direct observation by the LAMP method are that after the reaction is finished, a cover is opened, a fluorescent dye is added for color development reaction, and whether color development exists is observed to judge the test result, so that aerosol pollution is caused, and specific amplification and non-specific amplification cannot be distinguished, so that the probability of false positive diagnosis results is increased; and for weak positive reaction, the negative is probably misjudged by the way of artificial visual interpretation.
Disclosure of Invention
In view of the above, the present invention aims to provide a primer combination, a detection method and a kit for typing detection LAMP, which have the advantages of high speed, high sensitivity and strong specificity and can distinguish porcine parvovirus types 1-7, aiming at the problems in the prior art.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a primer combination for parting detection LAMP capable of distinguishing porcine parvovirus types 1-7 comprises a primer group 1-7;
the primer group 1 comprises 2 outer primers shown by SEQ ID NO.1 and SEQ ID NO.2, 2 inner primers shown by SEQ ID NO.3 and SEQ ID NO.4 and 2 loop primers shown by SEQ ID NO.5 and 6;
the primer group 2 comprises 2 outer primers shown by SEQ ID NO.7 and SEQ ID NO.8, 2 inner primers shown by SEQ ID NO.9 and SEQ ID NO.10 and 2 loop primers shown by SEQ ID NO.11 and 12;
the primer group 3 comprises 2 outer primers shown by SEQ ID NO.13 and SEQ ID NO.14, 2 inner primers shown by SEQ ID NO.15 and SEQ ID NO.16 and 2 loop primers shown by SEQ ID NO.17 and 18;
the primer group 4 comprises 2 outer primers shown by SEQ ID NO.19 and SEQ ID NO.20, 2 inner primers shown by SEQ ID NO.21 and SEQ ID NO.22 and 2 loop primers shown by SEQ ID NO.23 and 24;
the primer group 5 comprises 2 outer primers shown by SEQ ID NO.25 and SEQ ID NO.26, 2 inner primers shown by SEQ ID NO.27 and SEQ ID NO.28, and 2 loop primers shown by SEQ ID NO.29 and SEQ ID NO. 30;
the primer group 6 comprises 2 outer primers shown by SEQ ID NO.31 and SEQ ID NO.32, 2 inner primers shown by SEQ ID NO.33 and SEQ ID NO.34, and 2 loop primers shown by SEQ ID NO.35 and 36;
the primer group 7 comprises 2 outer primers shown by SEQ ID NO.37 and SEQ ID NO.38, 2 inner primers shown by SEQ ID NO.39 and SEQ ID NO.40, and 2 loop primers shown by SEQ ID NO.41 and 42.
Wherein the primer group 1 is a primer group for loop-mediated isothermal amplification of porcine parvovirus type 1, and is specifically as follows (5 '→ 3'):
outer primer PPV 1-F3: 5'-CTTGGTTGGTAAAGAAAGGT-3' (SEQ ID NO.1)
Outer primer PPV 1-B3: 5'-AGCTAAATCCAGGTCCTC-3' (SEQ ID NO.2)
Inner primer PPV 1-FIP: 5'-TGGGGTTTGCATTTTTGGCGGCTAGCTATATGC ATCATTG-3' (SEQ ID NO.3)
Inner primer PPV 1-BIP: 5'-TACACCAACAGACTCTCAGATTTCATTGGAGT TGCTGCGTA-3' (SEQ ID NO.4)
Loop primer PPV 1-LF: 5'-GACCAATCAGGTACATTTCC-3' (SEQ ID NO.5)
The loop primer PPV 1-LB: 5'-ACATCAGTGAAAACTTCGC-3' (SEQ ID NO. 6);
the primer set 2 is a primer set for loop-mediated isothermal amplification of porcine parvovirus type 2, and is specifically as follows (5 '→ 3'):
outer primer PPV 2-F3: 5'-GCAAATGGAGCCCAGCAG-3' (SEQ ID NO.7)
Outer primer PPV 2-B3: 5'-TGATCGCCTTCCCACCAG-3' (SEQ ID NO.8)
Inner primer PPV 2-FIP: 5'-TTCAGCCACCCCCCCATCTCTTCACCTTCATCT CGCAGTG-3' (SEQ ID NO.9)
Inner primer PPV 2-BIP: 5'-ATGATAGAGCACAGGCCAGGCGAGCGGTCTT CCTCATCTCA-3' (SEQ ID NO.10)
Loop primer PPV 2-LF: 5'-CTCGGGCCTTTGTGTCG-3' (SEQ ID NO.11)
The loop primer PPV 2-LB: 5'-GCCCCCCCCTTGTACTT-3' (SEQ ID NO. 12).
The primer set 1 is a primer set for loop-mediated isothermal amplification of porcine parvovirus type 3, and is specifically as follows (5 '→ 3'):
outer primer PPV 3-F3: 5'-AGCAACTGGTTGAAGAGATGG-3' (SEQ ID NO.13)
Outer primer PPV 3-B3: 5'-GCAGCCTCAAATCTCTCCATG-3' (SEQ ID NO.14)
Inner primer PPV 3-FIP: 5'-AGTCTCCCTTGTCTGGTCTTCCTCAACATTCCT AACTCGCCACA-3' (SEQ ID NO.15)
Inner primer PPV 3-BIP: 5'-ATCAGTGCGACCTGACCTTTGTCAAGCATACC AGACATGCTCTA-3' (SEQ ID NO.16)
Loop primer PPV 3-LF: 5'-GATATCCCACAGAATGCTCCAAC-3' (SEQ ID NO.17)
The loop primer PPV 3-LB: 5'-AAGGTATCTACTGCCTAAAGTACCA-3' (SEQ ID NO. 18).
The primer set 1 is a primer set for loop-mediated isothermal amplification of porcine parvovirus type 4, and is specifically as follows (5 '→ 3'):
outer primer PPV 4-F3: 5'-GACATTATGAACTGCCCTAT-3' (SEQ ID NO.19)
Outer primer PPV 4-B3: 5'-AGGGATGAATTCAGTCTCAG-3' (SEQ ID NO.20)
Inner primer PPV 4-FIP: 5'-TTCATTTCTTTTGGGGGTGGGTAAAAAAGGAC CTGCCAGT-3' (SEQ ID NO.21)
Inner primer PPV 4-BIP: 5'-AGAGCGGAACCAGATGAAATTCGTTTCTTCT TTCTCGGTGC-3' (SEQ ID NO.22)
Loop primer PPV 4-LF: 5'-TCTCTCCAACAGGAACTAAA-3' (SEQ ID NO.23)
The loop primer PPV 4-LB: 5'-AATCCAGAAGAACTGGACC-3' (SEQ ID NO. 24).
The primer set 1 is a primer set for loop-mediated isothermal amplification of porcine parvovirus type 5, and specifically comprises the following primer sets (5 '→ 3'):
outer primer PPV 5-F3: 5'-GGAACATCACCAATCAAGA-3' (SEQ ID NO.25)
Outer primer PPV 5-B3: 5'-GAGAGATCAATTTCATTGCG-3' (SEQ ID NO.26)
Inner primer PPV 5-FIP: 5'-AGTTCTTTCTGTTCTCGGTGACCTATTAGAACA CGACTCAAGC-3' (SEQ ID NO.27)
Inner primer PPV 5-BIP: 5'-ATCAAACATGGAGCGGGAGATTTCCAGGACC TGTGTAG-3' (SEQ ID NO.28)
Loop primer PPV 5-LF: 5'-GCTTCTTCTGGTTGTTCTTC-3' (SEQ ID NO.29)
The loop primer PPV 5-LB: 5'-CGGAACCGGTATCAACT-3' (SEQ ID NO. 30).
The primer set 1 is a primer set for loop-mediated isothermal amplification of porcine parvovirus type 6, and specifically comprises the following primer sets (5 '→ 3'):
outer primer PPV 6-F3: 5'-TCAAGATCAGGGAGTCATT-3' (SEQ ID NO.31)
Outer primer PPV 6-B3: 5'-TAGTGCTCACAATACGCT-3' (SEQ ID NO.32)
Inner primer PPV 6-FIP: 5'-GTAGTCACATCATTATCACACATCCACTGCTCC TACTTTTAAAGC-3' (SEQ ID NO.33)
Inner primer PPV 6-BIP: 5'-CCTGTACTCTTTCCTTGCTACCGCAGCTCTAA GACACTGT-3' (SEQ ID NO.34)
Loop primer PPV 6-LF: 5'-CATTTTATCACCCGTAGCC-3' (SEQ ID NO.35)
The loop primer PPV 6-LB: 5'-CCAGCTGGTACTCATTTG-3' (SEQ ID NO. 36).
The primer set 1 is a primer set for porcine parvovirus type 7 loop-mediated isothermal amplification, and is specifically as follows (5 '→ 3'):
outer primer PPV 7-F3: 5'-GGTACCACAACTGGTACGAAG-3' (SEQ ID NO.37)
Outer primer PPV 7-B3: 5'-TGGTGTTCCATGTCCATGTC-3' (SEQ ID NO.38)
Inner primer PPV 7-FIP: 5'-CGGTGGTTGGTTTAGGGACGAGACACGCTCTG GAGAAGCT-3' (SEQ ID NO.39)
Inner primer PPV 7-BIP: 5'-ACCAAAGAAGGGGTCGGAAACAGGTTTCGG GTGCCGTGATG-3' (SEQ ID NO.40)
Loop primer PPV 7-LF: 5'-CCTTCTTTGTAGGCCACGT-3' (SEQ ID NO.41)
The loop primer PPV 7-LB: 5'-TGGAGCAGACTTACACTCCC-3' (SEQ ID NO. 42).
The invention also provides a LAMP kit for parting detection capable of parting porcine parvovirus types 1-7, which comprises the primer combination, wherein each primer in the primer combination is independently packaged.
Preferably, the kit of the present invention further comprises a reaction solution containing a fluorescent dye and/or sterilized pure water.
In some embodiments, the reaction solution is a product of boao bio mass ltd, catalog No. cp.440020. The reaction solution contains a fluorescent dye.
Furthermore, in each primer group of the primer combination in the kit of the present invention, the reaction mol numbers of the 2 outer primers are the same, the reaction mol numbers of the 2 inner primers are the same, the reaction mol numbers of the 2 loop primers are the same, and the molar ratio of the outer primers to the inner primers to the loop primers in each primer group is (0.58-0.62): (4.6-5.0): (1.8-2.2). For example, the molar ratio of the primer F3, the primer B3, the primer FIP, the primer BIP, the primer LF and the primer LB is (0.29-0.31): (0.29-0.31):(2.3-2.5):(2.3-2.5): (0.9-1.1): (0.9-1.1).
In some embodiments, the molar ratio of outer primer to inner primer to loop primer in each primer set is 0.6:4.8: 2.
in some embodiments, the molar ratio of primer F3, primer B3, primer FIP, primer BIP, primer LF, and primer LB is 0.3: 0.3: 2.4: 2.4: 1: 1.
the invention also provides a LAMP detection method capable of carrying out parting detection for distinguishing 1-7 porcine parvovirus types, the primer combination is used for LAMP amplification, and the detection result is judged by observing the time when the S-shaped amplification curve of the real-time fluorescent PCR instrument begins to appear.
In the detection method of the invention, the LAMP amplification reaction system comprises 0.12 muL of 0.3mM outer primers, 0.96 muL of 2.4mM inner primers, 0.4 muL of 1mM loop primers, and 2 muL of sample DNA to be detected containing 10 muL and 2 muL of fluorescent dye reaction solution, and sterile pure water is added to make up to 20 muL.
In some embodiments, the reaction system is 0.12 μ L of each of 0.3mM outer primers F3 and B3; 0.96 mu L of each of 2.4mM inner primers FIP and BIP; 0.4. mu.L of each of 1mM loop primers LF and LB; 2 × 10 μ L of reaction solution; 2. mu.L of the DNA of the sample to be tested was supplemented with sterile purified water to 20. mu.L.
Preferably, the LAMP detection reaction condition in the detection method is constant temperature of 60-65 ℃ for 50 min. More preferably, the LAMP detection reaction condition is constant temperature of 65 ℃ for 50 min.
According to the technical scheme, the LAMP primer combination, the detection method and the kit can be used for carrying out parting detection on the porcine parvovirus types 1-7. According to the invention, multiple sets of LAMP primers are designed by respectively utilizing conserved segments of the porcine parvovirus NS1 gene and the NP2 gene, and finally, the LAMP primer combination which has good specificity and high sensitivity and can distinguish the typing detection of the porcine parvovirus 1-7 is screened out. The primer combination can be used for respectively detecting porcine parvovirus types 1-7, and has no cross reaction with other porcine viruses (such as circovirus type 2 (PCV2), pseudorabies virus (PRV), Classical Swine Fever Virus (CSFV), Swine Influenza Virus (SIV), highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and the like). According to the invention, the fluorescent dye is added into the LAMP reaction system, the one-step method detection of porcine parvovirus types 1-7 can be realized, the whole-process real-time monitoring is realized by combining fluorescence quantification, the result can be obtained within 1 hour, the detection sensitivity can reach 100 copies/mu L, and a powerful tool is provided for the research of porcine parvovirus typing. The detection method disclosed by the invention has the advantages of simplicity, convenience, rapidness, sensitivity and specificity, and also avoids errors caused by artificial judgment and environmental pollution caused by uncovering.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIGS. 1-3 are amplification curves for primer set PPV 1-1-PPV 1-3, respectively;
FIGS. 4-6 are amplification curves for primer set PPV 2-1-PPV 2-3, respectively;
FIGS. 7-9 are amplification curves for primer set PPV 3-1-PPV 3-3, respectively;
FIGS. 10-12 are amplification curves for primer set PPV 4-1-PPV 4-3, respectively;
FIGS. 13-15 are amplification curves for primer set PPV 5-1-PPV 5-3, respectively;
FIGS. 16-18 are amplification curves for primer set PPV 6-1-PPV 6-3, respectively;
FIGS. 19-21 are amplification curves for primer set PPV 7-1-PPV 7-3, respectively;
FIG. 22 shows the genomic copy number of 10 using the primer set PPV1-1 4 、10 3 Amplification curve at the time of detection;
FIG. 23 shows the genomic copy number of 10 using the primer set PPV1-2 4 、10 3 Amplification curve at the time of detection;
FIG. 24 shows the genomic copy number of 10 using the primer set PPV2-1 4 、10 3 Amplification curve at the time of detection;
FIG. 25 shows the genomic DNA sequence of the primer set PPV3-1Copy number of 10 4 、10 3 Amplification curve at the time of detection;
FIG. 26 shows the genomic copy number of 10 using the primer set PPV4-2 4 、10 3 Amplification curve at the time of detection;
FIG. 27 shows the genomic copy number of 10 using the primer set PPV4-3 4 、10 3 Amplification curve at the time of detection;
FIG. 28 shows the genomic copy number of 10 using the primer set PPV5-3 4 、10 3 Amplification curve at the time of detection;
FIG. 29 shows the genomic copy number of 10 using the primer set PPV6-2 4 、10 3 Amplification curve at the time of detection;
FIG. 30 shows the genomic copy number of 10 using the primer set PPV7-1 4 、10 3 Amplification curve at the time of detection;
FIG. 31 shows the genomic copy number of 10 using the primer set PPV7-2 4 、10 3 Amplification curve at the time of detection;
FIGS. 32-34 show the copy number of genome at 10 3 、10 2 、10 1 An amplification curve of a primer group PPV1 is adopted;
FIGS. 35-37 are graphs showing the copy number of 10 in genome, respectively 3 、10 2 、10 1 An amplification curve of a primer group PPV2 is adopted;
FIGS. 38-40 are at genome copy number 10 3 、10 2 、10 1 An amplification curve of a primer group PPV3 is adopted;
FIGS. 41-43 show the copy number of genome at 10 3 、10 2 、10 1 An amplification curve of a primer group PPV4 is adopted;
FIGS. 44-46 are graphs showing the copy number of genome at 10 3 、10 2 、10 1 An amplification curve of a primer group PPV5 is adopted;
FIGS. 47-49 show the copy number of genome at 10 3 、10 2 、10 1 An amplification curve of a primer group PPV6 is adopted;
FIGS. 50-52 are graphs showing the copy number of 10 in genome, respectively 3 、10 2 、10 1 An amplification curve of a primer group PPV7 is adopted;
FIGS. 53 to 59 are amplification curves at the time of specificity verification using the primer set PPV 1-PPV 7, respectively;
wherein, A-H in the figure is the color of the fluorescence curve corresponding to each reaction pool, and E4 in the figure is the copy number of the genome of 10 4 Amplification curve at the time of detection, E3 is at a genomic copy number of 10 3 Amplification curve in detection.
Detailed Description
The invention discloses a LAMP primer combination, a detection method and a detection kit capable of distinguishing the 1-7 porcine parvovirus types for parting detection. Those skilled in the art can modify the process parameters appropriately in view of the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products and are commercially available. The experimental procedures in the examples are conventional unless otherwise specified. Wherein the reaction solution containing the fluorescent dye is a product of Boo biological group, Inc., and the catalog number is CP.440020.
The DNA copy number was calculated as follows:
1a260 absorbance value ═ dsDNA 50 μ g/ml;
nucleic acid concentration (OD260) × (dilution factor) × (50) × ng/μ l;
average Molecular Weight (MW) represents grams per mole in daltons (dolton), i.e., 1dolton ═ 1 g/mol;
molar ratio of 6.02X 10 23 ；
Average Molecular Weight (MW) dsDNA ═ (number of bases) x (660 daltons/base);
copy number calculation formula:
(6.02×10 23 copies/mole) × (x ng/. mu.l.times.10 -9 ) /(DNA length. times.660) ═ copies/. mu.l.
Example 1 screening preparation of primer combinations
First, screening of primer combination
1. The primer sequences used are synthesized by the company of Producers, and a plurality of primer sets are designed aiming at the porcine parvovirus NS1 or VP2 genes, and the sequences of the primer sets are shown in Table 1.
TABLE 1 primer set sequences
In the above primer combination, each single-stranded DNA is packed independently.
In the above primer combinations, the molar ratios of the primer F3, the primer B3, the primer FIP, the primer BIP, the primer LF, and the primer LB in each primer set are all 0.3: 0.3: 2.4: 2.4: 1: 1.
2. and (2) taking porcine parvovirus type 1-7 detection gene plasmid DNA as a template, and respectively adopting the primer group prepared in the step (1) to carry out loop-mediated isothermal amplification detection on the template.
Porcine parvovirus type 6 detection gene plasmid: a DNA molecule with a conserved sequence compressed by KF999681.1, KP245953.1, KR709262.1 and KX384813.1 in Genebank number is inserted into a SacI site of a pUC57 plasmid (biological engineering (Shanghai) Co., Ltd.) to obtain a recombinant plasmid, and the plasmid is a porcine parvovirus type 6 detection gene plasmid.
Porcine parvovirus type 7 detection gene plasmid: a DNA molecule with a conserved sequence compressed by Genbank numbers of KY996756.1, KY996757.1, KY996758.1 and KU563733.1 is inserted into a SacI site of a pUC57 plasmid (a biological engineering (Shanghai) GmbH) to obtain a recombinant plasmid, wherein the plasmid is a porcine parvovirus 7 type detection gene plasmid.
Reaction system (20 μ L): 10. mu.L of a reaction solution containing a fluorescent dye (product of Boo Bio Inc., Catalogue No.: CP.440020), 2.96. mu.L of the primer mixture, 2. mu.L of the template DNA (5pg-50pg), and sterile purified water to 20. mu.L. The primer mixture is a mixture of each primer in the primer combination. In the reaction system, 0.12. mu.L of each of 0.3mM of the outer primers F3 and B3; 0.96 mu L of each of 2.4mM inner primers FIP and BIP; 0.4. mu.L each of 1mM loop primers LF and LB.
Reaction conditions are as follows: keeping the temperature at 65 ℃ for 50 min.
In the reaction process, a fluorescence PCR instrument is adopted to detect fluorescence signals.
Each reaction system was set up for 3 replicates.
If a positive amplification curve (i.e., an amplification curve that is typically a "sigmoid" amplification curve) appears within 45min, it indicates that the corresponding genomic content in the reaction system can be detected. If no positive amplification curve (i.e. amplification curve is typical "S-type" amplification curve) appears within 45min, it indicates that the corresponding genome content in the reaction system cannot be detected.
The screening experiment result of the porcine parvovirus type 1 primer is as follows: the results of the detection of the primer set 1 are shown in FIG. 1, the results of the detection of the primer set 2 are shown in FIG. 2, and the results of the detection of the primer set 3 are shown in FIG. 3, showing that the primer sets 1 and 2 are preferable.
The screening experiment result of the porcine parvovirus type 2 primer is as follows: the results of the detection of the primer set 1 are shown in FIG. 4, the detection of the primer set 2 is shown in FIG. 5, and the detection of the primer set 3 is shown in FIG. 6, showing that the primer set 1 is preferable.
The screening experiment result of the porcine parvovirus type 3 primer is as follows: the results of the detection of the primer set 1 are shown in FIG. 7, the detection of the primer set 2 is shown in FIG. 8, and the detection of the primer set 3 is shown in FIG. 9, showing that the primer set 1 is preferable.
The screening experiment result of the porcine parvovirus type 4 primer is as follows: the detection results of the primer set 1 are shown in FIG. 10, the detection results of the primer set 2 are shown in FIG. 11, and the detection results of the primer set 3 are shown in FIG. 12. The results showed that primer sets 2 and 3 were preferable.
The results of the porcine parvovirus type 5 primer screening experiment: the detection results of the primer set 1 are shown in FIG. 13, the detection results of the primer set 2 are shown in FIG. 14, and the detection results of the primer set 3 are shown in FIG. 15. The results showed that primer set 3 was preferable.
The screening experiment result of the porcine circovirus type 6 primer is as follows: the detection results of the primer set 1 are shown in FIG. 16, the detection results of the primer set 2 are shown in FIG. 17, and the detection results of the primer set 3 are shown in FIG. 18. The results showed that primer set 2 was preferable.
The screening experiment result of the porcine circovirus type 7 primer is as follows: the detection results of the primer set 1 are shown in FIG. 19, the detection results of the primer set 2 are shown in FIG. 20, and the detection results of the primer set 3 are shown in FIG. 21. The results showed that primer sets 1 and 2 were preferable.
Example 2 sensitivity prescreening
A sample to be detected: porcine parvovirus type 1-7 plasmids prepared in example 1.
1. And extracting plasmid DNA of a sample to be detected, and performing gradient dilution by using sterile water to obtain each diluent.
2. The diluent obtained in step 1 was used as a template for loop-mediated isothermal amplification using the primer combination prepared in example 1.
Reaction system (20 μ L): 10. mu.L of the reaction mixture (product catalog No. CP.440020, product of Boo Bio Inc.), 2.96. mu.L of the primer mixture, and 2. mu.L of the template diluent (each 1. mu.L of the diluent contains 10 copies of the genome 4 And 10 3 ) Water was added to 20. mu.L. The primer mixture is a mixture of each primer in the primer combination. In the reaction system, 0.12. mu.L of each of 0.3mM of the outer primers F3 and B3; 0.96 mu L of each of 2.4mM inner primers FIP and BIP; 0.4. mu.L each of 1mM loop primers LF and LB.
Reaction conditions are as follows: keeping the temperature at 65 ℃ for 50 min.
In the reaction process, a fluorescence PCR instrument is adopted to detect fluorescence signals.
The following 2 reaction systems were used in total, depending on the number of copies of the genome in the dilution:
reaction system 1: the number of copies of the genome contained in 1. mu.L of the dilution was 10 4 ；
Reaction system 2: the number of copies of the genome contained in 1. mu.L of the dilution was 10 3 ；
Each reaction system was set up for 3 replicates.
The genome copy number of the primer group PPV1-1 detection target gene in 1 mu L diluent is 10 4 And 10 3 (FIG. 22) 3 tests were detected with good reproducibility, and the primer set PPV1-2 had a gene copy number of 10 4 (FIG. 23) 3 tests showed complete peak-forming and good reproducibility, but the gene copy number was 10 3 In time, 3 detections cannot completely peak and have poor repeatability, so the primer group PPV1-1 is selected for sensitivity rescreening.
The genome copy number of the primer group PPV2-1 detection target gene in 1 mu L diluent is 10 4 And 10 3 (FIG. 24) all 3 assays were detectable with good reproducibility.
The genome copy number of the primer group PPV3-1 detection target gene in 1 mu L diluent is 10 4 And 10 3 (FIG. 25), 3 assays were detectable with good reproducibility.
Primer group PPV4-2 for detecting target gene, the genome copy number of which in 1 mu L of diluent is 10 4 And 10 3 (FIG. 26), 3 detections were all detected with good reproducibility, and the primer set PPV4-3 had a gene copy number of 10 4 (FIG. 27) 3 tests showed complete peak-forming and good reproducibility, but the gene copy number was 10 3 In time, 3 detections cannot completely peak and have poor repeatability, so the primer group PPV4-2 is selected for sensitivity rescreening.
The genome copy number of the primer group PPV5-3 detected target gene in 1 mu L diluent is 10 4 And 10 3 (FIG. 28), 3 assays were detectable with good reproducibility.
Primer group PPV6-2 for detecting target gene, the genome copy number of which in 1 mu L of diluent is 10 4 And 10 3 (FIG. 29), 3 assays were detectable with good reproducibility.
Primer group PPV7-1 testThe genome copy number of the target gene in 1 mu L of diluent is 10 4 And 10 3 (FIG. 30) 3 tests were detected with good reproducibility, and the primer set PPV7-2 had a gene copy number of 10 4 (FIG. 31) 3 tests showed complete peak-forming and good reproducibility, but the gene copy number was 10 3 In time, 3 detections cannot completely peak and have poor repeatability, so the primer group PPV7-1 is selected for sensitivity rescreening.
The results show that some screened primers have poor amplification effect after the concentration of the template is reduced, and the primers qualified in the primary screening are subjected to secondary screening to further reduce the sensitivity.
Example 3 sensitivity rescreening
A sample to be detected: porcine parvovirus type 1-7 plasmids prepared in example 1.
1. And extracting plasmid DNA of a sample to be detected, and performing gradient dilution by using sterile water to obtain each diluent.
2. The diluted solution obtained in step 1 was used as a template, and the primer combinations prepared in examples 1 and 2 were used for loop-mediated isothermal amplification.
Reaction system (20 μ L): 10. mu.L of the reaction mixture (product catalog No. CP.440020, product of Boo Bio Inc.), 2.96. mu.L of the primer mixture, and 2. mu.L of the template diluent (each 1. mu.L of the diluent contains 10 copies of the genome 3 、10 2 Or 10 1 ) Water was added to 20. mu.L. The primer mixture is a mixture of each primer in the primer combination. In the reaction system, 0.12. mu.L of each of 0.3mM of the outer primers F3 and B3; 0.96 mu L of each of 2.4mM inner primers FIP and BIP; 0.4. mu.L each of 1mM loop primers LF and LB.
The reaction conditions are as follows: keeping the temperature at 65 ℃ for 50 min.
In the reaction process, a fluorescence PCR instrument is adopted to detect fluorescence signals.
The total of 3 reaction systems are as follows according to the difference of genome copy number in the dilution liquid:
reaction system 1: the number of copies of the genome contained in 1. mu.L of the dilution was 10 3 ；
Reaction system 2: the number of copies of the genome contained in 1. mu.L of the dilution was 10 2 ；
Reaction system 3: the number of copies of the genome contained in 1. mu.L of the dilution was 10 1 ；
Each reaction system was set up for 20 replicates.
If a positive amplification curve (i.e., an amplification curve that is typically a "sigmoid" amplification curve) appears within 45min, it indicates that the corresponding genomic content in the reaction system can be detected. If no positive amplification curve (i.e. amplification curve is typical "S-type" amplification curve) appears within 45min, it indicates that the corresponding genome content in the reaction system cannot be detected.
The genome copy number of the primer group PPV1-1 detection target gene in 1 mu L diluent is 10 3 (FIG. 32) and 10 2 (FIG. 33) 20 assays were all detectable with good reproducibility, 10 1 (FIG. 34) 20 detections were incomplete and poor in reproducibility, so the sensitivity of the primer set was 100 copies/. mu.L
The genome copy number of the primer group PPV2-1 detection target gene in 1 mu L diluent is 10 3 (FIG. 35) and 10 2 (FIG. 36) 20 assays were all detectable with good reproducibility, 10 1 (FIG. 37) 20 detections were not completely peaked and were poorly reproducible, so the sensitivity of the primer set was 100 copies/. mu.L.
The genome copy number of the primer group PPV3-1 detection target gene in 1 mu L diluent is 10 3 (FIG. 38) and 10 2 (FIG. 39) 20 assays were all detectable with good reproducibility, 10 1 (FIG. 40) 20 detections were incomplete and poor in reproducibility, so the sensitivity of the primer set was 100 copies/. mu.L
Primer group PPV4-2 for detecting target gene, the genome copy number of which in 1 mu L of diluent is 10 3 (FIG. 41) and 10 2 (FIG. 42) 20 assays were all detectable with good reproducibility, 10 1 (FIG. 43) 20 measurements did not completely peak and were less reproducible, so the sensitivity of the primer set was 100 copies/. mu.L.
The genome copy number of the primer group PPV5-3 detected target gene in 1 mu L diluent is 10 3 (FIG. 44) and 10 2 (FIG. 45) 20 at the time ofAll the detection can be detected, and the repeatability is good, 10 1 (FIG. 46) 20 measurements did not completely peak and were less reproducible, so the sensitivity of the primer set was 100 copies/. mu.L.
Primer group PPV6-2 for detecting target gene, the genome copy number of which in 1 mu L of diluent is 10 3 (FIG. 47) and 10 2 (FIG. 48) 20 assays were all detectable with good reproducibility, 10 1 (FIG. 49) 20 detections were not completely peaked and were poorly reproducible, so the sensitivity of the primer set was 100 copies/. mu.L.
The genome copy number of the primer group PPV7-1 detection target gene in 1 mu L diluent is 10 3 (FIG. 50) and 10 2 (FIG. 51) 20 assays were all detectable with good reproducibility, 10 1 (FIG. 52) 20 measurements did not completely peak and were less reproducible, so the sensitivity of the primer set was 100 copies/. mu.L.
Example 4 specificity test
Classical swine fever virus Shimen strain AV1411(04/08/87) and highly pathogenic porcine reproductive and respiratory syndrome Virus (PRRSV-JXA1) were purchased from Chinese veterinary drug Authority, Swine influenza Virus (R) ((R))
VR-333 TM ) Purchased from ATCC, porcine pseudorabies virus (BNCC 129649), porcine parvovirus (BNCC124946) and porcine circovirus type 2 (BNCC128527) from BNCC, banna organisms; the porcine parvovirus type 2-7 plasmid is constructed by the production.
Each sample to be detected is respectively subjected to the following steps:
1. and extracting the genome DNA or RNA of the sample to be detected.
2. And (2) performing loop-mediated isothermal amplification by respectively adopting the primer groups obtained by screening in the example 3 by taking the genomic nucleic acid extracted in the step 1 as a template.
Reaction system (20 μ L): 10. mu.L of the reaction mixture (product catalog No. CP.440020 from Boo Bio Inc.), 2.96. mu.L of the primer mixture, 2. mu.L of the genomic template (5pg-50pg), 0.5. mu.L of AMV reverse transcriptase (added when the template is RNA), and 20. mu.L of RNase-free water. The primer mixture is a mixture of each primer in the primer combination. In the reaction system, 0.12. mu.L of each of 0.3mM of the outer primers F3 and B3; 0.96 mu L of 2.4mM of each of the inner primers FIP and BIP; 0.4. mu.L each of 1mM loop primers LF and LB.
Reaction conditions are as follows: keeping the temperature at 65 ℃ for 50 min.
In the reaction process, a fluorescence PCR instrument is adopted to detect fluorescence signals.
The result obtained using the primer set PPV1-1 is shown in FIG. 53, and a positive amplification curve (i.e., an amplification curve that is a typical "S-type" amplification curve) is shown only when the sample to be tested is a porcine parvovirus type 1 genomic DNA (sample to be tested 1). When the sample to be detected is porcine parvovirus type 2-7 plasmid DNA or porcine circovirus type 2 (PCV2), Classical Swine Fever Virus (CSFV), porcine pseudorabies virus (PRV), Swine Influenza Virus (SIV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), the sample does not show a positive amplification curve.
The results using the primer set PPV2-1 are shown in FIG. 54, and only when the sample to be tested is a porcine parvovirus type 2 plasmid DNA (sample 2 to be tested) shows a positive amplification curve (i.e., the amplification curve is a typical "S-type" amplification curve). When the sample to be detected is other types of porcine parvovirus or porcine circovirus type 2 (PCV2), Classical Swine Fever Virus (CSFV), porcine pseudorabies virus (PRV), Swine Influenza Virus (SIV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), the sample does not show a positive amplification curve.
The results using the primer set PPV3-1 are shown in FIG. 55, and only when the sample to be tested is a porcine parvovirus type 3 plasmid DNA (sample to be tested 3) shows a positive amplification curve (i.e., the amplification curve is a typical "S-type" amplification curve). When the sample to be detected is other types of porcine parvovirus or porcine circovirus type 2 (PCV2), Classical Swine Fever Virus (CSFV), porcine pseudorabies virus (PRV), Swine Influenza Virus (SIV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), the sample does not show a positive amplification curve.
The results using the primer set PPV4-2 are shown in FIG. 56, and only when the sample to be tested is a swine parvovirus type 4 plasmid DNA (sample to be tested 4) shows a positive amplification curve (i.e., the amplification curve is a typical "S-type" amplification curve). When the sample to be detected is other types of porcine parvovirus or porcine circovirus type 2 (PCV2), Classical Swine Fever Virus (CSFV), porcine pseudorabies virus (PRV), Swine Influenza Virus (SIV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), the sample does not show a positive amplification curve.
The results using the primer set PPV5-3 are shown in FIG. 57, and only when the sample to be tested is a porcine parvovirus type 5 plasmid DNA (sample to be tested 5) shows a positive amplification curve (i.e., the amplification curve is a typical "S-type" amplification curve). When the sample to be detected is other types of porcine parvovirus or porcine circovirus type 2 (PCV2), Classical Swine Fever Virus (CSFV), porcine pseudorabies virus (PRV), Swine Influenza Virus (SIV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), the sample does not show a positive amplification curve.
The results using the primer set PPV6-2 are shown in FIG. 58, and only when the sample to be tested is a swine parvovirus type 6 plasmid DNA (sample to be tested 6) shows a positive amplification curve (i.e., the amplification curve is a typical "S-type" amplification curve). When the sample to be detected is other types of porcine parvovirus or porcine circovirus type 2 (PCV2), Classical Swine Fever Virus (CSFV), porcine pseudorabies virus (PRV), Swine Influenza Virus (SIV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), the sample does not show a positive amplification curve.
The results using the primer set PPV7-1 are shown in FIG. 59, and only when the sample to be tested is a porcine parvo type 7 plasmid DNA (sample to be tested 7) shows a positive amplification curve (i.e., the amplification curve is a typical "S-type" amplification curve). When the sample to be detected is other types of porcine parvovirus or porcine circovirus type 2 (PCV2), Classical Swine Fever Virus (CSFV), porcine pseudorabies virus (PRV), Swine Influenza Virus (SIV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), the sample does not show a positive amplification curve.
The results show that the primer group has high specificity to the target gene.
The Applicant modified the reaction system in the same manner as in the above examples, and in some examples, the reaction system was 0.12. mu.L each of the 0.29mM outer primers F3 and B3; 0.96 mu L of each of 2.3mM of inner primers FIP and BIP; 0.9mM of loop primers LF and LB were each 0.4. mu.L. In other embodiments, the reaction system is 0.12. mu.L of each of 0.31mM outer primers F3 and B3; 0.96 mu L of each of 2.5mM inner primers FIP and BIP; 0.4. mu.L each of 1.1mM loop primers LF and LB. The results show that the specificity and sensitivity are similar to the results of the above examples.
In summary, the optimal primer set for detecting porcine parvovirus type 1 was selected for the porcine parvovirus type 1 NS1 gene as follows (5 '→ 3'):
outer primer PPV 1-F3: 5'-cttggttggtaaagaaaggt-3' (SEQ ID NO.1)
Outer primer PPV 1-B3: 5'-agctaaatccaggtcctc-3' (SEQ ID NO.2)
Inner primer PPV 1-FIP: 5'-tggggtttgcatttttggcggctagctatatgcatcattg-3' (SEQ ID NO.3)
Inner primer PPV 1-BIP: 5'-tacaccaacagactctcagatttcattggagttgctgcgta-3' (SEQ ID NO.4)
Loop primer PPV 1-LF: 5'-gaccaatcaggtacatttcc-3' (SEQ ID NO.5)
The loop primer PPV 1-LB: 5'-acatcagtgaaaacttcgc-3' (SEQ ID NO.6)
The optimal primer set for detecting porcine parvovirus type 2 was selected against the porcine parvovirus type 2 NS1 gene as follows (5 '→ 3'):
outer primer PPV 2-F3: 5'-gcaaatggagcccagcag-3' (SEQ ID NO.7)
Outer primer PPV 2-B3: 5'-tgatcgccttcccaccag-3' (SEQ ID NO.8)
Inner primer PPV 2-FIP: 5'-ttcagccacccccccatctcttcaccttcatctcgcagtg-3' (SEQ ID NO.9)
Inner primer PPV 2-BIP: 5'-atgatagagcacaggccaggcgagcggtcttcctcatctca-3' (SEQ ID NO.10)
Loop primer PPV 2-LF: 5'-ctcgggcctttgtgtcg-3' (SEQ ID NO.11)
The loop primer PPV 2-LB: 5'-gccccccccttgtactt-3' (SEQ ID NO.12)
The optimal primer set for detecting porcine parvovirus type 3 was selected against the porcine parvovirus type 3 NS1 gene as follows (5 '→ 3'):
outer primer PPV 3-F3: 5'-agcaactggttgaagagatgg-3' (SEQ ID NO.13)
Outer primer PPV 3-B3: 5'-gcagcctcaaatctctccatg-3' (SEQ ID NO.14)
Inner primer PPV 3-FIP: 5'-agtctcccttgtctggtcttcctcaacattcctaactcgccaca-3' (SEQ ID NO.15)
Inner primer PPV 3-BIP: 5'-atcagtgcgacctgacctttgtcaagcataccagacatgctcta-3' (SE Q ID NO.16)
Loop primer PPV 3-LF: 5'-gatatcccacagaatgctccaac-3' (SEQ ID NO.17)
The loop primer PPV 3-LB: 5'-aaggtatctactgcctaaagtacca-3' (SEQ ID NO.18)
The optimal primer set for detecting porcine parvovirus type 4 was selected against the porcine parvovirus type 4 NS1 gene as follows (5 '→ 3'):
outer primer PPV 4-F3: 5'-gacattatgaactgccctat-3' (SEQ ID NO.19)
Outer primer PPV 4-B3: 5'-agggatgaattcagtctcag-3' (SEQ ID NO.20)
Inner primer PPV 4-FIP: 5'-ttcatttcttttgggggtgggtaaaaaaggacctgccagt-3' (SEQ ID NO.21)
Inner primer PPV 4-BIP: 5'-agagcggaaccagatgaaattcgtttcttctttctcggtgc-3' (SEQ ID NO.22)
Loop primer PPV 4-LF: 5'-tctctccaacaggaactaaa-3' (SEQ ID NO.23)
The loop primer PPV 4-LB: 5'-aatccagaagaactggacc-3' (SEQ ID NO.24)
The optimal primer set for detecting porcine parvovirus type 5 was selected against the porcine parvovirus type 5 VP2 gene as follows (5 '→ 3'):
outer primer PPV 5-F3: 5'-ggaacatcaccaatcaaga-3' (SEQ ID NO.25)
Outer primer PPV 5-B3: 5'-gagagatcaatttcattgcg-3' (SEQ ID NO.26)
Inner primer PPV 5-FIP: 5'-agttctttctgttctcggtgacctattagaacacgactcaagc-3' (SEQ ID NO.27)
Inner primer PPV 5-BIP: 5'-atcaaacatggagcgggagatttccaggacctgtgtag-3' (SEQ ID NO.28)
Loop primer PPV 5-LF: 5'-gcttcttctggttgttcttc-3' (SEQ ID NO.29)
The loop primer PPV 5-LB: 5'-cggaaccggtatcaact-3' (SEQ ID NO.30)
The optimal primer set for detecting porcine parvovirus type 6 was selected against the porcine parvovirus type 6 NS1 gene as follows (5 '→ 3'):
outer primer PPV 6-F3: 5'-tcaagatcagggagtcatt-3' (SEQ ID NO.31)
Outer primer PPV 6-B3: 5'-tagtgctcacaatacgct-3' (SEQ ID NO.32)
Inner primer PPV 6-FIP: 5'-gtagtcacatcattatcacacatccactgctcctacttttaaagc-3' (SE Q ID NO.33)
Inner primer PPV 6-BIP: 5'-cctgtactctttccttgctaccgcagctctaagacactgt-3' (SEQ ID NO.34)
Loop primer PPV 6-LF: 5'-cattttatcacccgtagcc-3' (SEQ ID NO.35)
The loop primer PPV 6-LB: 5'-ccagctggtactcatttg-3' (SEQ ID NO.36)
The optimal primer set for detecting porcine parvovirus type 7 was selected against the porcine parvovirus type 7 VP2 gene as follows (5 '→ 3'):
outer primer PPV 7-F3: 5'-ggtaccacaactggtacgaag-3' (SEQ ID NO.37)
Outer primer PPV 7-B3: 5'-tggtgttccatgtccatgtc-3' (SEQ ID NO.38)
Inner primer PPV 7-FIP: 5'-cggtggttggtttagggacgagacacgctctggagaagct-3' (SEQ ID NO.39)
Inner primer PPV 7-BIP: 5'-accaaagaaggggtcggaaacaggtttcgggtgccgtgatg-3' (S EQ ID NO.40)
Loop primer PPV 7-LF: 5'-ccttctttgtaggccacgt-3' (SEQ ID NO.41)
The loop primer PPV 7-LB: 5'-tggagcagacttacactccc-3' (SEQ ID NO. 42).
Sequence listing
<110> Boao bionts Ltd
Beijing animal disease prevention and control center
<120> LAMP primer combination, detection method and kit capable of distinguishing 1-7 porcine parvovirus types for typing detection
<130> MP1905396
<160> 42
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agctaaatcc aggtcctc 18
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<211> 40
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<400> 3
tggggtttgc atttttggcg gctagctata tgcatcattg 40
<210> 4
<211> 41
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<400> 4
tacaccaaca gactctcaga tttcattgga gttgctgcgt a 41
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acatcagtga aaacttcgc 19
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ttcagccacc cccccatctc ttcaccttca tctcgcagtg 40
<210> 10
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<400> 10
atgatagagc acaggccagg cgagcggtct tcctcatctc a 41
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<400> 11
ctcgggcctt tgtgtcg 17
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gcccccccct tgtactt 17
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agcaactggt tgaagagatg g 21
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gcagcctcaa atctctccat g 21
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agtctccctt gtctggtctt cctcaacatt cctaactcgc caca 44
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atcagtgcga cctgaccttt gtcaagcata ccagacatgc tcta 44
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aaggtatcta ctgcctaaag tacca 25
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ttcatttctt ttgggggtgg gtaaaaaagg acctgccagt 40
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agagcggaac cagatgaaat tcgtttcttc tttctcggtg c 41
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ggaacatcac caatcaaga 19
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atcaaacatg gagcgggaga tttccaggac ctgtgtag 38
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<400> 29
gcttcttctg gttgttcttc 20
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cggaaccggt atcaact 17
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tcaagatcag ggagtcatt 19
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gtagtcacat cattatcaca catccactgc tcctactttt aaagc 45
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cctgtactct ttccttgcta ccgcagctct aagacactgt 40
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cattttatca cccgtagcc 19
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ggtaccacaa ctggtacgaa g 21
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cggtggttgg tttagggacg agacacgctc tggagaagct 40
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<213> Artificial Sequence (Artificial Sequence)
<400> 40
accaaagaag gggtcggaaa caggtttcgg gtgccgtgat g 41
<210> 41
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ccttctttgt aggccacgt 19
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Claims (5)
1. A LAMP primer combination capable of distinguishing the typing detection of porcine parvovirus types 1-7 comprises a primer group 1-7;
the primer group 1 comprises 2 outer primers shown by SEQ ID NO.1 and SEQ ID NO.2, 2 inner primers shown by SEQ ID NO.3 and SEQ ID NO.4 and 2 loop primers shown by SEQ ID NO.5 and 6;
the primer group 2 comprises 2 outer primers shown by SEQ ID NO.7 and SEQ ID NO.8, 2 inner primers shown by SEQ ID NO.9 and SEQ ID NO.10, and 2 loop primers shown by SEQ ID NO.11 and 12;
the primer group 3 comprises 2 outer primers shown by SEQ ID NO.13 and SEQ ID NO.14, 2 inner primers shown by SEQ ID NO.15 and SEQ ID NO.16 and 2 loop primers shown by SEQ ID NO.17 and 18;
the primer group 4 comprises 2 outer primers shown by SEQ ID NO.19 and SEQ ID NO.20, 2 inner primers shown by SEQ ID NO.21 and SEQ ID NO.22 and 2 loop primers shown by SEQ ID NO.23 and 24;
the primer group 5 comprises 2 outer primers shown by SEQ ID NO.25 and SEQ ID NO.26, 2 inner primers shown by SEQ ID NO.27 and SEQ ID NO.28, and 2 loop primers shown by SEQ ID NO.29 and SEQ ID NO. 30;
the primer group 6 comprises 2 outer primers shown by SEQ ID NO.31 and SEQ ID NO.32, 2 inner primers shown by SEQ ID NO.33 and SEQ ID NO.34, and 2 loop primers shown by SEQ ID NO.35 and 36;
the primer group 7 comprises 2 outer primers shown by SEQ ID NO.37 and SEQ ID NO.38, 2 inner primers shown by SEQ ID NO.39 and SEQ ID NO.40, and 2 loop primers shown by SEQ ID NO.41 and 42.
2. A LAMP kit for typing detection capable of distinguishing porcine parvovirus types 1 to 7, comprising the primer combination of claim 1, wherein each primer in the primer combination is independently packaged.
3. The kit according to claim 2, further comprising a reaction solution containing a fluorescent dye and/or sterilized pure water.
4. The kit according to claim 2, wherein the mole numbers of the 2 outer primers, the 2 inner primers and the 2 loop primers in each primer group in the primer combination are the same, and the mole numbers of the 2 loop primers are the same, and the mole ratio of the outer primers to the inner primers to the loop primers in each primer group is (0.58-0.62): (4.6-5.0): (1.8-2.2).
5. The kit according to claim 2, wherein the molar ratio of the outer primer to the inner primer to the loop primer in each primer set is 0.6:4.8: 2.
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