CN112795670A - Specific PCR amplification primer pair for detecting tricholoma alveorum, detection method and application - Google Patents

Specific PCR amplification primer pair for detecting tricholoma alveorum, detection method and application Download PDF

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CN112795670A
CN112795670A CN202110134048.1A CN202110134048A CN112795670A CN 112795670 A CN112795670 A CN 112795670A CN 202110134048 A CN202110134048 A CN 202110134048A CN 112795670 A CN112795670 A CN 112795670A
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沈克飞
罗文华
徐登峰
曹兰
杨柳
高丽娇
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Chongqing Academy of Animal Sciences
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Abstract

The invention belongs to the technical field of biological pathogen detection, and particularly relates to a specific PCR amplification primer pair for detecting trichomonas alvei, a detection method and application. The detection method comprises the steps of firstly extracting bee larva DNA, and then carrying out detection by using a primer pair WxlF 2: 5'-ACTGATCCAACAGATCCAAC-3', WxlR2: 5'-TCCCTTAAAGTCAGACAAGC-3'; or WxlF 3: 5'-AACAGATCCAACTGGTCAAA-3', WxlR 3: 5'-CTTAAAGTCAGACAAGCCGA-3' PCR amplification is carried out, and finally electrophoresis is carried out to observe the amplification condition so as to judge whether the trichotheca is carried. The detection method does not need to separate the single colony of the honeycomb coccus, is simple and easy to implement, can control the PCR amplification time within 1h, and can meet the requirement of quickly detecting European foul brood.

Description

Specific PCR amplification primer pair for detecting tricholoma alveorum, detection method and application
Technical Field
The invention belongs to the technical field of biological pathogen detection, and particularly relates to a specific PCR amplification primer pair for detecting trichomonas alvei, a detection method and application.
Technical Field
European Foul Brood (EFB), a malignant bacterial digestive tract disease occurring in bee larvae and pupae, occurs worldwide. The etiological agent of EFB is mainly the bacterium Behcet (Melissococcus pluton). EFB is defined as two kinds of animal epidemic diseases by the world animal health Organization (OIE), and Chinese bees (Chinese bees for short) are more susceptible than western bees and have more serious disease degree than the western bees. The larvae ingest food contaminated with the nidus Vespae causing infection, and subsequently the nidus Vespae colonizes the larval gut. The nidus Vespae mainly attacks digestive system of larvae of 1-2 days old, and mostly dies when the larvae of 3-4 days old are not covered, dead bodies have acid odor, and dead bodies are white or light yellow, and become brown after being dried. Chinese bee diseases are common in China, and are mostly generated in two seasons of spring and autumn, the disease condition is light in autumn, and the disease is less generated in summer, so that spring reproduction and autumn reproduction are influenced. The disease is spread rapidly and has a quick onset. Infected larvae and contaminated environments are the main sources of infection, adult worker bees become transmitters during activity, and sometimes bee-keeping persons also transmit during feeding and cleaning operations. The nidus Vespae can continue to survive for several years in bee colony, and mainly takes prevention and treatment combination and prevention-oriented measures in production. Therefore, detection of early infection by pathogens is critical. The nidus Vespae is difficult to separate out single colonies due to the strict requirement on culture conditions, and the detection period is long according to the traditional routes of bacteria separation, culture, identification and the like, so that the timeliness requirement of bacteria identification cannot be met. Nucleic acid detection is a commonly used method for rapid detection of pathogens.
The patent CN101792799A discloses a PCR rapid detection method for bee European foul brood, which adopts a semi-nested PCR amplification method, firstly, primers EFB1 and EFB2 are designed according to 16S rRNA gene of the bee hive coccus to carry out first round PCR amplification, the amplification result can distinguish the bee hive coccus from other mixed bacteria, but can not distinguish enterococcus faecalis which is close to the bee hive coccus; and then designing a third primer EFB3 by taking the obtained amplification product as a template, wherein the position of the third primer EFB3 is positioned between the EFB1 primer and the EFB2 primer, carrying out second round PCR amplification by taking the EFB1 primer and the EFB3 primer, and detecting the PCR amplification product by agarose gel electrophoresis. The invention uses two times of PCR amplification for distinguishing different bacteria, and the detection process is relatively complicated.
The patent CN107177690A discloses a dual TaqMan fluorescence quantitative PCR detection method for pathogens of bee larva putrid disease, which utilizes two probes, wherein the 5 ' end of one probe adopts relatively mature FAM as a fluorescence reporter group, the 5 ' end of the other probe selects VIC as a marker, and the 3 ' ends of the two probes select BHQ1 non-fluorescence quenching groups.
The invention discloses a fluorescent quantitative PCR detection kit for detecting bee-hive cocci and a detection method thereof in patent CN106755494A, wherein the fluorescent quantitative PCR detection kit and the method for detecting the bee-hive cocci are prepared by utilizing a specific conserved sequence design primer and a probe of the bee-hive cocci 16SrRNA and optimizing a reaction system.
The existing conventional PCR and fluorescence PCR are adopted in the aspect of the bee honeycomb cocci nucleic acid detection technology, although the sensitivity of the fluorescence PCR detection method is higher than that of the conventional PCR, a fluorescence PCR instrument is required, the requirement on the instrument is relatively high, and the basic layer has defects in instrument configuration. In addition, the cost of fluorescent PCR is 1/5 to 1/3 higher than that of conventional PCR. One technology in the prior art is biased to progressively distinguish different pathogens layer by layer to finally identify the meliccos alvei, and the other technology does not progressively distinguish monosomyia alvei layer by layer, but has the problems of long detection time, complex detection process and low detection sensitivity in the detection process.
Disclosure of Invention
In view of this, the present invention searches for putative fibronectin/fibrinogen-binding protein (Fib), WxL-domain-containing protein (WxL domain-binding protein, WxL), bacterial immunoglobulin-like domain-containing protein (bacterial Ig-protein-binding protein, Ig), siderophore ABC transporter substrate-binding protein (iron-siderophore ABC transporter substrate-binding protein, Sid), iron-siderophore ABC transporter family permease subunit (iron-siderophore ABC transporter family XT, protein), bacteriocin export transporter (cell wall containing ABC anchor protein, LPprotein-LPprotein), and the like of the nidus Vespae, based on the genome (GenBank NO. DAT1033) information of the nidus Vespae, LPXTG), designing primers according to the gene sequence by adopting a Primer-BLAST method, and screening a short-time-consuming, specific and sensitive PCR detection method for the trichomonas from the following 4 aspects: (1) in the PCR reaction, the time can be reduced by the extension time in the cycle, the extension time in the PCR cycle is set to be 10sec, and the method which takes short time is selected from the PCR method based on 17 pairs of primers; (2) the source of the used template DNA is a larva sample which also contains larva resident microorganisms, and a healthy larva sample in a non-morbidity stage is detected instead of pathogen DNA which is subjected to purification culture, so that the PCR amplification has higher requirements on methodability, and the specificity of the method is also tested; (3) comparing the influence of the sample genome DNA extraction method on the PCR amplification result, and screening a specific PCR method with strong adaptability; (4) the sensitivity of the PCR method is screened and tested by using diluted sample genomic DNA instead of specific cloned DNA fragments, so that the sensitive method is screened from the perspective of sample attributes (target copy number, sample purity and the like) rather than pure technology; (5) sequencing and sequence alignment of the PCR amplified bands from 50 samples was performed to ensure that the amplified bands were specific DNA for M.alvei. From the detection result, a specific and sensitive PCR detection method for the trichomonas alvei is established, and the PCR amplification time of the detection method is within 1 h.
The invention aims to provide a specific PCR amplification primer pair for detecting a Melissococcus pluton.
The primer pair is one of the following primer pairs:
(1)FibF2：5′-TAACTATCCGTGCAAATGGG-3′，
FibR2：5′-AGATCATTGCCTTGCTGTTT-3′；
(2)FibF3：5′-ATCCGTGCAAATGGGAAAAA-3′，
FibR3：5′-AGGGGATCAGCTAGAGAAAG-3′；
(3)WxlF1：5′-GTTCAAGCAGAAAACACGAC-3′，
WxlR1：5′-AACTGCCCATTCCAGTATCT-3′；
(4)WxlF2：5′-ACTGATCCAACAGATCCAAC-3′，
WxlR2：5′-TCCCTTAAAGTCAGACAAGC-3′；
(5)WxlF3：5′-AACAGATCCAACTGGTCAAA-3′，
WxlR3：5′-CTTAAAGTCAGACAAGCCGA-3′；
(6)IgF1：5′-ATGGGAAATGGCGAAAAATG-3′，
IgR1：5′-TTAATGGCTGCTTTTTGTGG-3′；
(7)IgF2：5′-ATCAAATGGGAAATGGCGAA-3′，
IgR2：5′-GCCGTAAATCCCCAATAGAC-3′；
(8)IgF3：5′-ATTGGGGATTTACGGCATCA-3′，
IgR3：5′-ATGGTAATCGCCCAGTCTTT-3′；
(9)SidF1：5′-TCGCCTTATCCTCTTTTCCA-3′，
SidR1：5′-GTTAGGTGCTGTTACGGTTG-3′；
(10)SidF2：5′-CGCCTTATCCTCTTTTCCAG-3′，
SidR2：5′-AAATGTTAGGCCGTTAGGTG-3′；
(11)SidF3：5′-GGTTCCTCCTTGATAACCAC-3′，
SidR3：5′-TCAATGGAAGATGCAGTAGC-3′；
(12)CheF1：5′-CTGTCTACCGCTTTAGTTGG-3′，
CheR1：5′-TTTGTTGCTTCCTTTCACCT-3′；
(13)CheF2：5′-CGTGGAAAACGACTAATTGCT-3′，
CheR2：5′-TCCAACTAAAGCGGTAGACA-3′；
(14)BacF1：5′-GCGAGATGATTGCCATTGTT-3′，
BacR1：5′-TTCGTCTGCCAAAATCACAC-3′；
(15)BacF2：5′-GATGATTGCCATTGTTGGAG-3′，
BacR2：5′-TAATGACTGTTTTGCCCTGT-3′；
(16)BacF3：5′-AACCTAACAGCAAACTAGCA-3′，
BacR3：5′-CGATAATGACTGTTTTGCCC-3′；
(17)LPXTGF1：5′-TTGGAGAAGCTGTGACTTAC-3′，
LPXTGR1：5′-TTACGCCTGGTATTGTTTCA-3′。
the invention also aims to provide a method for detecting the honeycomb cocci of the bee larvae, which is simple and easy to implement, does not need to separate out a single colony for detection, and has high detection sensitivity and accurate detection result.
The method comprises the following steps: (1) extracting genome DNA from a sample to be detected; (2) performing PCR amplification by using one of the following specific PCR amplification primer pairs;
(1)FibF2：5′-TAACTATCCGTGCAAATGGG-3′，
FibR2：5′-AGATCATTGCCTTGCTGTTT-3′；
(2)FibF3：5′-ATCCGTGCAAATGGGAAAAA-3′，
FibR3：5′-AGGGGATCAGCTAGAGAAAG-3′；
(3)WxlF1：5′-GTTCAAGCAGAAAACACGAC-3′，
WxlR1：5′-AACTGCCCATTCCAGTATCT-3′；
(4)WxlF2：5′-ACTGATCCAACAGATCCAAC-3′，
WxlR2：5′-TCCCTTAAAGTCAGACAAGC-3′；
(5)WxlF3：5′-AACAGATCCAACTGGTCAAA-3′，
WxlR3：5′-CTTAAAGTCAGACAAGCCGA-3′；
(6)IgF1：5′-ATGGGAAATGGCGAAAAATG-3′，
IgR1：5′-TTAATGGCTGCTTTTTGTGG-3′；
(7)IgF2：5′-ATCAAATGGGAAATGGCGAA-3′，
IgR2：5′-GCCGTAAATCCCCAATAGAC-3′；
(8)IgF3：5′-ATTGGGGATTTACGGCATCA-3′，
IgR3：5′-ATGGTAATCGCCCAGTCTTT-3′；
(9)SidF1：5′-TCGCCTTATCCTCTTTTCCA-3′，
SidR1：5′-GTTAGGTGCTGTTACGGTTG-3′；
(10)SidF2：5′-CGCCTTATCCTCTTTTCCAG-3′，
SidR2：5′-AAATGTTAGGCCGTTAGGTG-3′；
(11)SidF3：5′-GGTTCCTCCTTGATAACCAC-3′，
SidR3：5′-TCAATGGAAGATGCAGTAGC-3′；
(12)CheF1：5′-CTGTCTACCGCTTTAGTTGG-3′，
CheR1：5′-TTTGTTGCTTCCTTTCACCT-3′；
(13)CheF2：5′-CGTGGAAAACGACTAATTGCT-3′，
CheR2：5′-TCCAACTAAAGCGGTAGACA-3′；
(14)BacF1：5′-GCGAGATGATTGCCATTGTT-3′，
BacR1：5′-TTCGTCTGCCAAAATCACAC-3′；
(15)BacF2：5′-GATGATTGCCATTGTTGGAG-3′，
BacR2：5′-TAATGACTGTTTTGCCCTGT-3′；
(16)BacF3：5′-AACCTAACAGCAAACTAGCA-3′，
BacR3：5′-CGATAATGACTGTTTTGCCC-3′；
(17)LPXTGF1：5′-TTGGAGAAGCTGTGACTTAC-3′，
LPXTGR1：5′-TTACGCCTGGTATTGTTTCA-3′。
because insects have fat bodies, the influence of fat residues on PCR amplification needs to be considered when extracting the DNA of the bee sample.
As a scheme, the sample to be detected in the step (1) is bee larva exudate which is frozen bee larva thawed exudate or bee larva cadaver exudate.
Further, the method for gene extraction includes, but is not limited to, a concentrated salt method, an anionic detergent method, and a proteinase K method, and the existing methods for extracting DNA are all suitable for the detection method, and the existing DNA extraction kit can also be used for DNA extraction.
Further, the genome DNA obtained by gene extraction of the bee larva exudate is diluted by 10 multiplied by or 100 multiplied by or 1000 multiplied by or 10000 multiplied by the DNA, and then PCR amplification is carried out by using one of the following specific PCR amplification primer pairs:
(4)WxlF2：5′-ACTGATCCAACAGATCCAAC-3′，
WxlR2：5′-TCCCTTAAAGTCAGACAAGC-3′；
(5)WxlF3：5′-AACAGATCCAACTGGTCAAA-3′，
WxlR3：5′-CTTAAAGTCAGACAAGCCGA-3′；
(6)IgF1：5′-ATGGGAAATGGCGAAAAATG-3′，
IgR1：5′-TTAATGGCTGCTTTTTGTGG-3′；
(7)IgF2：5′-ATCAAATGGGAAATGGCGAA-3′，
IgR2：5′-GCCGTAAATCCCCAATAGAC-3′；
(8)IgF3：5′-ATTGGGGATTTACGGCATCA-3′，
IgR3：5′-ATGGTAATCGCCCAGTCTTT-3′；
(9)SidF1：5′-TCGCCTTATCCTCTTTTCCA-3′，
SidR1：5′-GTTAGGTGCTGTTACGGTTG-3′；
(11)SidF3：5′-GGTTCCTCCTTGATAACCAC-3′，
SidR3：5′-TCAATGGAAGATGCAGTAGC-3′；
(12)CheF1：5′-CTGTCTACCGCTTTAGTTGG-3′，
CheR1：5′-TTTGTTGCTTCCTTTCACCT-3′；
(13)CheF2：5′-CGTGGAAAACGACTAATTGCT-3′，
CheR2：5′-TCCAACTAAAGCGGTAGACA-3′；
(14)BacF1：5′-GCGAGATGATTGCCATTGTT-3′，
BacR1：5′-TTCGTCTGCCAAAATCACAC-3′；
(15)BacF2：5′-GATGATTGCCATTGTTGGAG-3′，
BacR2：5′-TAATGACTGTTTTGCCCTGT-3′。
as another scheme, the method for detecting the honeycomb coccus comprises the following steps (1): carrying out gene extraction on the bee larva tissue or bee larva exudate by using a proteinase K method to obtain genome DNA, wherein the bee larva tissue is in vitro bee larva tissue. The bee larva exudate is frozen bee larva thawed exudate or bee larva cadaver exudate.
As another scheme, the method for detecting the trichotheca apiacea further comprises the following steps: the step (1) is as follows: carrying out gene extraction on the bee larva tissues or bee larva exudates by using a modified proteinase K method to obtain genome DNA, wherein the modified proteinase K method comprises the following steps: prolonging the digestion time of the proteinase K to be not less than 6h, and keeping other steps unchanged; or the improved proteinase K method is as follows: after digestion with proteinase K, the digest was extracted with chloroform and then subjected to the next step. The bee larva tissue is in vitro bee larva tissue. The bee larva exudate is frozen bee larva thawed exudate or bee larva cadaver exudate.
As another aspect, the method for detecting a pneumococcal bacterium includes: the method comprises the following steps: carrying out gene extraction on the bee larva tissues to obtain larva genome DNA, and carrying out PCR amplification by using the following specific PCR amplification primer pairs:
(4)WxlF2：5′-ACTGATCCAACAGATCCAAC-3′
WxlR2: 5'-TCCCTTAAAGTCAGACAAGC-3'; or
(5)WxlF3：5′-AACAGATCCAACTGGTCAAA-3′，
WxlR 3: 5'-CTTAAAGTCAGACAAGCCGA-3' are provided. The bee larva tissue is in vitro bee larva tissue. The detection time of the detection method is within 1h, and the rapid detection requirement of detecting the bee larva and the honeycomb coccus can be met.
Further, the PCR amplification process in all the above-mentioned schemes of the method for detecting M.mellifera comprises: 94 ℃ for 2min and 94 ℃ for 30 s; 30s at 57 ℃; 72 ℃ for 10 s. The number of cycles is carried out as the case requires.
In certain embodiments, the PCR amplification is specifically: 94 ℃ for 2 min; 30 cycles of 94 ℃ for 30s, 57 ℃ for 30s and 72 ℃ for 10 s; 3min at 72 ℃; storing at room temperature.
Further, the reaction system of the PCR amplification is as follows: 2 XPCR Mix 10. mu.L; 0.5 mu L of upstream primer (10 mu mol/L); 0.5 mu L of downstream primer (10 mu mol/L); 0.5. mu.L of DNA; deionized water 8.5. mu.L.
Further, all the aforementioned schemes of the method for detecting the bee larva honeycomb coccus also include the judgment standard: obtaining an amplification product after the PCR amplification, carrying out electrophoresis on the amplification product, and if a specific band is amplified, determining that the amplification product is positive; if no specific band is amplified, the amplification result is negative.
In certain embodiments, the electrophoresis is specifically: adding the amplification product into the sample adding hole, using 1 XTAE buffer solution as electrophoresis solution, carrying out 1.2% agarose gel electrophoresis under constant pressure of 120V, after 20min of electrophoresis, placing the gel in a gel imager to photograph so as to observe the PCR result.
The invention aims to further provide an application of Wxl primer pairs in detecting the trichotheca alvei, wherein the Wxl primer pair has the following sequence:
WxlF2：5′-ACTGATCCAACAGATCCAAC-3′，WxlR2:5′-TCCCTTAAAGTCAGACAAGC-3′；
or WxlF 3: 5'-AACAGATCCAACTGGTCAAA-3' the flow of the air in the air conditioner,
WxlR3：5′-CTTAAAGTCAGACAAGCCGA-3′。
the invention also aims to provide a kit for detecting the trichomonas campestris by using the specific PCR amplification primer pair. The kit can quickly and accurately detect the nidus vespae cocci.
The sample to be detected of the kit comprises: bee larva tissue and bee larva exudate; the bee larva tissue is in vitro bee larva tissue; the bee larva exudate is frozen bee larva thawed exudate or bee larva cadaver exudate.
Specifically, when the kit contains a primer pair
WxlF2：5′-ACTGATCCAACAGATCCAAC-3′，
WxlR2:5′-TCCCTTAAAGTCAGACAAGC-3′；
Or WxlF 3: 5'-AACAGATCCAACTGGTCAAA-3' the flow of the air in the air conditioner,
WxlR 3: 5'-CTTAAAGTCAGACAAGCCGA-3', the test sample is bee larva tissue or bee larva exudate, the DNA extraction method includes but is not limited to salt concentration method, anion detergent method, proteinase K method, the existing DNA extraction method is suitable for the test method, and the existing DNA extraction kit can be used for DNA extraction.
When the kit comprises one of the following primer pairs:
(4)WxlF2：5′-ACTGATCCAACAGATCCAAC-3′，
WxlR2：5′-TCCCTTAAAGTCAGACAAGC-3′；
(5)WxlF3：5′-AACAGATCCAACTGGTCAAA-3′，
WxlR3：5′-CTTAAAGTCAGACAAGCCGA-3′；
(6)IgF1：5′-ATGGGAAATGGCGAAAAATG-3′，
IgR1：5′-TTAATGGCTGCTTTTTGTGG-3′；
(7)IgF2：5′-ATCAAATGGGAAATGGCGAA-3′，
IgR2：5′-GCCGTAAATCCCCAATAGAC-3′；
(8)IgF3：5′-ATTGGGGATTTACGGCATCA-3′，
IgR3：5′-ATGGTAATCGCCCAGTCTTT-3′；
(9)SidF1：5′-TCGCCTTATCCTCTTTTCCA-3′，
SidR1：5′-GTTAGGTGCTGTTACGGTTG-3′；
(11)SidF3：5′-GGTTCCTCCTTGATAACCAC-3′，
SidR3：5′-TCAATGGAAGATGCAGTAGC-3′；
(12)CheF1：5′-CTGTCTACCGCTTTAGTTGG-3′，
CheR1：5′-TTTGTTGCTTCCTTTCACCT-3′；
(13)CheF2：5′-CGTGGAAAACGACTAATTGCT-3′，
CheR2：5′-TCCAACTAAAGCGGTAGACA-3′；
(14)BacF1：5′-GCGAGATGATTGCCATTGTT-3′，
BacR1：5′-TTCGTCTGCCAAAATCACAC-3′；
(15)BacF2：5′-GATGATTGCCATTGTTGGAG-3′，
BacR2：5′-TAATGACTGTTTTGCCCTGT-3′。
the sample to be detected is bee larva exudate, and the DNA extraction is that 10 multiplied by or 100 multiplied by or 1000 multiplied by or 10000 multiplied by the genome DNA obtained by extracting bee larva exudate gene.
The term "bee larva exudate" in the present invention refers to exudate after thawing of frozen larvae.
The "proteinase K method" in the present invention is a conventional proteinase K method for extracting DNA, and in some embodiments, the following methods are specifically used: (1) collecting larval tissue 0.1 g; adding 200ul of the solution A into the thalli, oscillating or blowing by a pipette to enable the thalli to be fully suspended, adding lysozyme with the final concentration of 20mg/ml, adding 20ul of RNase A (10mg/ml) into the suspension, fully reversing and uniformly mixing, and standing at room temperature for 15-30 min; (2) adding 20ul protease K (10mg/ml) into the tube, fully mixing, digesting at 55 ℃ for 30-60min, reversing the centrifuge tube during digestion, mixing for several times until the sample is completely digested, and then, the bacteria liquid is clear and sticky; (3) adding 200ul of the solution B into the tube, fully reversing and uniformly mixing, if white precipitate appears, standing at 75 ℃ for 15-30min, and eliminating the precipitate without influencing subsequent experiments. If the solution is not clear, the digestion of the sample is not thorough, the extraction amount and purity of the DNA can be reduced, and an adsorption column can be blocked; (4) adding 200ul of anhydrous ethanol into the tube, mixing, adding solution and flocculent precipitate into adsorption column, standing for 2min, wherein flocculent precipitate may appear at the time without affecting DNA extraction; (5) centrifuging at 12000rpm for 2min, discarding waste liquid, and placing the adsorption column into the collection tube; (6) adding 600ul rinsing solution (before use, checking whether absolute ethanol is added or not), centrifuging at 12000rpm for 1min, discarding the waste liquid, and placing the adsorption column into a collecting tube; (7) adding 600ul rinsing liquid into the adsorption column, centrifuging at 12000rpm for l min, discarding waste liquid, and placing the adsorption column into a collecting tube. (8) Centrifuging at 12000rpm for 2min, placing the adsorption column in an open room temperature or 50 deg.C incubator for several minutes to remove residual rinsing liquid in the adsorption column, otherwise ethanol in the rinsing liquid will affect subsequent experiments such as enzyme digestion, PCR, etc.; (9) placing the adsorption column into a clean centrifuge tube, suspending and dripping 50-200ul of eluent preheated by 65 deg.C water bath into the center of the adsorption membrane, standing at room temperature for 5min, and centrifuging at 12000rpm for 1 min; (10) adding the eluate into adsorption column, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to obtain high-quality bacterial genome DNA. Wherein the solution A is mainly used for cracking cells and bacteria and is generally pH7.4Tris-Cl buffer solution. In some embodiments, a commercial DNA kit is used for DNA extraction, and solution a belongs to kit reagents and is provided by a DNA kit providing party; the solution B is mainly used for adjusting the pH value to be in the optimal range of the adsorption column and is also Tris-Cl buffer solution. In some embodiments, a commercial DNA kit is used for DNA extraction, and solution a belongs to kit reagents and is provided by a DNA kit providing party.
The nucleotide positions and product sizes corresponding to the "17 pairs of primers" in the present invention are as follows:
Figure BDA0002919217980000091
Figure BDA0002919217980000101
the invention has the beneficial effects that:
the detection method provided by the invention does not need to separate the single colony of the honeycomb coccus, is simple and feasible, and can easily obtain a detection sample.
The detection method provided by the invention has high detection sensitivity, and the PCR amplification time is within 1h, so that the rapid detection requirement of the trichomonas can be met.
Drawings
FIG. 1 shows the result of PCR amplification using sample DNA extracted by direct extraction as a template.
FIG. 2 shows the PCR amplification results using DNA extracted from the larvae exudate as a template.
FIG. 3 shows the result of PCR amplification using DNA extracted by chloroform extraction as a template.
FIG. 4 shows the result of PCR amplification using DNA extracted by extending the protease digestion time as a template.
FIG. 5 shows the PCR amplification results of DNA templates from samples extracted from clinically healthy larvae by direct extraction.
FIG. 6 shows the PCR amplification results of DNA extracted from the larva exudate of clinical healthy larva.
FIG. 7 shows the PCR amplification results of DNA extracted from clinical healthy larvae by chloroform extraction.
FIG. 8 shows the PCR amplification of DNA extracted from clinically healthy larvae with prolonged protease digestion time as template.
FIG. 9 shows the result of PCR amplification of sample DNA at 10000 Xdilution.
FIG. 10 shows the results of WxlF2/WxlR 2-PCR.
FIG. 11 shows the results of WxlF3/WxlR 3-PCR.
In fig. 1 to 9, M: marker DL 2000; 1-17: sequentially obtaining PCR results of the primer pairs 1-17; in fig. 10-11, Marker DL 2000; 1-7: sequentially suspected European larva foul brood disease onset larva; 8: healthy larvae; 9: and (5) negative control.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Samples used in the examples of the present invention: the diseased larva sample is collected from Chinese bee colony in spring 2020 with bee larva death, the healthy larva sample is collected from healthy Chinese bee colony in summer 2020 without clinical symptoms, and the sample is stored at-20 deg.C for use.
Reagents in the examples of the present invention: PCR Mix, pMD18-T vectors were purchased from Baori physician article of technology (Beijing) Ltd; the bacterial DNA extraction kit is purchased from Solebao Biotechnology Ltd, Cat #: d1600; the gel recovery kit was purchased from Tiangen Biotechnology (Beijing) Ltd. Solution A and solution B used in the bacterial DNA extraction kit are both provided by the company, and belong to kit matching reagents.
EXAMPLE 1 sample genomic DNA extraction
(1) Direct extraction method
DNA was extracted from the larval tissue using a bacterial DNA extraction kit, the extraction method was performed according to the kit instructions, as follows:
(1) collecting larval tissue 0.1 g; adding 200ul of the solution A into the thalli, oscillating or blowing by a pipette to enable the thalli to be fully suspended, adding lysozyme with the final concentration of 20mg/ml, adding 20ul of RNase A (10mg/ml) into the suspension, fully reversing and uniformly mixing, and standing at room temperature for 15-30 min;
(2) adding 20ul protease K (10mg/ml) into the tube, fully mixing, digesting at 55 ℃ for 30-60min, reversing the centrifuge tube during digestion, mixing for several times until the sample is completely digested, and then, the bacteria liquid is clear and sticky;
(3) adding 200ul of the solution B into the tube, fully reversing and uniformly mixing, if white precipitate appears, standing at 75 ℃ for 15-30min, and eliminating the precipitate without influencing subsequent experiments. If the solution is not clear, the digestion of the sample is not thorough, the extraction amount and purity of the DNA can be reduced, and an adsorption column can be blocked;
(4) adding 200ul of anhydrous ethanol into the tube, mixing, adding solution and flocculent precipitate into adsorption column, standing for 2min, wherein flocculent precipitate may appear at the time without affecting DNA extraction;
(5) centrifuging at 12000rpm for 2min, discarding waste liquid, and placing the adsorption column into the collection tube;
(6) adding 600ul rinsing solution (before use, checking whether absolute ethanol is added or not), centrifuging at 12000rpm for 1min, discarding the waste liquid, and placing the adsorption column into a collecting tube;
(7) adding 600ul rinsing liquid into the adsorption column, centrifuging at 12000rpm for l min, discarding the waste liquid, and placing the adsorption column into a collecting pipe;
(8) centrifuging at 12000rpm for 2min, placing the adsorption column in an open room temperature or 50 deg.C incubator for several minutes to remove residual rinsing liquid in the adsorption column, otherwise ethanol in the rinsing liquid will affect subsequent experiments such as enzyme digestion, PCR, etc.;
(9) placing the adsorption column into a clean centrifuge tube, suspending and dripping 50-200ul of eluent preheated by 65 deg.C water bath into the center of the adsorption membrane, standing at room temperature for 5min, and centrifuging at 12000rpm for 1 min;
(10) adding the eluate into adsorption column, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to obtain high-quality bacterial genome DNA.
(2) DNA extraction from frozen larva exudate
To reduce the effect of larval components such as fat bodies, proteins, etc., DNA was extracted from frozen larval exudates. Frozen larva samples were taken out and thawed, 50-100 μ L of sample exudate was aspirated for DNA extraction, and other steps were performed according to kit instructions (direct extraction method).
(3) Chloroform extraction method
In order to reduce the residual lipid in the extracted DNA, the cathepsin K digest was extracted with chloroform. Extracting DNA from larva tissue by using a bacterial DNA extraction kit, wherein the extraction method refers to a direct extraction method, after proteinase K is added in the step (2) of the direct extraction method for digestion, larva tissue digestive juice is extracted by using chloroform with the same volume, the larva tissue digestive juice is centrifuged at 12000rpm at 4 ℃ for 10min, supernatant is taken for extracting DNA, and then the step (3) of the direct extraction method is accessed for carrying out.
(4) Extraction method for prolonging digestion time of protease
To reduce tissue proteins in the extracted DNA, proteinase K digestion time was extended. Extracting DNA from larva tissue by using bacterial DNA extraction kit, wherein the extraction method refers to direct extraction method, the digestion time of proteinase K in the step (2) in the direct extraction method is prolonged, the digestion time is not less than 6h, and other methods and steps are still performed according to the direct extraction method.
Example 2 primer design
According to the genome (GenBank NO. DAT1033) information of the nidus, the putative protein fibronectin/fibrinogen binding protein Fib, the protein WxL containing WxL structural domain, the protein Ig containing bacterial immunoglobulin-like structural domain, the substrate binding protein Sid of siderophore ABC transporter, the permease subunit Che of iron chelate uptake ABC transporter family, the bacteriocin export ABC transporter Bac and the protein LPXTG containing LPXTG cell wall anchoring structural domain of the nidus are searched, and the Primer pair is designed by the Primer-BLAST method according to the gene sequence, and the information of the Primer pair is shown in the table 1. The primer pair was synthesized by Biotechnology engineering (Shanghai) Ltd.
TABLE 1 primer information for amplification of genes from Beehive bee
Figure BDA0002919217980000121
Figure BDA0002919217980000131
Figure BDA0002919217980000141
Example 3 PCR amplification and electrophoretic analysis
(1) PCR amplification
The DNA extracted by the 4 methods in example 1 was used as a template, and PCR amplification was performed using the primers designed in example 2, and deionized water was used as a negative control. The PCR reaction system was 20. mu.L, and the reaction system is shown in Table 2. The PCR reaction parameters are as follows: 94 ℃ for 2 min; 30 cycles at 94 ℃ for 30sec, 57 ℃ for 30sec, 72 ℃ for 10 sec; 3min at 72 ℃; storing at room temperature.
TABLE 2 PCR reaction System
Composition of Dosage (mu L)
2×PCR Mix 10
Upstream primer (10. mu. mol/L) 0.5
Downstream primer (10. mu. mol/L) 0.5
DNA 0.5
Deionized water 8.5
(2) Electrophoretic analysis
mu.L of PCR amplification product (the amplification product of PCR amplification using 17 pairs of primers in Table 1 according to the reaction system of Table 2 and the reaction parameters) was added to the well, 1 XTAE buffer solution was used as the electrophoresis solution, 1.2% agarose gel electrophoresis was performed at constant pressure of 120V, and after 20min of electrophoresis, the gel was photographed in a gel imager to observe the PCR results.
The electrophoresis results show that the sizes of fragments amplified by the primers of the 4 th pair (WxlF2/WxlR2) and the 5 th pair (WxlF3/WxlR3) match the expected sizes by using the sample DNA extracted by the direct extraction method as a template, and the results are shown in FIG. 1;
the DNA of the sample extracted from the larvae exudate was used as a template, and the 17 pairs of primers amplified a band, and the sizes of the fragments were consistent with the expected sizes, and the results are shown in FIG. 2.
The sample DNA extracted by the chloroform extraction method is used as a template, 17 pairs of primers amplify bands, and the sizes of the fragments are consistent with the expected sizes, and the result is shown in figure 3.
The sample DNA extracted by the extraction method with the prolonged protease digestion time is taken as a template, 17 pairs of primers amplify bands, and the sizes of the fragments are consistent with the expected sizes, and the results are shown in FIG. 4.
From the PCR amplification results of DNA extracted by the 4 methods, DNA extracted by using thawed larva exudate and DNA extracted by the improved method I and the improved method II can better meet the PCR amplification requirement. However, from the viewpoint of simplification of operation, the method for extracting DNA from the thawed larva exudate is simpler and takes less time. From the viewpoint of adaptability to the conditions for extracting the sample DNA, the primer pairs WxlF2/WxlR2 and WxlF3/WxlR3 have relaxed requirements on the extraction conditions.
(3) PCR amplification and electrophoresis analysis of clinical healthy larva
DNA samples were extracted from the clinically healthy larvae by the 4 methods described above, PCR amplification was performed under the same conditions, and electrophoresis was performed, and 17 pairs of primers did not amplify bands from the clinically healthy larvae samples, and the results are shown in FIGS. 5 to 8.
Example 4 sensitivity detection
And (3) diluting the DNA extracted from the thawed larva exudate by 10X, 100X, 1000X and 10000X, and carrying out PCR amplification by using the diluted DNA as a template and adopting the primer pair, the PCR reaction system and the reaction parameters so as to test the sensitivity of different primers to PCR. Add 5. mu.L of PCR amplification product into the well, use 1 XTAE buffer as electrophoresis solution, perform 1.2% agarose gel electrophoresis at constant pressure 120V, after electrophoresis for 20min, photograph the gel in the gel imager to observe the PCR result.
Electrophoresis results show that PCR using primer pairs of 4(WxlF2/WxlR2), 5(WxlF3/WxlR3), 6(IgF1/IgR1), 7(IgF2/IgR2), 8(IgF3/IgR3), 9(SidF1/SidR1), 11(SidF2/SidR2), 12(SidF3/SidR3), 13(CheF2/CheR2), 14(BacF1/BacR1) and 15(BacF2/BacR2) can still amplify bands in 10000 times diluted templates, and has better sensitivity compared with other primer pairs, and the results are shown in FIG. 9.
EXAMPLE 5 sample testing
The method is applied to detecting diseased larva samples in the same bee field, larva samples with the same clinical symptoms in other bee fields and healthy larva samples by selecting WxlF2/WxlR2-PCR, WxlF3/WxlR3-PCR, and observing results by agarose gel electrophoresis by taking no DNA template and sterilized deionized water as negative controls.
The results showed that WxlF2/WxlR2-PCR (FIG. 10) and WxlF3/WxlR3-PCR (FIG. 11) amplified specific bands from 50 diseased larva samples, while healthy larvae did not amplified bands, and both showed 100% positive and negative agreement.
Example 6 sequence determination and analysis
The PCR amplified band of interest was recovered as described above (in example 3), cloned into pMD18-T, transformed into DH 5. alpha. and positive clones selected for bi-directional sequencing of the insert in the recombinant plasmid and BLST search at NCBI for coverage and homology alignment.
The result shows that the coverage rate of the target band sequence amplified by all 17 pairs of primers to the corresponding part of the genome of the honeycomb cocci reaches 100 percent, and the coverage rate with other sequences is lower than 25 percent; homology with the corresponding portion of the genome of M.melittis was higher than 98%, indicating that the amplified band is the M.melittis genome sequence.
By comparing the influence of the sample genome DNA extraction method on the PCR amplification result and the PCR amplification efficiency of different primer pairs on the diluted sample genome DNA, the optimal nucleic acid detection method of 2 specific sensitive species of the honey bee nidus is screened out, namely WxlF2/WxlR2-PCR and WxlF3/WxlR3-PCR, the detection time of the 2 methods is within 1h, and the rapid detection requirement of European foul brood can be met.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims (10)

1. A specific PCR amplification primer pair for detecting a Melissococcus pluton (Melissococcus pluton), wherein the primer pair is one of the following primer pairs:
(1)FibF2：5′-TAACTATCCGTGCAAATGGG-3′，
FibR2：5′-AGATCATTGCCTTGCTGTTT-3′；
(2)FibF3：5′-ATCCGTGCAAATGGGAAAAA-3′，
FibR3：5′-AGGGGATCAGCTAGAGAAAG-3′；
(3)WxlF1：5′-GTTCAAGCAGAAAACACGAC-3′，
WxlR1：5′-AACTGCCCATTCCAGTATCT-3′；
(4)WxlF2：5′-ACTGATCCAACAGATCCAAC-3′，
WxlR2：5′-TCCCTTAAAGTCAGACAAGC-3′；
(5)WxlF3：5′-AACAGATCCAACTGGTCAAA-3′，
WxlR3：5′-CTTAAAGTCAGACAAGCCGA-3′；
(6)IgF1：5′-ATGGGAAATGGCGAAAAATG-3′，
IgR1：5′-TTAATGGCTGCTTTTTGTGG-3′；
(7)IgF2：5′-ATCAAATGGGAAATGGCGAA-3′，
IgR2：5′-GCCGTAAATCCCCAATAGAC-3′；
(8)IgF3：5′-ATTGGGGATTTACGGCATCA-3′，
IgR3：5′-ATGGTAATCGCCCAGTCTTT-3′；
(9)SidF1：5′-TCGCCTTATCCTCTTTTCCA-3′，
SidR1：5′-GTTAGGTGCTGTTACGGTTG-3′；
(10)SidF2：5′-CGCCTTATCCTCTTTTCCAG-3′，
SidR2：5′-AAATGTTAGGCCGTTAGGTG-3′；
(11)SidF3：5′-GGTTCCTCCTTGATAACCAC-3′，
SidR3：5′-TCAATGGAAGATGCAGTAGC-3′；
(12)CheF1：5′-CTGTCTACCGCTTTAGTTGG-3′，
CheR1：5′-TTTGTTGCTTCCTTTCACCT-3′；
(13)CheF2：5′-CGTGGAAAACGACTAATTGCT-3′，
CheR2：5′-TCCAACTAAAGCGGTAGACA-3′；
(14)BacF1：5′-GCGAGATGATTGCCATTGTT-3′，
BacR1：5′-TTCGTCTGCCAAAATCACAC-3′；
(15)BacF2：5′-GATGATTGCCATTGTTGGAG-3′，
BacR2：5′-TAATGACTGTTTTGCCCTGT-3′；
(16)BacF3：5′-AACCTAACAGCAAACTAGCA-3′，
BacR3：5′-CGATAATGACTGTTTTGCCC-3′；
(17)LPXTGF1：5′-TTGGAGAAGCTGTGACTTAC-3′，
LPXTGR1：5′-TTACGCCTGGTATTGTTTCA-3′。
2. a method of detecting a. alvei, the method comprising the steps of: (1) extracting genome DNA from a sample to be detected; (2) performing PCR amplification by using one of the following specific PCR amplification primer pairs;
(1)FibF2：5′-TAACTATCCGTGCAAATGGG-3′，
FibR2：5′-AGATCATTGCCTTGCTGTTT-3′；
(2)FibF3：5′-ATCCGTGCAAATGGGAAAAA-3′，
FibR3：5′-AGGGGATCAGCTAGAGAAAG-3′；
(3)WxlF1：5′-GTTCAAGCAGAAAACACGAC-3′，
WxlR1：5′-AACTGCCCATTCCAGTATCT-3′；
(4)WxlF2：5′-ACTGATCCAACAGATCCAAC-3′，
WxlR2：5′-TCCCTTAAAGTCAGACAAGC-3′；
(5)WxlF3：5′-AACAGATCCAACTGGTCAAA-3′，
WxlR3：5′-CTTAAAGTCAGACAAGCCGA-3′；
(6)IgF1：5′-ATGGGAAATGGCGAAAAATG-3′，
IgR1：5′-TTAATGGCTGCTTTTTGTGG-3′；
(7)IgF2：5′-ATCAAATGGGAAATGGCGAA-3′，
IgR2：5′-GCCGTAAATCCCCAATAGAC-3′；
(8)IgF3：5′-ATTGGGGATTTACGGCATCA-3′，
IgR3：5′-ATGGTAATCGCCCAGTCTTT-3′；
(9)SidF1：5′-TCGCCTTATCCTCTTTTCCA-3′，
SidR1：5′-GTTAGGTGCTGTTACGGTTG-3′；
(10)SidF2：5′-CGCCTTATCCTCTTTTCCAG-3′，
SidR2：5′-AAATGTTAGGCCGTTAGGTG-3′；
(11)SidF3：5′-GGTTCCTCCTTGATAACCAC-3′，
SidR3：5′-TCAATGGAAGATGCAGTAGC-3′；
(12)CheF1：5′-CTGTCTACCGCTTTAGTTGG-3′，
CheR1：5′-TTTGTTGCTTCCTTTCACCT-3′；
(13)CheF2：5′-CGTGGAAAACGACTAATTGCT-3′，
CheR2：5′-TCCAACTAAAGCGGTAGACA-3′；
(14)BacF1：5′-GCGAGATGATTGCCATTGTT-3′，
BacR1：5′-TTCGTCTGCCAAAATCACAC-3′；
(15)BacF2：5′-GATGATTGCCATTGTTGGAG-3′，
BacR2：5′-TAATGACTGTTTTGCCCTGT-3′；
(16)BacF3：5′-AACCTAACAGCAAACTAGCA-3′，
BacR3：5′-CGATAATGACTGTTTTGCCC-3′；
(17)LPXTGF1：5′-TTGGAGAAGCTGTGACTTAC-3′，
LPXTGR1：5′-TTACGCCTGGTATTGTTTCA-3′。
3. the method according to claim 2, wherein the sample to be tested in step (1) is a bee larva exudate, said bee larva exudate being a frozen bee larva thawed exudate or a bee larva cadaver exudate.
4. The method of claim 3, wherein the genomic DNA is diluted 10 Xor 100 Xor 1000 Xor 10000 Xand then PCR amplified using one of the following specific PCR amplification primer pairs:
(4)WxlF2：5′-ACTGATCCAACAGATCCAAC-3′，
WxlR2：5′-TCCCTTAAAGTCAGACAAGC-3′；
(5)WxlF3：5′-AACAGATCCAACTGGTCAAA-3′，
WxlR3：5′-CTTAAAGTCAGACAAGCCGA-3′；
(6)IgF1：5′-ATGGGAAATGGCGAAAAATG-3′，
IgR1：5′-TTAATGGCTGCTTTTTGTGG-3′；
(7)IgF2：5′-ATCAAATGGGAAATGGCGAA-3′，
IgR2：5′-GCCGTAAATCCCCAATAGAC-3′；
(8)IgF3：5′-ATTGGGGATTTACGGCATCA-3′，
IgR3：5′-ATGGTAATCGCCCAGTCTTT-3′；
(9)SidF1：5′-TCGCCTTATCCTCTTTTCCA-3′，
SidR1：5′-GTTAGGTGCTGTTACGGTTG-3′；
(11)SidF3：5′-GGTTCCTCCTTGATAACCAC-3′，
SidR3：5′-TCAATGGAAGATGCAGTAGC-3′；
(12)CheF1：5′-CTGTCTACCGCTTTAGTTGG-3′，
CheR1：5′-TTTGTTGCTTCCTTTCACCT-3′；
(13)CheF2：5′-CGTGGAAAACGACTAATTGCT-3′，
CheR2：5′-TCCAACTAAAGCGGTAGACA-3′；
(14)BacF1：5′-GCGAGATGATTGCCATTGTT-3′，
BacR1：5′-TTCGTCTGCCAAAATCACAC-3′；
(15)BacF2：5′-GATGATTGCCATTGTTGGAG-3′，
BacR2：5′-TAATGACTGTTTTGCCCTGT-3′。
5. the method of claim 2, wherein step (1) is: and (3) carrying out gene extraction on the bee larva tissues or bee larva exudates by using a proteinase K method to obtain genome DNA.
6. The method of claim 2, wherein step (1) is: carrying out gene extraction on the bee larva tissues or bee larva exudates by using a modified proteinase K method to obtain genome DNA, wherein the modified proteinase K method comprises the following steps: prolonging the digestion time of the proteinase K to be not less than 6h, and keeping other steps unchanged; or the improved proteinase K method is as follows: after digestion with proteinase K, the digest was extracted with chloroform and then subjected to the next step.
7. The method of claim 2, wherein the method comprises: carrying out gene extraction on the bee larva tissues to obtain larva genome DNA, and carrying out PCR amplification by using the following specific PCR amplification primer pairs:
(4) WxlF 2: 5'-ACTGATCCAACAGATCCAAC-3', WxlR2: 5'-TCCCTTAAAGTCAGACAAGC-3'; or
(5)WxlF3：5′-AACAGATCCAACTGGTCAAA-3′，
WxlR3：5′-CTTAAAGTCAGACAAGCCGA-3′。
8. The method of any one of claims 2 to 7, wherein the PCR amplification procedure comprises: 94 ℃ for 2min and 94 ℃ for 30 s; 30s at 57 ℃; 72 ℃ for 10 s.
The application of primer pair Wxl in detecting the nidus vespae is characterized in that the sequence of the primer pair Wxl is as follows:
WxlF2：5′-ACTGATCCAACAGATCCAAC-3′，WxlR2:5′-TCCCTTAAAGTCAGACAAGC-3′；
or WxlF 3: 5'-AACAGATCCAACTGGTCAAA-3' the flow of the air in the air conditioner,
WxlR3：5′-CTTAAAGTCAGACAAGCCGA-3′。
10. a nidulans bacterium detection kit comprising the specific PCR amplification primer pair according to claim 1.
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OKUMURA,K.等: "Melissococcus plutonius DAT1033 DNA, complete genome", 《GENBANK》 *
席伟军等: "环介导等温扩增(LAMP)技术检测蜜蜂球囊菌", 《中国农业科学》 *
杨东来等: "蜂蜜中蜜蜂欧洲幼虫腐臭病的检测方法研究", 《中国口岸科学技术》 *
王丽君等: "欧洲幼虫腐臭病病原检测及防控建议", 《蜜蜂杂志》 *

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