CN104651496A - Method for rapidly detecting salmonella based on immunomagnetic bead-multiplex PCR - Google Patents
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Abstract
The invention discloses a method for rapidly detecting salmonella based on immune magnetic bead-multiplex PCR. The method comprises the following steps: enriching salmonella in a to-be-detected sample by salmonella specific immune magnetic beads and extracting bacteria DNA; respectively synthesizing primer pairs for specifically amplifying salmonella, salmonella enteritidis and salmonella pullorum, and carrying out PCR amplification; and detecting amplified products by gel electrophoresis, and judging whether salmonella is contained. By virtue of the method, salmonella, salmonella enteritidis and salmonella pullorum can be simultaneously detected; the method is short in detection time, high in specificity, high in sensitivity, low in cost, and simple in result judgment.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a salmonella rapid detection method based on immunomagnetic bead-multiplex PCR.
Background
Salmonella bacteria (A), (B)Salmonella) Is an important zoonotic, gram-negative pathogen in enterobacteriaceae. The animal-derived salmonella easily causes acute diarrhea of a person taking food, even outbreak of food poisoning, causes gastroenteritis, typhoid fever and paratyphoid fever, and poses a threat to human health.
Salmonella pullorum can cause pullorum disease. The disease is mainly prevalent in chicks of 2-3 weeks, but the trend of susceptible day age increase and clinical disease type diversification is generated at present, so that the whole chicken raising period is harmed by the disease. Pullorum chicken usually shows drowsiness, weakness and loss of appetite, gathers into a group, has two wings drooping, gives out sharp screaming when defecating, and has 20-30% of death rate due to white or green excrement attached to anus and periphery. The death rate of 10-20% can be caused in the bred chickens, and the diseased chickens are mostly caused by the fact that the diseased chickens are not completely cured and become chronic or are infected during the brooding period. Adult chickens do not exhibit the characteristics of acute infection, often presenting with asymptomatic infection.
The salmonella enteritidis is very similar to the chicken group disease symptoms caused by the salmonella pullorum, but the host of the salmonella enteritidis is wider than the salmonella pullorum, is one of the main pathogenic bacteria causing human food poisoning at present, and the infection is mainly caused by eating animal food polluted by the salmonella enteritidis, especially meat and eggs of poultry, so the research on the salmonella enteritidis is concerned by scholars at home and abroad.
Because salmonella serotypes are various and various biochemical reactions are complex, various salmonella detection methods such as a traditional culture method, an enzyme-linked immunosorbent assay, a biosensor technology and a nucleic acid amplification technology are established at present, but the methods or procedures are tedious, time-consuming and labor-consuming, or low in specificity, poor in sensitivity, or high in cost, and the like, and are not suitable for rapid detection and popularization and application, so that the establishment of the method capable of rapidly and accurately detecting salmonella, salmonella pullorum and salmonella enteritidis simultaneously is very important.
The Multiplex PCR (Multiplex PCR) was first proposed by Chamberland et al in 1988, also called Multiplex primer PCR or Multiplex PCR, and has had incomparable advantages over the single PCR technology because it can amplify multiple target genes simultaneously in the same system: saving time, reducing cost, reducing workload, and particularly saving precious experimental samples. However, in practical application, the components of the sample to be detected are often complex, so that the PCR detection result has high interference, and the sensitivity and accuracy of the detection result are influenced. According to the invention, the immunomagnetic beads are adopted to enrich the salmonella in the sample, so that other components except the target bacteria in the sample can be removed, and the interference on the subsequent PCR amplification is avoided or reduced, thus the detection sensitivity and accuracy are improved, and the salmonella, the salmonella enteritidis and the salmonella pullorum can be simultaneously identified, so that the method is economical and simple.
Disclosure of Invention
The invention aims to provide a salmonella rapid detection method based on immunomagnetic beads-multiplex PCR aiming at the defects of the prior art. The detection method for detecting the salmonella has the advantages of short detection time, high sensitivity, strong specificity and low cost.
The invention is realized by the following technical scheme.
The invention designs a salmonella rapid detection method based on immunomagnetic bead-multiplex PCR, which comprises the following steps:
(1) respectively synthesizing primer pairs for specifically amplifying salmonella, salmonella enteritidis and salmonella pullorum;
(2) enriching salmonella in a sample to be detected by using salmonella specific immunomagnetic beads, and extracting bacterial DNA;
(3) amplifying by using the primer pair synthesized in the step (1) by adopting a multiplex PCR method;
(4) detecting the amplification product by gel electrophoresis, and judging whether the sample contains salmonella enteritidis, salmonella pullorum or other salmonella;
(5) and (5) judging a result: taking 5 mu L of PCR amplification product, carrying out electrophoresis analysis by using 1% agarose gel, observing under the irradiation of an ultraviolet lamp, and if a single amplification band appears at the position of 796bp in the electrophoresis result, indicating that the sample contains salmonella, but not salmonella enteritidis and salmonella pullorum; if the electrophoresis result shows that amplified bands appear at positions of 796bp and 600bp simultaneously, the sample contains the salmonella pullorum; if the electrophoresis result shows that amplified bands appear at positions of 796bp and 304bp simultaneously, the sample contains salmonella enteritidis; if no amplified band appears at the positions of 796bp, 600bp and 304bp as a result of electrophoresis, the sample does not contain salmonella.
The method for enriching salmonella by using immunomagnetic beads in the step (1) is disclosed in the following patents: a method for enriching salmonella based on immunomagnetic beads (patent number: 201410772937.0).
Compared with the prior art, the invention has the following beneficial effects:
(1) and (3) shortening the detection time: the immunomagnetic beads are used for enriching the salmonella, so that the bacterium increasing operation step in the salmonella detection process can be effectively replaced, and the detection time is shortened.
(2) The detection sensitivity and accuracy are improved: when the salmonella specific immunomagnetic beads enrich trace salmonella in the sample, other components except target bacteria in the sample can be removed, so that interference on subsequent multiplex PCR is prevented, false positive is effectively avoided or reduced, and the detection sensitivity and accuracy are improved.
(3) Simple operation and low cost: the detection method designed by the invention does not need large instruments and specially trained professionals, does not need expensive reagents, and has low detection cost.
Drawings
FIG. 1 is a gel electrophoresis result diagram of an experiment for evaluating specificity of a Salmonella rapid detection method based on immunomagnetic bead-multiplex PCR in example 2.
FIG. 2 is a gel electrophoresis result diagram of the sensitivity evaluation experiment of the immunomagnetic bead-multiplex PCR-based salmonella rapid detection method in example 3.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Habor Laboratory Press, 1989), or according to the manufacturer's recommendations.
Example 1 establishment of a Salmonella rapid detection method based on immunomagnetic bead-multiplex PCR
(1) Synthesizing primer pairs for specifically amplifying salmonella, salmonella enteritidis and salmonella pullorum;
according to the literature reports, respectively synthesizeinvA、fliCAndsdfIthe three primer pairs, primer sequences and related information are as follows:
salmonella specific primer sequence:
invA-F： 5’-CGGTGGTTTTAAGCGTACTCTT-3’；
invA-R： 5’-CGAATATGCTCCACAAGGTTA-3’；
salmonella enteritidis specific primer sequences:
sdfI –F：5’-TGTGTTTTATCTGATGCAAGAGG-3’；
sdfI –R：5’-TGAACTACGTTCGTTCTTCTGG-3’；
the specific primer sequence of the salmonella pullorum is as follows:
fliC –F：5’-GCGGAAAATAACACTGCGG-3’；
fliC –R：5’-TGCCCCCAGAGAAGAACGAA-3’；
wherein,sdfIprimer sequences are described in the following documents: PG Agron, RL Walker. identification by reactive hybridization of sequences specific for Salmonella enterica Enterprise entity]. Am Soc Microbiol，2001，,67（11）：4984-4991；
fliCPrimer sequences are described in the following documents: construction and application of PCR detection technology for Xue Jun dragon, Zhang Wei industry, salmonella pullorum]Stock veterinary magazine, 2011,30 (6): 23-27;
invAprimer sequences are described in the following documents: PM Fratamico, TP Strobaugh.Simultaneous detection of Salmonella spp and Escherichia coli O157H 7 by multiplex PCR [ J]. Journal of Industrial Microbiology and Biotechnology，1998, 21（3）: 92-98。
(2) Immunomagnetic bead enriched salmonella
200. mu.L of each of the 12h liquid cultures containing Salmonella pullorum (ATCC 13036) and Salmonella enteritidis (ATCC 13076) was added to 300. mu.L of Salmonella-specific immunomagnetic beads for Salmonella enrichment.
The method of enriching salmonella with immunomagnetic beads has been described in the following patents: method for enriching salmonella based on immunomagnetic beads (patent number: 201410772937.0)
(3) DNA template preparation
And (3) putting the immunomagnetic beads adsorbing the salmonella into a 1.5ml centrifuge tube, adding 100 mu L of sterile double-distilled water for resuspension, centrifuging at 12,000r/min for 5min, and discarding the supernatant. Resuspending the mycelia in 100 μ L sterile double distilled water, boiling in boiling water bath for 10min, immediately taking out, standing at-80 deg.C for 30min, thawing in boiling water bath, centrifuging at 12,000r/min for 5min, and collecting the supernatant and standing at-20 deg.C for use.
(4) Establishment of multiplex PCR detection method
The 25. mu.L reaction system of multiplex PCR is, specifically, 2.5. mu.L of 10 XPCR reaction buffer, 25mmol/L Mg2+ 2.0 μ L, 4.5 μ L of 2.5mmol/L dNTP, 1.2 μ L of 10 μ M invA primer pair, 0.4 μ L of 10 μ M fliC primer pair, 0.8 μ L of 10 μ MsdfI primer pair, 0.5U/μ L Taq enzyme, 2 μ L of template solution, and finally supplementing to 25 μ L with sterile double distilled water; the PCR detection system amplification parameters are specifically as follows: pre-denaturation at 94 ℃ for 5 min; thereafter, amplification cycles were started, and the procedure for each cycle was: denaturation at 94 deg.C for 1min, annealing at 58 deg.C for 50s, and extension at 72 deg.C for 1 min; a total of 35 cycles; and finally, extending for 10min at 72 ℃, cooling to 12 ℃, and finishing.
(5) Determination of results
The judgment is specifically as follows: taking 5 mu L of PCR amplification product, carrying out electrophoresis analysis by using 1% agarose gel, observing under the irradiation of an ultraviolet lamp, and if a single amplification band appears at the position of 796bp in the electrophoresis result, indicating that the sample contains salmonella, but not salmonella enteritidis and salmonella pullorum; if the electrophoresis result shows that amplified bands appear at positions of 796bp and 600bp simultaneously, the sample contains the salmonella pullorum; if the electrophoresis result shows that amplified bands appear at positions of 796bp and 304bp simultaneously, the sample contains salmonella enteritidis; if no amplified band appears at the positions of 796bp, 600bp and 304bp as a result of electrophoresis, the sample does not contain salmonella.
As a result: taking 5 mu L of PCR amplification product, carrying out electrophoresis on 1% agarose gel, and then, under the irradiation of an ultraviolet lamp, observing amplification bands of the liquid culture containing the salmonella pullorum (ATCC 13036) at positions of 796bp and 600bp at the same time, which indicates that the established PCR can successfully detect the salmonella pullorum; amplification bands were observed simultaneously at the 796bp and 304bp positions in the liquid culture containing Salmonella enteritidis (ATCC 13076), indicating that the established PCR was successful in detecting Salmonella enteritidis.
Example 2 Salmonella rapid detection method specificity evaluation experiment based on immunomagnetic bead-multiplex PCR
(1) Immunomagnetic bead enriched salmonella
Respectively taking 200ul of 12h liquid culture of salmonella pullorum, salmonella enteritidis, salmonella paratyphi A, salmonella typhimurium, escherichia coli, proteus, enterobacter cloacae, staphylococcus, pseudomonas aeruginosa and klebsiella pneumoniae, adding the liquid culture into 300 ul of immunomagnetic beads, reacting for 30min at 37 ℃, putting the immunomagnetic beads on a magnet for acting for 2-3 min to fully adsorb the magnetic beads, and discarding the suspension.
(2) DNA template preparation
The genomic DNAs of Salmonella pullorum, Salmonella enteritidis, Salmonella paratyphi A, Salmonella typhimurium, Escherichia coli, Proteus, Enterobacter cloacae, Staphylococcus, Pseudomonas aeruginosa and Klebsiella pneumoniae were each extracted according to the step (3) of example 1 and stored at-20 ℃ for further use.
(3) Salmonella immunomagnetic bead-multiplex PCR detection method specificity evaluation test
According to the PCR reaction system in the step (4) of the embodiment 1, 2 μ L of each strain DNA template prepared in the step (2) is taken as a PCR reaction template and is respectively added into the PCR reaction system, and 2 μ L of sterile double distilled water is taken as a template for a blank control; then, PCR amplification reaction was performed according to the PCR cycle parameters in step (4) of example 1.
(4) Determination of results
Taking 5 mu L of PCR amplification product, carrying out electrophoresis analysis by using 1% agarose gel, observing under the irradiation of an ultraviolet lamp, and if a single amplification band appears at the position of 796bp in the electrophoresis result, indicating that the sample contains salmonella, but not salmonella enteritidis and salmonella pullorum; if the electrophoresis result shows that amplified bands appear at positions of 796bp and 600bp simultaneously, the sample contains the salmonella pullorum; if the electrophoresis result shows that amplified bands appear at positions of 796bp and 304bp simultaneously, the sample contains salmonella enteritidis; if no amplified band appears at the positions of 796bp, 600bp and 304bp as a result of electrophoresis, the sample does not contain salmonella.
As a result: FIG. 1: lane M: DNA marker 2000; lane 1: negative control; lanes 2-11: salmonella pullorum, Salmonella enteritidis, Salmonella paratyphi A, Salmonella typhimurium, Escherichia coli, Proteus, Enterobacter cloacae, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae.
As shown in FIG. 1, after the DNA of Salmonella pullorum is amplified by PCR and detected by gel electrophoresis, amplified bands can be observed at 796bp and 600bp positions under the irradiation of an ultraviolet lamp; PCR amplification of salmonella enteritidis DNA, and simultaneous observation of amplified bands at 769bp and 304bp positions under irradiation of an ultraviolet lamp after gel electrophoresis detection; PCR amplification is carried out on the DNA of the salmonella paratyphi A and the salmonella typhimurium, and after gel electrophoresis detection, a single amplification strip is only observed at the position of 796bp under the irradiation of an ultraviolet lamp; PCR amplifying colibacillus, proteus, enterobacter cloacae, staphylococcus, pseudomonas aeruginosa and klebsiella pneumoniae DNA and blank control, after gel electrophoresis detection, no amplification band is observed at 796bp, 600bp and 304bp positions under the irradiation of an ultraviolet lamp. The results show that the established immunomagnetic bead-multiplex PCR detection method can specifically detect salmonella, salmonella pullorum and salmonella enteritidis.
Example 3 Immunomagnetic bead-multiplex PCR-based Salmonella rapid detection method sensitivity evaluation test
(1) Immunomagnetic bead enriched salmonella
The content of Salmonella in the 12h liquid culture containing Salmonella pullorum (ATCC 13036) was determined by plate counting method, and the result showed that the concentration of the original bacterial liquid was 5.7X 107cfu/ml, 10-fold gradient dilution with physiological saline, 9 gradients in total. Respectively taking 200ul of diluted bacteria liquid, adding into 300 ul of magnetic beads, reacting at 37 ℃ for 30min, placing on a magnet for 2-3 min to make the magnetic beads fully adsorb, and discarding the suspension.
(2) DNA template preparation
Bacterial DNA was extracted from each of the 9 dilution gradients according to step (3) of example 1 and stored at-20 ℃ until use.
(3) Determination of results
Taking 5 mu L of PCR amplification product, carrying out electrophoresis analysis by using 1% agarose gel, observing under the irradiation of an ultraviolet lamp, and if a single amplification band appears at the position of 796bp in the electrophoresis result, indicating that the sample contains salmonella, but not salmonella enteritidis and salmonella pullorum; if the electrophoresis result shows that amplified bands appear at positions of 796bp and 600bp simultaneously, the sample contains the salmonella pullorum; if the electrophoresis result shows that amplified bands appear at positions of 796bp and 304bp simultaneously, the sample contains salmonella enteritidis; if no amplified band appears at the positions of 796bp, 600bp and 304bp as a result of electrophoresis, the sample does not contain salmonella.
As a result: FIG. 2: lane M: DNA marker 2000; lane 1: negative control; lanes 2-10: 5.7X 106 CUF/ml、5.7×105 CUF/ml、5.7×104 CUF/ml、5.7×103 CUF/ml、5.7×102 CUF/ml、5.7×101 CUF/ml、5.7×100 CUF/ml、5.7×10-1 CUF/ml、5.7×10-2 CUF/ml。
As can be seen from FIG. 2, clear bands were observed in lanes 2 to 6, and the concentrations of the bacterial liquids were 5.7X 106 CUF/ml 、5.7×105 CUF/ml、5.7×104 CUF/ml、5.7×103 CUF/ml、5.7×102 CUF/ml, no amplified band was seen after lane 6. Therefore, the PCR detection sensitivity was judged to be 5.7X 102 CUF/ml, and has high sensitivity.
Claims (3)
1. A salmonella rapid detection method based on immunomagnetic bead-multiplex PCR is characterized in that: after the salmonella specific immunomagnetic beads are adopted to enrich the salmonella in the sample, the final detection and evaluation of the salmonella are realized by a multiple PCR method.
2. The immunomagnetic bead-multiplex PCR-based rapid detection method for Salmonella according to claim 1, wherein the detection of Salmonella, Salmonella pullorum, and Salmonella enteritidis can be performed simultaneously on the sample to be detected.
3. A salmonella rapid detection method based on immunomagnetic bead-multiplex PCR is characterized in that:
(1) enriching trace salmonella in a sample to be detected by using immunomagnetic beads, and extracting bacterial DNA by using a boiling method;
(2) performing multiplex PCR identification; the multiplex PCR reaction system is 25 mu L, and specifically comprises: 10 XPCR reaction buffer 2.5. mu.L, 25mmol/L Mg2+ 2.0. mu.L, 2.5mmol/L dNTP 4.5. mu.L, 10. mu.M invAPrimer pair 1.2. mu.L, 10. mu.MfliCPrimer pair 0.4. mu.L, 10. mu.MsdfIPrimer pair 0.8 μ L, 2.5U/μ L Taq enzyme 0.5U, template solution 2 μ L, finally supplement to 25 μ L with sterile double distilled water; the PCR detection system amplification parameters are specifically as follows: pre-denaturation at 94 ℃ for 5 min; thereafter, amplification cycles were started, and the procedure for each cycle was: denaturation at 94 deg.C for 1min, annealing at 58 deg.C for 50s, and extension at 72 deg.C for 1 min; a total of 35 cycles; finally, extending for 10min at 72 ℃, cooling to 12 ℃, and finishing;
(3) and (4) judging the result: taking 5 mu L of PCR amplification product, carrying out electrophoresis analysis by using 1% agarose gel, observing under the irradiation of an ultraviolet lamp, and if a single amplification band appears at the position of 796bp in the electrophoresis result, indicating that the sample contains salmonella, but not salmonella enteritidis and salmonella pullorum; if the electrophoresis result shows that amplified bands appear at positions of 796bp and 600bp simultaneously, the sample contains the salmonella pullorum; if the electrophoresis result shows that amplified bands appear at positions of 796bp and 304bp simultaneously, the sample contains salmonella enteritidis; if no amplified band appears at the positions of 796bp, 600bp and 304bp as a result of electrophoresis, the sample does not contain salmonella.
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| CN102816850A (en) * | 2012-08-28 | 2012-12-12 | 无锡中德伯尔生物技术有限公司 | Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis |
-
2015
- 2015-01-19 CN CN201510022943.9A patent/CN104651496A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816850A (en) * | 2012-08-28 | 2012-12-12 | 无锡中德伯尔生物技术有限公司 | Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis |
Non-Patent Citations (4)
| Title |
|---|
| PM FRATAMICO等: "Simultaneous detection of Salmonella spp and Escherichia coli O157:H7 by multiplex PCR", 《JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| 杨林等: "多重PCR方法快速鉴别肠炎、鼠伤寒、鸡白痢及鸡伤寒沙门菌", 《畜牧兽医学报》 * |
| 王晓闻等: "免疫磁珠捕获 -PCR 检测牛乳中沙门氏菌的研究", 《山西农业大学学报（自然科学版）》 * |
| 薛俊龙等: "鸡白痢沙门氏菌PCR检测技术的建立与应用", 《畜牧兽医杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988207A (en) * | 2017-12-26 | 2018-05-04 | 济南凯晨科技有限公司 | Extracted from complex samples and purify the kit and method of salmonella DNA |
| CN107988207B (en) * | 2017-12-26 | 2021-04-06 | 济南凯晨生物科技有限公司 | Kit and method for extracting and purifying salmonella DNA from complex sample |
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