CN101880711A - Nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes - Google Patents
Nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes Download PDFInfo
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- CN101880711A CN101880711A CN2010101262840A CN201010126284A CN101880711A CN 101880711 A CN101880711 A CN 101880711A CN 2010101262840 A CN2010101262840 A CN 2010101262840A CN 201010126284 A CN201010126284 A CN 201010126284A CN 101880711 A CN101880711 A CN 101880711A
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Abstract
The invention discloses a nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes, belonging to the field of biological assay. In the method, by virtue of a loop-mediated isothermal amplification (LAMP) technology and a specific primer, the specific area of target genes is amplified by an LAMP technical platform; and with the aid of a positive and negative quality control system and an internal control detection system, nucleic acid screening or detection is carried out on the staphylococcus aureus, the salmonella, the shigella and the listeria monocytogenes from the molecular level. The nucleic acid screening method of the invention has the characteristics of being simple, convenient, economical, fast, sensitive and specific, and the method has wide application prospect.
Description
Technical field
The invention belongs to the Micro biological Tests field, be specifically related to a kind of streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes nucleic acid screening method of developing based on isothermal duplication (LAMP) know-why of encircling mediation.
Background technology
The detection method of streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes mainly is that microorganism culturing is identified and the multiplex PCR detection technique at present.(1) microbial culture, credible result degree height, and can do the bacterium drug sensitive test, but take long, application is restricted, because hazardness such as streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes are big, laboratory safety is had relatively high expectations simultaneously, be unfavorable for the general examination of above-mentioned bacterium.(2) polymerase chain reaction (PCR), specificity is relatively poor, and advantage is that susceptibility can reach 98%～100%., but it needs relevant valuable equipments such as pcr amplification instrument, gel electrophoresis, is unfavorable for primary care epidemic prevention organization and fast-field evaluation.
At the beginning of 21 century, Notomi T etc. has developed a kind of constant temperature nucleic acid amplification theory of novelty, be so-called loop-mediated isothermal amplification method (Loop-Mediated Isothermal Amplification, LAMP), be characterized in 6/4 kind of special primer of 8/6 zone design at target gene, utilize a kind of strand displacement Bst archaeal dna polymerase in constant temperature (about 65 ℃) insulation dozens of minutes, can finish nucleic acid amplification reaction, the short period of time amplification efficiency can reach 10
9-10
10Individual copy.Directly judge whether to react by amplified production electrophoresis or color reaction.This method does not need processes such as the thermally denature of template, long-time temperature cycle, loaded down with trivial details electrophoresis, ultraviolet visualization, the LAMP method has high specific, high efficiency, quick, cheap (do not need specific valuable test set, only need the thermostat container of simple structure), the easy characteristics such as (directly estimating judgement) that detect.
Summary of the invention
The purpose of this invention is to provide a kind of nucleic acid detection method that is applied to streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes, the one, fill up the blank aspect streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes LAMP (loop-mediated isothermal amplification method) detection technique, make in the detection technique method of streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes and reach advanced world standards; The 2nd, detectivity further improves, and the detectability of LAMP detection technique is higher than conventional sense method 100-200 doubly, has so just effectively overcome the problem of the traditional microorganism culturing authentication method recall rate end of than; The 3rd, because classical polymerase chain reaction (PCR) amplification technique is had relatively high expectations (as the pcr amplification instrument to plant and instrument, agargel electrophoresis system etc.), technology is comparatively complicated, be unfavorable for that it prevents epidemic at primary care, the widespread usage of inspection and quarantine mechanism, and the LAMP detection technique has plant and instrument requirement detection (thermostat container that only needs simple structure), detection time short (0.5-1.0 hour), the specificity height is (at 6/4 kind of special primer of 8/6 zone design of target gene,) etc. characteristics, can use the gene amplification technology in primary care epidemic prevention organization and field monitoring, to be extensive use of.
The present invention realizes with following technical scheme: a kind ofly be applied to the examination streptococcus aureus, Salmonellas, the detection method of Shigellae and Listeria monocytogenes nucleic acid, utilize ring mediated isothermal amplification (LAMP) technology to carry out, utilize the specific region of LAMP technology platform by using the primer amplified target gene (respectively at streptococcus aureus clfA gene (the Genebank accession number is EF207779), Salmonellas invA gene (the Genebank accession number is M90846), Shigellae ipaH gene (the Genebank accession number is M76444), singly increase Liszt's hly gene (the GeneBank accession number is M24199)), positive and negative Quality Control and internal reference detection architecture auxiliary time, from molecular level to streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes carry out nucleic acid screening or detection.
Further, in the examination detection method of streptococcus aureus of the present invention, Salmonellas, Shigellae and Listeria monocytogenes nucleic acid, in LAMP examination process, introduced yin and yang attribute control test system.The positive control quality control product is the DNA that is same as the amplified target gene, the negative control quality control product is the DNA in the amplified target gene inequality, and uses electrophoretic analysis or PicoGreen fluorescence dye development process to detect internal reference and streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes target gene amplified production simultaneously.
Further, in streptococcus aureus of the present invention, Salmonellas, Shigellae and Listeria monocytogenes nucleic acid detection method, at streptococcus aureus clfA gene (the Genebank accession number is EF207779), Salmonellas invA gene (the Genebank accession number is M90846), Shigellae ipaH gene (the Genebank accession number is M76444), the hly gene (the GeneBank accession number is M24199) that singly increases the Liszt designs loop-mediated isothermal amplification (LAMP) primer respectively.
Further, in streptococcus aureus of the present invention, Salmonellas, Shigellae and Listeria monocytogenes nucleic acid detection method, it is characterized in that used LAMP reaction system is a series of reacted constituent compositions by optimizing, and has specific response procedures.
The present invention has strict discriminating sieving and diagnosis system, to guarantee the accuracy of detected result.This differentiates the sieving and diagnosis system by positive and negative quality control product, and internal reference quality control product and relevant amplification and detection architecture are formed.
Whether effectively the positive and the negative quality control product of differentiating the sieving and diagnosis system among the present invention are every batch of laboratory test results important symbols.For every batch of experiment, must introduce the detection of a negative quality control product, to guarantee that reaction system false positive results can not occur; For every batch of experiment, must introduce the detection of streptococcus aureus, Salmonellas, Shigellae and a Listeria monocytogenes positive quality control product, to guarantee that reaction system false negative reaction can not occur.At the positive quality control product of streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes is the plasmid DNA of artificial constructed above-mentioned bacterium target sequence to be checked.
Specific amplification products in the differential diagnosis of the present invention system designs by the contriver, prepares by the nucleic acid synthesis device.The nucleic acid synthesis device can be selected different manufacturers for use, and the product of different model also can entrust relevant genome company synthetic.All primers are according to LAMP design of primers principle, utilize ICBgyrB database and RIDOM database to seek the LAMP gene order of streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes, utilize software to choose its specific sequence, utilize PrimerExplorerIV software further to analyze, design 4 cover primers are respectively at above-mentioned 4 kinds of bacteriums.LAMP system and response procedures in the differential diagnosis of the present invention system design by the contriver.The nucleic acid specificity primer sequence of streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes, LAMP reaction system and response procedures are as follows:
One, loop-mediated isothermal amplification (LAMP) primer
Table 1 streptococcus aureus clfA gene LAMP detects primer
The LAMP reaction primer sequence of (GeneBank EF207779) is formed
Table 2 Salmonellas invA gene LAMP detects primer
The LAMP reaction primer sequence of (GeneBank M90846) is formed
Table 3 Shigellae ipaH gene LAMP detects primer
6
The LAMP reaction primer sequence of (GeneBank M76444) is formed
The hly gene LAMP that table 4 singly increases the Liszt detects primer
The LAMP reaction primer sequence of (GeneBank M24199) is formed
The LAMP reaction system of the streptococcus aureus that two, the present invention includes, Salmonellas, Shigellae and Listeria monocytogenes is composed as follows:
10 * Bst buffer (200mM Tris-HCl (pH8.8,25 ℃), 100mM KCl, 100mM (NH
4)
2SO
4, 20mMMgSO
4, 0.1%Triton-100) 2.5 μ l; Betaine (4M) 5 μ l; Primer (F3: B3: FIP: BIP=5 μ M: 5 μ M: 40 μ M: 1 μ l 40 μ M); Bst enzyme (8U) 1 μ l; DNTPs (10mM) 3.5 μ l; MgCl
2(25mM) 6 μ l; Template 1 μ l; Sterilization deionized water 4 μ l.。
Three, amplification program: 65 ℃ of 1 hour → 80 ℃ 10 minutes → 10 ℃ preservations;
Four, reaction end detection method: after reaction finishes, add 1 μ L Pico Green dyestuff, whether mixing, observing response pipe color change or get the above-mentioned bacterium LAMP of 5 μ L reaction product, carry out electrophoresis detection on 2% sepharose.Whether observe has the scalariform band to produce.
The invention has the beneficial effects as follows: method is easy, economical, quick, sensitive.
Description of drawings
Fig. 1 is streptococcus aureus LAMP reaction product dyeing figure;
Among the figure: 1, positive plasmid pGEM-T-clfA; 2, golden yellow grape ball genomic dna; 3, negative control.
Fig. 2 is that Salmonellas (invA gene) LAMP detects dyeing figure;
Among the figure: 1: positive control pGEM-T-invA, 2: salmonella gene group DNA, 3: negative control.
Fig. 3 is Listeria monocytogenes (hly gene) LAMP amplified production gel electrophoresis figure;
Among the figure: M:DL2000; 1: positive plasmid pGEM-T-hly; 2: the Listeria monocytogenes genomic dna; 3: negative control.
Fig. 4 is Shigellae LAMP reaction product dyeing figure;
Among the figure: 1, positive plasmid pGEM-T-ipaH; 2, shigella dysenteriae 48097; 3, shigella flexneri 51302; 4, Shigella bogdii 51149; 5, the Song Shi Shigellae 51334; 6, negative control.
Embodiment
Embodiment
(1) nucleic acid extraction: from 10 hours bacteria suspension of 37 ℃ of shaking tables cultivations, draw 1ml bacterium liquid, the centrifugal 5min of 10000rpm, abandon supernatant, add the concussion of 100 μ l sterilized waters and shake up (or a picking 2-3 bacterium colony is in 100 μ l sterilized waters on the flat board of preserving) from 4 ℃ of refrigerators.100 ℃ are boiled 10min, and with the centrifugal 5min of 10000rpm, supernatant liquor is a genomic dna with desk centrifuge, can be directly as the template of nucleic acid amplification reaction.
(2) LAMP reaction: prepare above-mentioned reaction system, with the reaction system mixing, divide and install in the PCR reaction tubes of 0.2ml, every pipe 19ul, add the extractive template of (1) step, every pipe 1ul, positive reference substance and each 1ul of negative control product in the PCR reaction tubes respectively, make positive control, negative control respectively, mix.Put into constant water bath box, set reaction conditions: 65 ℃, 60 minutes, 80 ℃ of 10 minutes termination reactions, 10 ℃ of preservations, reaction finish the back and add 1 μ L Pico Green dyestuff, mixing, observing response pipe whether color changes or gets the above-mentioned bacterium LAMP of 5 μ L reaction product, carries out electrophoresis detection on 2% sepharose.
(3) result judges: the reaction tubes of positive for bacteria genomic dna template can be observed color and becomes green, and negative control then is orange.After agarose gel electrophoresis detected, the reaction product of positive for bacteria genomic dna template had specific scalariform band to occur, and negative control does not then have band and occurs.
It is shown in Figure 4 that it the results are shown in accompanying drawing 1-.
Sequence table
＜110〉Xuzhou Entry-Exit Inspection and Quarantine Bureau, People's Republic of China
＜120〉based on the streptococcus aureus, Salmonellas, Shigellae and the Listeria monocytogenes nucleic acid screening method that encircle the isothermal amplification technique that mediates
<160>4
<170>Primer?explorer?v4
<210>1
<211>18
<212>DNA
＜213〉primer
<400>1
TCAACTGAAG?CAACACCT 18
<210>2
<211>19
<212>DNA
＜213〉primer
<400>2
CAGTCGTACC?AGAGTCAAT 19
<210>3
<211>46
<212>DNA
＜213〉primer
<400>3
CGCACTTGTA?TTAACCGCTT?GATTATCAAA?CAATGAATCA?GCTCCA 46
<210>4
<211>45
<212>DNA
＜213〉primer
<400>4
AGAGCATTTA?GTTTAGCGGC?AGTAGCAACT?GTCACATTCG?TCAAC 45
<210>5
<211>18
<212>DNA
＜213〉primer
<400>5
GAACGTGTCG CGGAAGTC’ 18
<210>6
<211>19
<212>DNA
＜213〉primer
<400>6
CGGCAATAGC?GTCACCTT’ 19
<210>7
<211>38
<212>DNA
＜213〉primer
<400>7
GCGCGGCATC?CGCATCAATA?TCTGGATGGT?ATGCCCGG 38
<210>8
<211>39
<212>DNA
＜213〉primer
<400>8
GAACGGCGAA?GCGTACTGGA?CATCGCACCG?TCAAAGGAA 39
<210>9
<211>18
<212>DNA
＜213〉primer
<400>9
CCGGATTCCG?TGAACAGG 18
<210>10
<211>20
<212>DNA
＜213〉primer
<400>10
AGCGCCGGTA?TCATTATCGA 20
<210>11
<211>42
<212>DNA
＜213〉primer
<400>11
AGCAACAGCG?AAAGACTGCT?GTCTGCATGG?CTGGAAAAACTC 42
<210>12
<211>40
<212>DNA
＜213〉primer
<400>12
GCCACTGAGA?GCTGTGAGGA?CCTGATGGAC?CAGGAGGGTT 40
<210>13
<211>18
<212>DNA
＜213〉primer
<400>13
TAAACTTCGG?CGCAATCA 18
<210>14
<211>20
<212>DNA
＜213〉primer
<400>14
CAAATAAACT?TGACGGCCAT 20
<210>15
<211>46
<212>DNA
＜213〉primer
<400>15
GGAAGGTCTT?GTAGGTTCAT?TAACAGGAAA?ATGCAAGAAG?AAGTCA 46
<210>16
<211>44
<212>DNA
＜213〉primer
<400>16
TCGGCAAAGC?TGTTACTAAA?GAGCTTGAGA?TATATGCAGG?AGGA 44
Claims (4)
1. nucleic acid detection method that is used for streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes, utilize ring mediated isothermal amplification (LAMP) technology to carry out, it is characterized in that, by using Auele Specific Primer, utilize the specific region of LAMP technology platform amplified target gene, assisting down of positive and negative Quality Control and internal reference detection architecture, streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes are carried out nucleic acid screening or detection from molecular level; Described streptococcus aureus clfA gene LAMP detection specificity primer is:
The LAMP reaction primer sequence of (GeneBank EF207779) is formed
Described Salmonellas invA gene LAMP detects primer
The LAMP reaction primer sequence of (GeneBank M90846) is formed
Described Shigellae ipaH gene LAMP detects primer
The LAMP reaction primer sequence of (GeneBank M76444) is formed
The described hly gene LAMP that singly increases the Liszt detects primer and is
The LAMP reaction primer sequence of (GeneBank M24199) is formed
2. streptococcus aureus according to claim 1, Salmonellas, Shigellae and Listeria monocytogenes nucleic acid screening detection method is characterized in that utilizing the particular target nucleotide sequence zone of LAMP technology amplification streptococcus aureus, Salmonellas, Shigellae and Listeria monocytogenes.
3. streptococcus aureus according to claim 1, Salmonellas, Shigellae and Listeria monocytogenes nucleic acid detection method, it is characterized in that in the described yin and yang attribute control test system, the positive control quality control product is the DNA that is same as amplification bacterium target gene, the negative control quality control product is the DNA in amplification bacterium target gene inequality, and uses electrophoretic analysis, development process detects negative and positive contrast and streptococcus aureus simultaneously under the Pico Green dyestuff ultraviolet lamp, Salmonellas, Shigellae and Listeria monocytogenes target gene amplified production.
4. the nucleic acid detection method of streptococcus aureus according to claim 1, Salmonellas, Shigellae and Listeria monocytogenes, it is characterized in that, used LAMP reaction system is a series of reacted constituent compositions by optimizing, and has specific response procedures; Its LAMP reaction system is composed as follows: 10 * Bst buffer (200mM Tris-HCl (pH8.8,25 ℃), 100mM KCl, 100mM (NH
4)
2SO
4, 20mMMgSO
4, 0.1%Triton-100) 2.5 μ l; Betaine (4M) 5 μ l; Primer (F3: B3: FIP: BIP=5 μ M: 5 μ M: 40 μ M: 1 μ l 40 μ M); Bst enzyme (8U) 1 μ l; DNTPs (10mM) 3.5 μ l; MgCl
2(25mM) 6 μ l; Template 1 μ l; Sterilization deionized water 4 μ l; Amplification program is: 65 ℃ of 1 hour → 80 ℃ 10 minutes → 10 ℃ preservations.
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| CN102586439A (en) * | 2012-02-28 | 2012-07-18 | 盛司潼 | Method for simultaneously and quickly detecting multiple nucleic acids |
| CN102851381A (en) * | 2012-09-21 | 2013-01-02 | 武汉真福医药科技发展有限公司 | LAMP kit for rapid detection of Listeria monocytogenes |
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