CN104087654B - The identification with multi-plex PCR test kit of Salmonellas and five kinds of serotypes thereof - Google Patents
The identification with multi-plex PCR test kit of Salmonellas and five kinds of serotypes thereof Download PDFInfo
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Abstract
The invention discloses the identification with multi-plex PCR test kit of a kind of Salmonellas and five kinds of serotypes thereof.The invention provides the primer sets detecting Salmonellas and five kinds of serotypes thereof, be made up of primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5 and primer pair 6.The present invention is directed to Salmonellas and five kinds of important serotype (mouse typhus, enteritis, Argonne that, hog cholera and S. pullonum) specific fragment, be designed for six pairs of primers of specific amplified, amplified fragments size is respectively Salmonellas-425bp, mouse typhus-198bp, enteritis-302bp, that-145bp of Argonne, hog cholera-368bp, white dysentery-249bp, sample gene group, through multiplex PCR, electrophoresis detection, can make corresponding judgement.The present invention, by providing six pairs of Auele Specific Primers, utilizes a step multiplex PCR, can identify Salmonellas and five kinds of important serotypes, has fast, low cost, simple to operate and result are easy to the feature that judges.
Description
Technical field
The present invention relates to biological technical field, particularly relate to the identification with multi-plex PCR test kit of a kind of Salmonellas and five kinds of serotypes thereof.
Background technology
Salmonellas, as one of important pathogenic micro-organism, by food transmission to the mankind, can be caused the symptom such as gastro-enteritis, septicemia, become food borne pathogenic microorganism important in worldwide.Biochemical identification and serological typing are the EPDML common methods of research salmonellosis, because Salmonellas has 2600 various serotypes, conventional serological somatotype needs a large amount of diagnostic serums, those skilled in the art and loaded down with trivial details operation steps, due to the existence of rough type bacterium and single-phase bacterium, cause conventional serological somatotype cannot accurate somatotype to part Salmonellas.Therefore, more simply, authentication method has great importance in practice accurately and rapidly.At present, mouse typhus, enteritis, Argonne that, hog cholera and S. pullonum as common Salmonellas, set up most important in epidemiology survey and clinical diagnosis practice to its method identified fast.
Multiple PCR technique improves on the basis of Standard PCR, namely, in a PCR reaction system, by adding multipair primer, multiple target sequence is increased, detected, its reaction principle, reagent composition is consistent with Standard PCR with operating process, has more efficient, economic feature.
By finding the retrieval of existing document, have two kinds of thinkings to the PCR qualification of Salmonellas at present: one is the specific fragment for each serotype, design primer is identified; Two is the coding genes involveds for O antigen and H antigen, and design primer is identified.The people such as Liu Bin (2012) have delivered " DevelopmentofanovelmultiplexPCRassayfortheidentification ofSalmonellaentericaTyphimuriumandEnteritidis " at " FoodControl " magazine, specific fragment for mouse typhus and Salmonella enteritidis designs primer, it is identified, due to two kinds of serotypes only can be identified, there is certain limitation.
Summary of the invention
An object of the present invention is to provide the primer sets of a kind of detection or auxiliary detection Salmonellas or its five kinds of serotypes.
Primer sets provided by the invention, is made up of primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5 and primer pair 6;
Described primer pair 1 is made up of the single strand dna shown in sequence 8 in the single strand dna shown in sequence in sequence table 7 and sequence table;
Described primer pair 2 is made up of the single strand dna shown in sequence 10 in the single strand dna shown in sequence in sequence table 9 and sequence table;
Described primer pair 3 is made up of the single strand dna shown in sequence 12 in the single strand dna shown in sequence in sequence table 11 and sequence table;
Described primer pair 4 is made up of the single strand dna shown in sequence 14 in the single strand dna shown in sequence in sequence table 13 and sequence table;
Described primer pair 5 is made up of the single strand dna shown in sequence 16 in the single strand dna shown in sequence in sequence table 15 and sequence table;
Described primer pair 6 is made up of the single strand dna shown in sequence 18 in the single strand dna shown in sequence in sequence table 17 and sequence table.
In above-mentioned primer sets, in described primer sets, the ratio of each bar primer is equimolar ratio;
Described Salmonellas five kinds of serotypes are Salmonella typhimurium, Salmonella enteritidis, that Salmonellas of Argonne, Salmonella choleraesuls and S. pullonum.
Another object of the present invention is to provide the PCR reagent of a kind of detection or auxiliary detection Salmonellas or its five kinds of serotypes.
PCR reagent provided by the invention, is made up of above-mentioned primer sets, PCR damping fluid and water;
Above-mentioned PCR damping fluid is PremixExTaq
tMversion2.0 (Loadingdyemix), purchased from TaKaRa, D335A; Containing Taq enzyme, dNTP, Mg2+, Loadingbuffer; Wherein, the amount of Taq enzyme in reaction system is 0.5U, the final concentration of various dNTP in reaction system be 0.2mM, Mg
2+final concentration in reaction system is 2mM.
The final concentration of each bar primer in described PCR reagent in described primer sets is 0.1 μM;
Described Salmonellas five kinds of serotypes are specially Salmonella typhimurium, Salmonella enteritidis, that Salmonellas of Argonne, Salmonella choleraesuls and S. pullonum.
Above-mentioned water is sterilized water.
3rd object of the present invention is to provide the PCR kit of a kind of detection or auxiliary detection Salmonellas or its five kinds of serotypes.
Test kit provided by the invention, comprises above-mentioned primer sets or above-mentioned PCR reagent; Described Salmonellas five kinds of serotypes are specially Salmonella typhimurium, Salmonella enteritidis, that Salmonellas of Argonne, Salmonella choleraesuls and S. pullonum.
Above-mentioned primer sets or above-mentioned PCR reagent to detect and/or whether auxiliary detection tested bacteria is application in Salmonellas product in preparation;
Or the application in preparation detects and/or whether auxiliary detection tested bacteria is any one product in Salmonellas five kinds of serotypes of above-mentioned primer sets or above-mentioned PCR reagent is also the scope of protection of the invention; In above-mentioned application, described Salmonellas five kinds of serotypes are specially Salmonella typhimurium, Salmonella enteritidis, that Salmonellas of Argonne, Salmonella choleraesuls and S. pullonum.
Detection in above-mentioned application and/or auxiliary detection are carry out multiplexed PCR amplification by above-mentioned primer sets or above-mentioned PCR reagent to described tested bacteria.
In above-mentioned application, the annealing temperature of described multiplexed PCR amplification is 60 DEG C.
4th object of the present invention is to provide a kind of primer pair.
Primer pair provided by the invention is any one in following 6 kinds of primer pairs: primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5 and primer pair 6;
Described primer pair 1 is made up of the single strand dna shown in sequence 8 in the single strand dna shown in sequence in sequence table 7 and sequence table;
Described primer pair 2 is made up of the single strand dna shown in sequence 10 in the single strand dna shown in sequence in sequence table 9 and sequence table;
Described primer pair 3 is made up of the single strand dna shown in sequence 12 in the single strand dna shown in sequence in sequence table 11 and sequence table;
Described primer pair 4 is made up of the single strand dna shown in sequence 14 in the single strand dna shown in sequence in sequence table 13 and sequence table;
Described primer pair 5 is made up of the single strand dna shown in sequence 16 in the single strand dna shown in sequence in sequence table 15 and sequence table;
Described primer pair 6 is made up of the single strand dna shown in sequence 18 in the single strand dna shown in sequence in sequence table 17 and sequence table.
The present invention relates to a kind of food borne pathogenic microorganism Rapid Identification based on multiplex PCR (multiplexpolymerasechainreaction) technology.For Salmonellas and five kinds of important serotype (mouse typhus, enteritis, Argonne that, hog cholera and S. pullonum) specific fragment, be designed for six pairs of primers of specific amplified, amplified fragments size is respectively Salmonellas-425bp, mouse typhus-198bp, enteritis-302bp, that-145bp of Argonne, hog cholera-368bp, white dysentery-249bp, sample gene group, through multiplex PCR, electrophoresis detection, can make corresponding judgement.The present invention, by providing six pairs of Auele Specific Primers, utilizes a step multiplex PCR, can identify Salmonellas and five kinds of important serotypes, has fast, low cost, simple to operate and result are easy to the feature that judges.
Accompanying drawing explanation
Fig. 1 is the amplification figure of multiplex PCR
Fig. 2 is sensitivity technique result figure
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, for detecting the acquisition of the primer of Salmonellas and serotype thereof
Use bioinformatic analysis tools BLAST, in full-length genome, find out the specific sequence of Salmonellas specific sequence InvA gene (sequence 1) and five kinds of serotypes: Salmonella typhimurium distinguished sequence (sequence 2), Salmonella enteritidis distinguished sequence (sequence 3), that Salmonellas distinguished sequence of Argonne (sequence 4), Salmonella choleraesuls distinguished sequence (sequence 5), S. pullonum distinguished sequence (sequence 6); For six pairs of specific sequences, application PrimerPlex2.61 molecular biology software, optimum configurations: amplicon length 100bp-500bp, primer length 18-25bp, design six pairs of PCR primer, primer sequence is as follows:
Salmonellas specific primer sequences:
InvA-F:GCTCGTAATTCGCCGCCATT; (sequence 7)
InvA-R:CATCAGCAAGGTAGCAGTCAGTATT; (sequence 8)
Salmonella typhimurium specific primer sequences:
Sty-F:AGCCAGCCGATAACCTCAACTT; (sequence 9)
Sty-R:CGAAGCCATCTACCGTGAACTG; (sequence 10)
Salmonella enteritidis specific primer sequences:
Sen-F:CGCAAGGCTATTGTTGAAGTGAAG; (sequence 11)
Sen-R:CAGTACAGGCACAGCACATCAA; (sequence 12)
That Salmonellas specific primer sequences of Argonne:
Sao-F:AATTGTCTGCGTCATTGAGTTGGA; (sequence 13)
Sao-R:CGGCGGTTCTTCATCTATCTTCG; (sequence 14)
Salmonella choleraesuls specific primer sequences:
Sch-F:CATTACAAGGACGAAGACGCTTATC; (sequence 15)
Sch-R:CAACAGGTATTGCCAGACACAGT; (sequence 16)
S. pullonum specific primer sequences:
Sga-F:GACATCAGTACAGTTCTCCACCTC; (sequence 17)
Sga-R:CACTCCGCTTATTCACCGACAG; (sequence 18)
Embodiment 2, for detecting the application of the primer of Salmonellas and serotype thereof
One, primer is detecting the application in Salmonellas and serotype thereof
1, the extraction of genomic dna
Detect by 12 strain Salmonellass and 10 strain nonsalmonella, bacterial strain composition is as shown in table 1; Use genome to extract test kit (Tiangen company, article No. DP302), extract the genomic dna of 12 strain Salmonellass and 10 strain nonsalmonella in table 1.Wherein 12 strain Salmonellass comprise five kinds of serotype Salmonellass: Salmonella typhimurium, Salmonella enteritidis, that Salmonellas of Argonne, Salmonella choleraesuls and S. pullonum.
Wherein Klebsiella klebsiella S1, Serratia Serratia S2, Citrobacter citrobacter S3, Enterobactercloacae enterobacter cloacae S4 is all described below in document: He Tao. and zoological park primate enterobacteriaceae lactobacteriaceae Antimicrobial Resistance is studied. Beijing: animal medicine institute of China Agricultural University, 2011 summers;
Enterococcusfaecalis enterococcus faecalis JH2-2 is documented in as in Publication about Document: LiuY; WangY; WuC; ShenZ, SchwarzS, DuXD; DaiL; ZhangW, ZhangQ, ShenJ.FirstreportofthemultidrugresistancegenecfrinEntero coccusfaecalisofanimalorigin.AntimicrobAgentsChemother.2 012Mar; 56 (3): 1650-4;
Proteusvulgaris proteus vulgaris S5 is documented in as in Publication about Document: WangY; WangY; WuCM; SchwarzS; ShenZ; ZhangW, ZhangQ, ShenJZ.Detectionofthestaphylococcalmultiresistancegenecf rinProteusvulgarisoffoodanimalorigin.JAntimicrobChemothe r.2011Nov; 66 (11): 2521-6;
SalmonellaSaintpaul moscow' saint paul S6 is documented in as in Publication about Document: Li, R., Lai, J., Wang, Y., Liu, S., Li, Y., Liu, K., Shen, J., Wu, C.2013.PrevalenceandcharacterizationofSalmonellaspeciesi solatedfrompigs, ducksandchickensinSichuanProvince, China.InternationalJournalOfFoodMicrobiology163,14-18.
Above-mentioned each bacterium all can obtain from China Agricultural University.
Table 1 is bacterial strain to be detected and source thereof
2, multiplex PCR specific amplification
Six pairs of specific PCR primers embodiment 1 synthesized are diluted to 10 μMs respectively, and then six pairs of primer equimolar ratio examples mixing, becomes mix primer.
Each component in 20 μ L multi-PRC reaction systems and final concentration thereof are: [PremixExTaqTMVersion2.0 (Loadingdyemix), purchased from TaKaRa, D335A, containing Taq enzyme, dNTP, Mg for 10ulPCR damping fluid
2+, Loadingbuffer; Wherein, the amount of Taq enzyme in reaction system is 0.5U, the final concentration of various dNTP in reaction system be 0.2mM, Mg
2+final concentration in reaction system is 2mM], each 0.2 μ L(mix primer 2.4 μ L of every bar primer in six pairs of PCR primer of embodiment 1, the final concentration of each primer in reaction system is 0.1 μM), templet gene group DNA1 μ L, supply volume with sterilized water.Removing template, other component in reaction system can form pcr amplification reagent.
Above-mentioned template is respectively the genomic dna of 12 strain Salmonellass in table 1, the genomic dna of 10 strain nonsalmonella, positive control (the genomic dna equal-volume of five kinds of serotype Salmonellass mixes by hybrid template) and negative control (sterilized water).
20 μ l multi-PRC reaction systems are carried out pcr amplification according to following condition:
Multi-PRC reaction condition is: 95 DEG C of denaturation 5min, afterwards 32 circulations (94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s), and after loop ends, 72 DEG C extend 8min, 4 DEG C of preservations; Obtain PCR primer.
After amplification, 3% sepharose carries out electrophoresis, condition is 5V/cm, 25min, obtains picture by gel imaging system.
If the PCR primer obtained has the fragment of 425bp size, then test strains is or candidate is Salmonellas, otherwise test strains is not or candidate is not Salmonellas;
If the PCR primer obtained only has the fragment of 425bp and 198bp size, then test strains is or candidate is Salmonella typhimurium; Otherwise test strains is not or candidate is not Salmonella typhimurium;
If the PCR primer obtained only has the fragment of 425bp and 302bp size, then test strains is or candidate is Salmonella enteritidis; Otherwise test strains is not or candidate is not Salmonella enteritidis;
If the PCR primer obtained only has the fragment of 425bp and 145bp size, then test strains is or candidate is that Salmonellas of Argonne; Otherwise test strains is not or candidate is not that Salmonellas of Argonne;
If the PCR primer obtained only has the fragment of 425bp and 368bp size, then test strains is or candidate is Salmonella choleraesuls; Otherwise test strains is not or candidate is not Salmonella choleraesuls;
If the PCR primer obtained only has the fragment of 425bp and 249bp size, then test strains is or candidate is S. pullonum; Otherwise test strains is not or candidate is not S. pullonum.
Result as shown in Figure 1, swimming lane 1-25 is followed successively by respectively: DL500DNAMarker, Salmonella typhimurium ATCC70048, Salmonella typhimurium ATCC13311, Salmonella enteritidis ATCC13076, S. pullonum ATCC10398, that Salmonellas ATCC51957 of Argonne, Salmonella choleraesuls ATCC7001, Salmonella anatis ATCC9270, salmonella london ATCC8389, Indiana Salmonellas ATCC51957, Boulogne steps on Lu Pu Salmonellas ATCCBAA-664, the inferior Salmonellas ATCC6960 of Dare, intestinal bacteria ATCC25922, Pseudomonas aeruginosa ATCC27853, streptococcus aureus ATCC29213, animal campylobacter jejuni ATCC33560, enterococcus faecalis JH2-2, klebsiella S1, Serratia S2, citrobacter S3, enterobacter cloacae S4, proteus vulgaris S5, moscow' saint paul S6, positive control (hybrid template), negative control (without template), the product only having a 425bp of S6 shown in ATCC9270, ATCC8389, ATCC51957, ATCCBAA-664, the ATCC6960 shown in swimming lane 8-12 and swimming lane 23, illustrate that these bacterial strains are Salmonellas, and be not any one in five kinds of serotype Salmonellass, ATCC70048, ATCC13311 shown in swimming lane 2 and 3 has the product of 425bp and 198bp, illustrates that this two strains bacterial strain is Salmonella typhimurium, ATCC13076 shown in swimming lane 4 has the product of 425bp and 302bp, illustrates that this strain bacterial strain is Salmonella enteritidis, ATCC10398 shown in swimming lane 5 has the product of 425bp and 249bp, illustrates that this strain bacterial strain is S. pullonum, ATCC51957 shown in swimming lane 6 has the product of 425bp and 145bp, illustrates that this strain bacterial strain is that Salmonellas of Argonne, ATCC57001 shown in swimming lane 7 has the product of 425bp and 368bp, illustrates that this strain bacterial strain is Salmonella choleraesuls, positive control (hybrid template) has the product of all sizes.Other swimming lanes do not have the product of 425bp, are illustrated as nonsalmonella.
Above result shows, primer specificity is high, can be used for identifying Salmonellas or its five kinds of serotypes.
Two, sensitivity technique
PCR reaction is carried out by above-mentioned 20 μ l multi-PRC reaction systems and above-mentioned multi-PRC reaction condition, template is following five kinds of serotypes: the genomic dna of Salmonella typhimurium ATCC70048, Salmonella enteritidis ATCC13076, S. pullonum ATCC10398, Argonne that Salmonellas ATCC51957 and Salmonella choleraesuls ATCC57001 carries out PCR reaction, electrophoresis detection amplified production according to the amount of template amount 50ng, 10ng, 5ng, 1ng, 0.5ng, 0.1ng, 0.05ng and 0.01ng in every 20ul system respectively.
Observe electrophoresis result, as shown in Figure 2: every strain Salmonellas template amount is from left to right followed successively by 50ng, 10ng, 5ng, 1ng, 0.5ng, 0.1ng, 0.05ng and 0.01ng, swimming lane M:DL500DNAMarker, swimming lane sun: positive control (hybrid template, the amount equal-volume mixing of 50ng is often planted according to five kinds of serotypes), swimming lane is cloudy: negative control (sterilized water).As can be seen from Figure 2, the 5th swimming lane of every strain bacterium still can see band clearly, and corresponding DNA amount is 0.5ng, and therefore the detection sensitivity of this multiplex PCR is 0.5ng/PCR, has higher sensitivity.
Therefore, the present invention, by providing six pairs of Auele Specific Primers, utilizes a step multiplex PCR, can identify Salmonellas or five kinds of important serotypes, has fast, low cost, simple to operate and result are easy to the feature that judges.
Claims (7)
1. the primer sets of detection or auxiliary detection Salmonellas or its five kinds of serotypes, is made up of primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5 and primer pair 6;
Described primer pair 1 is made up of the single strand dna shown in sequence 8 in the single strand dna shown in sequence in sequence table 7 and sequence table;
Described primer pair 2 is made up of the single strand dna shown in sequence 10 in the single strand dna shown in sequence in sequence table 9 and sequence table;
Described primer pair 3 is made up of the single strand dna shown in sequence 12 in the single strand dna shown in sequence in sequence table 11 and sequence table;
Described primer pair 4 is made up of the single strand dna shown in sequence 14 in the single strand dna shown in sequence in sequence table 13 and sequence table;
Described primer pair 5 is made up of the single strand dna shown in sequence 16 in the single strand dna shown in sequence in sequence table 15 and sequence table;
Described primer pair 6 is made up of the single strand dna shown in sequence 18 in the single strand dna shown in sequence in sequence table 17 and sequence table;
Described Salmonellas five kinds of serotypes are Salmonella typhimurium, Salmonella enteritidis, that Salmonellas of Argonne, Salmonella choleraesuls and S. pullonum.
2. primer sets according to claim 1, is characterized in that:
In described primer sets, the ratio of each bar primer is equimolar ratio.
3. the PCR reagent of detection or auxiliary detection Salmonellas or its five kinds of serotypes, is made up of the primer sets described in claim 1 or 2, PCR damping fluid and water;
The final concentration of each bar primer in described PCR reagent in described primer sets is 0.1 μM;
Described Salmonellas five kinds of serotypes are specially Salmonella typhimurium, Salmonella enteritidis, that Salmonellas of Argonne, Salmonella choleraesuls and S. pullonum.
4. detect or the PCR kit of auxiliary detection Salmonellas or its five kinds of serotypes, comprise PCR reagent described in primer sets described in claim 1 or 2 or claim 3; Described Salmonellas five kinds of serotypes are Salmonella typhimurium, Salmonella enteritidis, that Salmonellas of Argonne, Salmonella choleraesuls and S. pullonum.
5. the application of PCR reagent described in the primer sets described in claim 1 or 2 or claim 3 in preparation detects and/or whether auxiliary detection tested bacteria is any one product in Salmonellas five kinds of serotypes; Described Salmonellas five kinds of serotypes are Salmonella typhimurium, Salmonella enteritidis, that Salmonellas of Argonne, Salmonella choleraesuls and S. pullonum.
6. apply according to claim 5, it is characterized in that:
Detection in described application and/or auxiliary detection are for carrying out multiplexed PCR amplification by PCR reagent described in the primer sets described in claim 1 or 2 or claim 3 to described tested bacteria.
7. apply according to claim 5 or 6, it is characterized in that:
The annealing temperature of described multiplexed PCR amplification is 60 DEG C.
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| Blind comparison of traditional serotyping with three multiplex PCRs for the identification of Salmonella serotypes;Herrera-Leon S et al.;《Res Microbiol》;20061220;第158卷(第2期);摘要，材料和方法，结果，图1，表1-3 * |
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