CN107201400B - Quintuple PCR detection method and detection kit for avian escherichia coli, salmonella gallinarum, salmonella pullorum and the like - Google Patents
Quintuple PCR detection method and detection kit for avian escherichia coli, salmonella gallinarum, salmonella pullorum and the like Download PDFInfo
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Abstract
The invention provides a multiplex PCR primer group for avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium, a quintuple PCR detection kit comprising the primer group and a quintuple PCR detection method. The primer group comprises: primer pairs ECphoA-F and ECphoA-R for specifically amplifying the avian escherichia coli phoA gene; a primer pair SGglgC-F and SGglgC-R for specifically amplifying the glgC gene of the salmonella gallinarum; a primer pair SGPspeC-F and SGPspeC-R of the speC genes of the salmonella gallinarum and the salmonella pullorum is specifically amplified; primer pairs SEsdf I-F and SEsdf I-R for specifically amplifying salmonella enteritidis sdf I gene; primer pairs STstm4495-F and STstm4495-R for specifically amplifying stm4495 gene of Salmonella typhimurium.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a multiplex PCR primer set for detecting avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium, a quintuple PCR detection kit comprising the primer set, and a quintuple PCR detection method using the primer set.
Background
With the rapid development of the large-scale poultry industry, the occurrence and the prevalence of epidemic diseases are correspondingly increased, which leads to the reduction of economic benefits of the poultry industry. The poultry industry is seriously harmed by important bacterial infectious diseases such as avian colibacillosis, pullorum disease, fowl typhoid and the like.
At present, the domestic prevention and treatment of bacterial diseases mainly depends on antibiotics, but the long-term and unreasonable use of the antibiotics causes that: on one hand, the wide drug resistance is generated, and on the other hand, the drug residue directly influences the safety of animal products. Therefore, the enhancement of long-term monitoring and research of these bacterial diseases is of great importance to the poultry industry and public health.
The diagnosis and monitoring technology for bacterial diseases at home and abroad is various, and mainly comprises the following steps: bacteria separation culture and biochemical identification, molecular biology technology, immunology technology and the like. The PCR technology is widely applied due to the advantages of strong specificity, high sensitivity, simple operation, rapid detection and the like.
At present, various detection methods are established for avian escherichia coli and different serotype salmonella, however, a rapid detection and differential diagnosis PCR method for avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium is not provided.
Disclosure of Invention
In order to solve the problems, the invention adopts the following technical scheme:
one object of the present invention is to provide a primer set for detecting avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis, and salmonella typhimurium, which is capable of accurately, rapidly, stably, specifically, and with high sensitivity, and is characterized by comprising: primer pairs ECphoA-F and ECphoA-R for specifically amplifying the avian escherichia coli phoA gene; a primer pair SGglgC-F and SGglgC-R for specifically amplifying the glgC gene of the salmonella gallinarum; a primer pair SGPspeC-F and SGPspeC-R of the speC genes of the salmonella gallinarum and the salmonella pullorum is specifically amplified; primer pairs SEsdf I-F and SEsdf I-R for specifically amplifying salmonella enteritidis sdf I gene; a primer pair STstm4495-F and STstm4495-R for specifically amplifying the stm4495 gene of the salmonella typhimurium,
the nucleotide sequences of the primers are respectively as follows:
ECphoA-F，5’-GGCAATACACTCACTATGCGCTG-3’,
ECphoA-R，5’-AGGATTCGCAGCATGATCCTG-3’；
SGglgC-F，5’-CGTCGCTATAAAGCGGAATATGTC-3’，
SGglgC-R，5’-CCTTTTCAAAACATACGCGAGTAG-3’；
SGPspeC-F，5’-GTTGCCGTACCGGTCGTAACG-3’，
SGPspeC-R，5’-TTCCTGACGGGCATCGACGGC-3’；
SEsdfⅠ-F，5’-GATGTGGTTGGTTCGTCACTG-3’，
SEsdfⅠ-R，5’-GCGAGACCTCAAACTTACTCAG-3’；
STstm4495-F，5’-CAGCGGTATGATGCGGTAGT-3’，
STstm4495-R，5’-TCACCGGTGGACATGCCTGC-3’。
the primer set provided by the invention also has the following characteristics: the size of the target fragment for amplification of the primer pair ECPhoA-F and ECPhoA-R is 761 bp; the size of a target fragment for amplification of the primer pair SGglgC-F and SGglgC-R is 83 bp; the size of a target fragment for amplification of the primer pair SGPspeC-F and SGPspeC-R is 249 bp; the size of a target fragment for amplification of a primer pair SEsdf I-F and SEsdf I-R is 409 bp; the target fragment size for amplification of primer pair STstm4495-F and STstm4495-R was 569 bp.
Another objective of the present invention is to provide a quintuple PCR assay kit for detecting avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium, comprising: 2 XPCR premixed solution and a primer group for establishing a PCR reaction system, wherein the PCR reaction system is used for specifically amplifying each primer pair in the primer group, and the primer group is the primer group.
The quintuple PCR detection kit provided by the invention also has the following characteristics: wherein the volume of 2 XPCR PreMix was 12.5. mu.L, in the primer set, the volumes of the primer pair ECphoA-F and ECphoA-R were 0.5. mu.L each, the volumes of the primer pair SGglgC-F and SGglgC-R were 0.2. mu.L each, the volumes of the primer pair SGPspeC-F and SGPspeC-R were 0.3. mu.L each, the volumes of the primer pair SEsdfI-F and SEsdfI-R were 0.4. mu.L each, and the volumes of the primer pair STm4495-F and STstm4495-R were 0.5. mu.L each.
The invention also aims to provide a quintuple PCR detection method for detecting avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium, which is characterized by comprising the following steps: step 1, preparing a PCR template based on bacteria to be detected; step 2, preparing a specific primer group, and preparing the specific primer group by using the primer group of any one of claims 1 or 2; step 3, carrying out PCR amplification reaction, based on the PCR template obtained in the step 1, establishing a PCR reaction system comprising the primer group by adopting the specific primer group obtained in the step 2, and then carrying out PCR amplification reaction by adopting the PCR reaction system under the condition of preset PCR reaction parameters to obtain a PCR amplification product; and 4, performing electrophoresis detection on the amplification product to obtain an electrophoresis result, and judging whether avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis or salmonella typhimurium appears according to the electrophoresis result.
The quintuple PCR detection method provided by the invention also has the following characteristics: the PCR template obtained in the step 1 is a bacterial liquid of bacteria to be detected, and the specific process is as follows: inoculating the strain of bacteria to be detected to LB liquid medium, and culturing at 37 deg.C to OD600The obtained bacterial liquid is the PCR template, which is 1.0.
The quintuple PCR detection method provided by the invention also has the following characteristics: the PCR template obtained in the step 1 is a bacteria crude extraction DNA, and the specific process is as follows: inoculating a bacterial strain to be detected to an LB liquid culture medium, culturing at 37 ℃ until OD600 is 1.0 to obtain bacterial liquid, centrifuging 1mL of the bacterial liquid at 10000-12000r/min for 1-2min, removing supernatant, adding 0.1-0.5mL of sterilized ultrapure water for resuspension, boiling for 5-10min, and centrifuging at 10000-12000r/min for 3-5min to obtain supernatant which is the crude bacterial DNA.
The quintuple PCR detection method provided by the invention also has the following characteristics: the PCR reaction system of the step 3 comprises: 12.5. mu.L of 2 XPCR PreMix, 1.0. mu.L of template, 7.7. mu.L of ultrapure water, and a primer set in which the volumes of the primer pair ECphoA-F and ECphoA-R were each 0.5. mu.L, the volumes of the primer pair SGglgC-F and SGglgC-R were each 0.2. mu.L, the volumes of the primer pair SGPspeC-F and SGPspeC-R were each 0.3. mu.L, the volumes of the primer pair SEsdf I-F and SEsdf I-R were each 0.4. mu.L, and the volumes of the primer pair STstm4495-F and STstm4495-R were each 0.5. mu.L.
The quintuple PCR detection method provided by the invention also has the following characteristics: when a specific primer set was prepared, the concentration of each primer used in the primer set was 10pM, and the dilutions of each primer used were stored at-20 ℃ and used.
The quintuple PCR detection method provided by the invention also has the following characteristics: pre-denaturing the PCR reaction parameters at 95 ℃ for 5 min; 35 cycles at 95 ℃ for 30s, 57 ℃ for 30s, and 72 ℃ for 50 s; 72 ℃ for 10 min.
The quintuple PCR detection method provided by the invention also has the following characteristics: taking 10 mu L of PCR amplification product, spotting the product in a hole of a 2.0% agarose gel electrophoresis plate, carrying out electrophoresis for 30-40min under the voltage of 80-100V, taking a picture under a gel imaging system to obtain an electrophoresis result, judging the electrophoresis result under the precondition that no band appears in negative control, judging the poultry escherichia coli when a 761bp band appears in the electrophoresis result, judging the chicken salmonella typhi when 83bp and 249bp bands appear in the electrophoresis result, judging the chicken salmonella typhimurium when a 249bp band appears in the electrophoresis result, judging the salmonella enteritis when a 409bp band appears in the electrophoresis result, and judging the salmonella typhimurium when a 569bp band appears in the electrophoresis result.
The invention also provides an application of the primer group in preparing a quintuple PCR detection kit for detecting avian Escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium.
Action and Effect of the invention
The multiplex PCR primer set for detecting the avian escherichia coli, the salmonella gallinarum, the salmonella pullorum, the salmonella enteritidis and the salmonella typhimurium, the quintuple PCR detection kit comprising the primer set and the quintuple PCR detection method using the primer set, provided by the invention, have the advantages of sensitivity, specificity, rapidness and stability for detecting the avian escherichia coli, the salmonella gallinarum, the salmonella pullorum, the salmonella enteritidis and the salmonella typhimurium by adopting the quintuple PCR detection method of the primer set or the quintuple PCR detection kit comprising the primer set, because the primer set has strong specificity and high sensitivity and good repeatability, and the effective means is provided for rapidly detecting and identifying the avian escherichia coli, the salmonella gallinargherium, the salmonella pullorum, the salmonella enteritidis and the salmonella typhimurium, meanwhile, the primer group can also be applied to the preparation of a quintuple PCR detection kit for detecting avian Escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium.
Drawings
FIG. 1 shows the result of electrophoresis of the PCR amplification product of example 1;
FIG. 2 is the test results of the specificity evaluation in evaluation example 1;
FIG. 3 shows the results of the sensitivity evaluation using the bacterial suspension as a template in evaluation example 2;
FIG. 4 shows the results of the sensitivity evaluation test using bacterial genomic DNA as a template in evaluation example 2.
Detailed Description
The following describes embodiments of the present invention with reference to the drawings. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.
The experimental methods used in the implementation are conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
Example 1 quintuple PCR detection method
In this embodiment, the primer set for multiplex PCR detection of avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis, and salmonella typhimurium is used for detection of avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis, and salmonella typhimurium, and specifically includes the following steps:
Inoculating bacterial strain to be detected into LB liquid culture medium, and culturing at 37 deg.C to OD600The bacterial solution was obtained as a template for PCR as 1.0.
Preparing a primer group for detecting avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium by multiplex PCR: the concentration of each primer group is 10pM, the diluent of the primer with the concentration is stored at minus 20 ℃ for standby, repeated freeze thawing is avoided,
the primer group comprises the following primer pairs:
primer pairs ECphoA-F and ECphoA-R for specifically amplifying the avian escherichia coli phoA gene; a primer pair SGglgC-F and SGglgC-R for specifically amplifying the glgC gene of the salmonella gallinarum; a primer pair SGPspeC-F and SGPspeC-R of the speC genes of the salmonella gallinarum and the salmonella pullorum is specifically amplified; primer pairs SEsdf I-F and SEsdf I-R for specifically amplifying salmonella enteritidis sdf I gene; a primer pair STstm4495-F and STstm4495-R for specifically amplifying the stm4495 gene of the salmonella typhimurium,
the nucleotide sequences of the primers are respectively as follows:
ECphoA-F，5’-GGCAATACACTCACTATGCGCTG-3’,
ECphoA-R，5’-AGGATTCGCAGCATGATCCTG-3’；
SGglgC-F，5’-CGTCGCTATAAAGCGGAATATGTC-3’，
SGglgC-R，5’-CCTTTTCAAAACATACGCGAGTAG-3’；
SGPspeC-F，5’-GTTGCCGTACCGGTCGTAACG-3’，
SGPspeC-R，5’-TTCCTGACGGGCATCGACGGC-3’；
SEsdfⅠ-F，5’-GATGTGGTTGGTTCGTCACTG-3’，
SEsdfⅠ-R，5’-GCGAGACCTCAAACTTACTCAG-3’；
STstm4495-F，5’-CAGCGGTATGATGCGGTAGT-3’，
STstm4495-R，5’-TCACCGGTGGACATGCCTGC-3’。
wherein the target fragment amplified by the primer pair ECPhoA-F and ECPhoA-R has the size of 761 bp; the size of a target fragment for amplification of the primer pair SGglgC-F and SGglgC-R is 83 bp; the size of a target fragment for amplification of the primer pair SGPspeC-F and SGPspeC-R is 249 bp; the size of a target fragment for amplification of a primer pair SEsdf I-F and SEsdf I-R is 409 bp; the target fragment size for amplification of primer pair STstm4495-F and STstm4495-R was 569 bp.
Based on the PCR template obtained in the step 1, adopting the specific primer group obtained in the step 2 to establish a PCR reaction system of the primer group: taking a PCR tube, adding 12.5 mu L of 2 XPCRPreMix, 0.5 mu L of each primer ECphoA-F/ECphoA-R, 0.2 mu L of each primer SGglgC-F/SGglgC-R, 0.3 mu L of each primer SGPspeC-F/SGPspeC-R, 0.4 mu L of each primer SEsdf I-F/SEsdf I-R, 0.5 mu L of each primer STstm4495-F/STstm4495-R, 1.0 mu L of template and 7.7 mu L of ultrapure water into the PCR tube, and mixing uniformly to obtain a 25 mu L PCR reaction system.
Then, the PCR reaction system is adopted to carry out PCR amplification reaction under the condition of preset PCR reaction parameters to obtain a PCR amplification product: pre-denaturing the PCR reaction parameters at 95 ℃ for 5 min; 35 cycles of 95 ℃ for 30s, 57 ℃ for 30s and 72 ℃ for 50 s; 72 ℃ for 10 min.
performing electrophoresis detection on the amplification product obtained in the step 3 to obtain an electrophoresis result, and judging whether avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis or salmonella typhimurium appear according to the electrophoresis result:
taking 10 mu L of PCR amplification product, spotting the product in a hole of a 2.0% agarose gel electrophoresis plate, carrying out electrophoresis for 30-40min under the voltage of 80-100V, taking a picture under a gel imaging system to obtain an electrophoresis result, and judging the electrophoresis result on the premise that no band appears in negative control:
when 761bp bands appear in the electrophoresis result, the poultry escherichia coli is judged,
when the band of 83bp and 249bp appears in the electrophoresis result, the salmonella gallinarum is judged,
when the 249bp band appears in the electrophoresis result, the salmonella pullorum is judged,
when the 409bp band appears in the electrophoresis result, the salmonella enteritidis is judged,
and judging the salmonella typhimurium when a 569bp band appears in the electrophoresis result.
FIG. 1 shows the result of electrophoresis of the PCR amplification product of example 1.
As shown in FIG. 1, lane 1 shows the quintuple PCR products of avian Escherichia coli, Salmonella gallinarum, Salmonella pullorum, Salmonella enteritidis, and Salmonella typhimurium; lane 2 is Salmonella gallinarum (83bp and 249 bp); lane 3 shows Salmonella pullorum (249 bp); lane 4 salmonella enteritidis (409 bp); lane 5 is Salmonella typhimurium (569 bp); lane 6 avian e.coli (761 bp); lane 7 is a negative control.
Example 2 detection by quintuple PCR detection kit
This example, consistent with the procedure of example 1, differs in that this example directly uses a quintuple PCR detection kit comprising the primer set mentioned in example 1 to establish the above-mentioned PCR reaction system.
The composition of the quintuple PCR detection kit at this time includes 2 XPCR PreMix and a primer set, wherein the volume of the 2 XPCR PreMix is 12.5. mu.L, the volumes of the primer pair ECphoA-F and ECphoA-R in the primer set are 0.5. mu.L each, the volumes of the primer pair SGglgC-F and SGglgC-R in the primer set are 0.2. mu.L each, the volumes of the primer pair SGPspeC-F and SGPspeC-R are 0.3. mu.L each, the volumes of the primer pair SEsdf I-F and SEsdf I-R are 0.4. mu.L each, and the volumes of the primer pair STm4495-F and STstm4495-R are 0.5. mu.L each.
Evaluation example 1 evaluation of specificity
In this example, the primer sets in example 1 and example 2 were specifically detected by using the quintuple PCR detection kit in example 2 as an example:
selecting bacteria such as riemerella anatipestifer, staphylococcus aureus, pseudomonas aeruginosa PA14, streptococcus suis, salmonella typhimurium, salmonella pullorum, listeria monocytogenes, pasteurella avicularis, avian pathogenic escherichia coli, salmonella gallinarum, mycoplasma gallisepticum, salmonella typhimurium, bordetella avium, staphylococcus aureus, salmonella enteritidis (CVCC 1805), mycoplasma gallisepticum and the like, carrying out PCR according to the operation of a quintuple PCR detection kit, and carrying out electrophoresis identification on the product in 2% agarose gel.
FIG. 2 shows the results of the test for evaluating specificity in evaluation example 1.
As shown in fig. 2, lane M: DNA marker; lane 1: carrying out quintuple PCR; lane 2: riemerella anatipestifer type 1 CH 3; lane 3: staphylococcus aureus (CVCC 543); lane 4: pseudomonas aeruginosa PA 14; lane 5: streptococcus suis; lane 6: riemerella anatipestifer type 2 Yb 2; lane 7: salmonella typhimurium SAT 52; lane 8: riemerella anatipestifer serotype 10 HXb 2; lane 9: salmonella gallinarum; lane 10: listeria monocytogenes 10403 s; lane 11: avian pasteurella (CVCC 493); lane 12: avian pathogenic escherichia coli; lane 13, riemerella anatipestifer (wg 4); lane 14: salmonella pullorum (CVCC 519); lane 15: avian mycoplasma (CVCC 274); lane 16: salmonella typhimurium SL 1344; lane 17: bordetella avicularis (IPDH 591-77); lane 18: staphylococcus aureus (CVCC 543); lane 19: salmonella enteritidis (CVCC 1805); lane 20: mycoplasma gallisepticum (CVCC 1651); lane 21: and (5) negative control.
The results show that the avian pathogenic escherichia coli, the salmonella gallinarum, the salmonella pullorum, the salmonella enteritidis and the salmonella typhimurium are respectively used as templates and can be amplified to form specific bands; and the PCR results are negative when other bacteria and other control samples are amplified. The primer sets in the embodiment 1 and the embodiment 2 are strong in specificity, and the established quintuple PCR detection method or the detection kit can be used for accurately, quickly, stably and specifically distinguishing avian Escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium from other bacteria.
Evaluation example 2 evaluation of sensitivity
This example uses the quintuple PCR detection kit of example 2 as an example to evaluate the sensitivity of the primer sets of example 1 and example 2:
in this example, PCR amplification was carried out using bacterial cultures and bacterial DNA as templates, which were diluted in multiple ratios, respectively.
(1) Sensitivity test using bacterial culture as template
Respectively inoculating avian Escherichia coli, Salmonella gallinarum, Salmonella pullorum, Salmonella enteritidis and Salmonella typhimurium to LB liquid culture medium, and shaking to OD at 37 deg.C6001.0, respectively centrifuging 1mL of bacterial solution at 12000r/min for 1min, removing supernatant, washing with sterile ultrapure water for 3 times, resuspending 1mL of sterile ultrapure water, respectively preparing bacterial solutions with a concentration of 106CFU/μL、105CFU/μL、104CFU/μL、103CFU/μL、102CFU/. mu.L, 10 CFU/. mu.L. Respectively sucking 1 mu L of the primer, adding the primer into a PCR reaction system, carrying out PCR according to the operational instructions of the quintuple PCR detection kit, and determining the sensitivity of the primer group to avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium according to the test result.
FIG. 3 shows the results of the evaluation of the sensitive strand using the bacterial suspension as a template in evaluation example 2.
As shown in FIG. 3, the results are analyzed in Table 1, which shows that 1000CFU of bacteria can be detected at the lowest in the PCR reaction system using the above primer set.
TABLE 1 sensitivity evaluation results using bacterial liquid as template
(+: occurrence of specific band; -, -: absence of specific band)
(2) Sensitivity test using bacterial genomic DNA as template
Respectively inoculating avian Escherichia coli, Salmonella gallinarum, Salmonella pullorum, Salmonella enteritidis and Salmonella typhimurium to LB liquid culture medium, and shaking to OD at 37 deg.C6001mL of each enrichment solution was taken and subjected to the procedure using a kit (Tiangen Biotech Co., Ltd.) to extract bacterial DNA. The dilution concentrations were 100 ng/. mu.L, 50 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 500 pg/. mu.L, 100 pg/. mu.L, and 10 pg/. mu.L, respectively. Adding 1 μ L of the extract into a PCR reaction system, and performing PCR according to the operational instructions of the quintuple PCR kitAccording to test results, the sensitivity of the primer group to avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium is determined.
FIG. 4 shows the results of the sensitivity evaluation using bacterial genomic DNA as a template in evaluation example 2.
As shown in FIG. 4, the results are analyzed in Table 2, and the five-fold PCR detection kit for avian Escherichia coli, Salmonella gallinarum, Salmonella pullorum, Salmonella enteritidis and Salmonella typhimurium established in the present invention can detect at least 100pg of avian Escherichia coli, Salmonella gallinarum, Salmonella enteritidis and Salmonella typhimurium, but only 500pg of Salmonella pullorum.
TABLE 2 sensitivity evaluation results using bacterial genomic DNA as template
(+: occurrence of specific band; -, -: absence of specific band)
The primer sets in the embodiment 1 and the embodiment 2 are strong in sensitivity, and the established quintuple PCR detection method or the quintuple PCR detection kit can be used for sensitively distinguishing avian Escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis, salmonella typhimurium from other bacteria.
Evaluation example 3 evaluation of reproducibility
In this embodiment, the reproducibility of the prepared quintuple PCR detection kit for avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium is evaluated by taking the quintuple PCR detection kit of example 2 as an example:
in this embodiment, the positive templates are detected under different time and different operating conditions (including PCR instrument and operator), and the detection results are shown in Table 3.
TABLE 3 results of repeated measurements
(+: occurrence of specific band; -, -: absence of specific band)
As shown in Table 3, according to the PCR detection results, the primer sets in the embodiments 1 and 2 have strong repeatability, and the established quintuple PCR detection method or the quintuple PCR detection kit can be used for distinguishing avian Escherichia coli, Salmonella gallinarum, Salmonella pullorum, Salmonella enteritidis and Salmonella typhimurium from other bacteria with good repeatability.
Effects and effects of example 1 and example 2
As can be seen from evaluation examples 1 to 3, the primer sets used for amplifying the positive strains stored in the laboratory all can amplify specific bands, and the control samples such as other bacterial strains and the like do not amplify bands, which indicates that the specificity of the primer sets is strong, and the primer sets used for detecting the positive samples with different dilutions can achieve the lowest detection sensitivity of 1000CFU bacterial liquid and 500pg bacterial genome DNA, which indicates that the sensitivity of the primer sets is high, and meanwhile, the primer sets have good repeatability, which indicates that the stability is strong. Therefore, the quintuple PCR detection method using the primer set or the quintuple PCR detection kit containing the primer set provided in the embodiment 1 and the embodiment 2 has the advantages of sensitivity, specificity, rapidness and stability for detection of avian Escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium, provides an effective means for rapid detection and identification of avian Escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium, and can be used for preparing the quintuple PCR detection kit for detection of avian Escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium.
In addition, in example 1 and example 2, the bacterial liquid of the bacteria to be detected cultured in step 1 is used as the PCR template, and as the present invention, the bacterial liquid in step 1 may be further extracted to obtain crude bacterial DNA as the PCR template, and the specific process at this time is as follows: inoculating a bacterial strain to be detected to an LB liquid culture medium, culturing at 37 ℃ until OD600 is 1.0 to obtain bacterial liquid, centrifuging 1mL of the bacterial liquid at 10000-12000r/min for 1-2min, removing supernatant, adding 0.1-0.5mL of sterilized ultrapure water for resuspension, boiling for 5-10min, and centrifuging at 10000-12000r/min for 3-5min to obtain supernatant which is the crude bacterial DNA.
The scope of the invention is not to be limited by the specific embodiments, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Modifications are also within the scope of the appended claims.
Claims (11)
1. A primer set for detecting multiple PCR of avian escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium is characterized by comprising the following components in parts by weight:
primer pairs ECphoA-F and ECphoA-R for specifically amplifying the avian escherichia coli phoA gene; a primer pair SGglgC-F and SGglgC-R for specifically amplifying the glgC gene of the salmonella gallinarum; a primer pair SGPspeC-F and SGPspeC-R of the speC genes of the salmonella gallinarum and the salmonella pullorum is specifically amplified; primer pairs SEsdf I-F and SEsdf I-R for specifically amplifying salmonella enteritidis sdf I gene; a primer pair STstm4495-F and STstm4495-R for specifically amplifying the stm4495 gene of the salmonella typhimurium,
the nucleotide sequences of the primers are respectively as follows:
ECphoA-F，5’-GGCAATACACTCACTATGCGCTG-3’,
ECphoA-R，5’-AGGATTCGCAGCATGATCCTG-3’；
SGglgC-F，5’-CGTCGCTATAAAGCGGAATATGTC-3’，
SGglgC-R，5’-CCTTTTCAAAACATACGCGAGTAG-3’；
SGPspeC-F，5’-GTTGCCGTACCGGTCGTAACG-3’，
SGPspeC-R，5’-TTCCTGACGGGCATCGACGGC-3’；
SEsdfⅠ-F，5’-GATGTGGTTGGTTCGTCACTG-3’，
SEsdfⅠ-R，5’-GCGAGACCTCAAACTTACTCAG-3’；
STstm4495-F，5’-CAGCGGTATGATGCGGTAGT-3’，
STstm4495-R，5’-TCACCGGTGGACATGCCTGC-3’；
the size of the target fragment for amplification of the primer pair ECPhoA-F and ECPhoA-R is 761 bp;
the size of a target fragment for amplification of the primer pair SGglgC-F and SGglgC-R is 83 bp;
the size of a target fragment for amplification of the primer pair SGPspeC-F and SGPspeC-R is 249 bp;
the size of a target fragment for amplification of a primer pair SEsdf I-F and SEsdf I-R is 409 bp;
the target fragment size for amplification of primer pair STstm4495-F and STstm4495-R was 569 bp.
2. The use of the primer set of claim 1 in the preparation of a quintuple PCR detection kit for detecting avian Escherichia coli, Salmonella gallinarum, Salmonella pullorum, Salmonella enteritidis, and Salmonella typhimurium.
3. A quintuple PCR detection kit is used for detecting avian Escherichia coli, Salmonella gallinarum, Salmonella pullorum, Salmonella enteritidis and Salmonella typhimurium, and is characterized by comprising the following components:
2 XPCR premixed solution and primer group for establishing PCR reaction system,
wherein the PCR reaction system is used for specific amplification of each primer pair in the primer group,
the primer set is the primer set of claim 1.
4. The detection kit according to claim 3, characterized in that:
wherein the volume of the 2 XPCR premix is 12.5 muL,
in the primer set, the volumes of the primer pair ECphoA-F and ECphoA-R were each 0.5. mu.L, the volumes of the primer pair SGglgC-F and SGglgC-R were each 0.2. mu.L, the volumes of the primer pair SGPspeC-F and SGPspeC-R were each 0.3. mu.L, the volumes of the primer pair SEsdf I-F and SEsdf I-R were each 0.4. mu.L, and the volumes of the primer pair STm4495-F and STm4495-R were each 0.5. mu.L.
5. A quintuple PCR detection method for detecting avian Escherichia coli, salmonella gallinarum, salmonella pullorum, salmonella enteritidis and salmonella typhimurium for non-disease diagnosis and treatment purposes is characterized by comprising the following steps:
step 1, preparing a PCR template based on bacteria to be detected;
step 2, preparing a specific primer group, and preparing the specific primer group by using the primer group in claim 1;
step 3, carrying out PCR amplification reaction, based on the PCR template obtained in the step 1, establishing a PCR reaction system comprising the primer group by adopting the specific primer group obtained in the step 2, and then carrying out PCR amplification reaction by adopting the PCR reaction system under the condition of preset PCR reaction parameters to obtain a PCR amplification product;
and 4, performing electrophoresis detection on the amplification product to obtain an electrophoresis result, and judging whether the avian escherichia coli, the salmonella gallinarum, the salmonella pullorum, the salmonella enteritidis or the salmonella typhimurium appears according to the electrophoresis result.
6. The quintuple PCR detection method of claim 5, wherein:
the PCR template obtained in the step 1 is a bacterial liquid of the bacteria to be detected, and the specific process is as follows:
inoculating the strain of the bacteria to be detected to an LB liquid culture medium, and culturing the strain to OD under the condition that the temperature is 37 DEG C600Obtaining the bacterial liquid as 1.0.
7. The quintuple PCR detection method of claim 5, wherein:
the PCR template obtained in the step 1 is a bacteria crude extraction DNA, and the specific process is as follows:
subjecting said to be examinedThe bacterial strains were inoculated into LB liquid medium and cultured at 37 ℃ to OD600Obtaining bacterial liquid 1.0, then centrifuging 1mL of the bacterial liquid at 10000-12000r/min for 1-2min, removing supernatant, adding 0.1-0.5mL of sterilized ultrapure water for resuspension, boiling for 5-10min, and then centrifuging at 10000-12000r/min for 3-5min to obtain supernatant which is the crude bacterial DNA.
8. The quintuple PCR detection method of any one of claims 5 to 7, wherein:
the PCR reaction system of the step 3 comprises:
12.5. mu.L of a 2 XPCR premix, 1.0. mu.L of the template, 7.7. mu.L of ultrapure water, and the primer set,
in the primer set, the volumes of the primer pair ECphoA-F and ECphoA-R were each 0.5. mu.L, the volumes of the primer pair SGglgC-F and SGglgC-R were each 0.2. mu.L, the volumes of the primer pair SGPspeC-F and SGPspeC-R were each 0.3. mu.L, the volumes of the primer pair SEsdf I-F and SEsdf I-R were each 0.4. mu.L, and the volumes of the primer pair STm4495-F and STm4495-R were each 0.5. mu.L.
9. The quintuple PCR detection method of any one of claims 5 to 7, wherein:
when a specific primer set was prepared, the concentration of each primer used in the primer set was 10pM, and the diluted solution of each primer used was stored at-20 ℃ and then used.
10. The quintuple PCR detection method of any one of claims 5 to 7, wherein:
the condition of the predetermined PCR reaction parameter is pre-denaturation at 95 ℃ for 5 min; 35 cycles at 95 ℃ for 30s, 57 ℃ for 30s, and 72 ℃ for 50 s; 72 ℃ for 10 min.
11. The quintuple PCR detection method of any one of claims 5 to 7, wherein:
the specific process of the step 4 is that,
taking 10 mu L of PCR amplification product, spotting the PCR amplification product in a hole of a 2.0% agarose gel electrophoresis plate, carrying out electrophoresis for 30-40min under the voltage of 80-100V, taking a picture under a gel imaging system to obtain an electrophoresis result, and judging the electrophoresis result on the premise that no band appears in negative control,
when 761bp bands appear in the electrophoresis result, the poultry escherichia coli is judged,
when the band of 83bp and 249bp appears in the electrophoresis result, the salmonella gallinarum is judged,
when the 249bp band appears in the electrophoresis result, the salmonella pullorum is judged,
when the 409bp band appears in the electrophoresis result, the salmonella enteritidis is judged,
and judging the salmonella typhimurium when the 569bp band appears in the electrophoresis result.
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