CN110592241A - Quadruple fluorescent quantitative PCR (polymerase chain reaction) detection method and detection kit for salmonella - Google Patents
Quadruple fluorescent quantitative PCR (polymerase chain reaction) detection method and detection kit for salmonella Download PDFInfo
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Abstract
The invention discloses a quadruple fluorescent quantitative PCR detection method and a detection kit for salmonella, in particular to a quadruple fluorescent quantitative PCR detection method and a detection kit for salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a quadruple fluorescent quantitative PCR primer set for detecting salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium, a quadruple fluorescent PCR detection method comprising the primer set, and a quadruple PCR detection kit comprising 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. Salmonella is a large group of gram-negative bacteria parasitizing in human and animal intestines, is a common zoonosis type pathogenic bacterium, and has more than 2500 serotypes reported at present. Among them, salmonella enteritidis, salmonella pullorum, salmonella gallinarum, and salmonella typhimurium can infect poultry and bring about serious economic loss. In addition, salmonella enteritidis, salmonella typhimurium and the like can enter human food chains through eggs and chicken, become important pathogens causing death due to food poisoning of people in various countries, and bring hidden troubles to food safety.
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 traditional bacteria detection method usually needs to separate and culture bacteria, so that the operation is complex, the time consumption is long, and the pollution is easy. In addition, the identification of the salmonella serotype mainly adopts the traditional methods of serological agglutination test, biochemical identification and the like, but has the defects of more serious cross reaction, more false positive, lower sensitivity and the like; the antigen structures of salmonella pullorum, salmonella gallinarum and salmonella enteritidis are similar, and serotypes are difficult to distinguish by a traditional method. In the process of serotype identification, the judgment standards of different operators are not completely consistent, so that the stability, repeatability and sensitivity of the result are very poor. Many rapid and sensitive molecular biology methods have been applied to detection and identification of bacteria, overcoming the defects of the traditional detection method, and the application of the conventional PCR technology to detection of pathogenic bacteria has a good effect. However, the conventional PCR method has the defect of low specificity, and certain components in a complex sample have inhibition effect on PCR amplification, so that false positive is easily generated during amplification. In addition, the conventional PCR method needs to determine the result through gel electrophoresis, so that the operation is complex and the cross contamination problem exists. And the fluorescent quantitative PCR adopts complete closed-tube detection, so that the post-treatment of PCR products is omitted, the cross contamination is avoided, and the sensitivity is obviously higher than that of the common PCR method. However, when multiple fluorescent PCR is used to simultaneously detect multiple target genes in the same reaction tube, there is interference between different primers and probes. Therefore, the multiple fluorescence PCR method with strong specificity and high sensitivity needs to be established by optimizing factors such as primer concentration, probe concentration, optimal reaction conditions and the like. Therefore, an efficient and rapid detection method for distinguishing and identifying common salmonella of poultry is urgently needed, and has great significance for effective prevention and control of epidemic diseases.
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 fluorescent quantitative primer set for detecting salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium, which is capable of accurately, rapidly, stably, specifically and sensitively detecting salmonella enteritidis, salmonella pullorum, salmonella typhimurium, and is characterized by comprising: a primer pair of lysgD-F, lygD-R and a probe of lysgD-P for specifically amplifying the lysgD gene of the salmonella enteritidis; specifically amplifying a primer pair SGP-F, SGP-R and a probe SGP-P of the speC gene of the salmonella gallinarum and the salmonella pullorum; a primer pair SG-F, SG-R and a probe SG-P for specifically amplifying the glgC gene of the salmonella gallinarum; a primer pair SAT-F, SAT-R and a probe SAT-P for specifically amplifying stm4495 gene of salmonella typhimurium,
the nucleotide sequences of the primers are respectively as follows:
lygD-F，5’-TCTGGGACGCCAAAAAGC-3’，(SEQ ID NO.1)
lygD-R，5’-TGACGGTAGATTGTGTCTCAAAGC-3’，(SEQ ID NO.2)
lygD-P，5’-TCAAACTTACTCAGGAGATCGCCGCTG-3’：(SEQ ID NO.3)
the reporter group of the probe lygD-P is CY5, and the quenching group is BHQ-2;
SGP-F，5’-GGATGTCCACGCTCATTTCTC-3’，(SEQ ID NO.4)
SGP-R，5’-TGAAAGCTGGCGTTACGGTTA-3’，(SEQ ID NO.5)
SGP-P，5’-CGTCAGGCCCACCGCCGACAG-3’；(SEQ ID NO.6)
a report group of the probe SGP-P is FAM, and a quenching group is BHQ-1;
SG-F，5’-CAGGCGATCATATCTACAAGCAGG-3’，(SEQ ID NO.7)
SG-R，5’-TCTTGTCGCTTTCATCGACCGC-3’，(SEQ ID NO.8)
SG-P，5’-ACTCGCGTATGTTTTGAAAAGGGC-3’；(SEQ ID NO.9)
the reporter group of the probe SG-P is JOE, and the quenching group is BHQ-1;
SAT-F，5’-GTTCAGCTCCGGTAAAGAGAA-3’，(SEQ ID NO.10)
SAT-R，5’-AGCAGCGGCACTACATATTC-3’，(SEQ ID NO.11)
SAT-P，5’-CGTTTGAGTGCCTGGTCTATCTGCA-3’(SEQ ID NO.12)
the reporter group of the probe SAT-P is CY3, and the quenching group is BHQ-2;
the invention also aims to provide a quadruple fluorescent PCR detection kit based on a TaqMan probe method, which is used for detecting salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium and is characterized by comprising the following components in parts by weight: 2 XPremix Ex Taq (Probe qPCR) Premix and primer set for establishing PCR reaction system.
The quadruple fluorescent PCR detection kit provided by the invention also has the following characteristics: wherein, in the primer set, the volume of 2 XPremix Ex Taq (Probe qPCR) is 10. mu.L, the volumes of the primer pair of lysD-F and lysD-R are each 0.2. mu.L, the volume of the Probe of lysD-P is 0.1. mu.L, the volumes of the primer pair of SGP-F and SGP-R are each 0.1. mu.L, the volume of the Probe of SGP-P is 0.1. mu.L, the volumes of the primer pair of SG-F and SG-R are each 0.2. mu.L, the volume of the Probe of SG-P is 0.1. mu.L, the volumes of the primer pair of SAT-F and SAT-R are each 0.4. mu.L, and the volume of the Probe of SAT-P is 0.8. mu.L.
The invention also aims to provide a quadruple fluorescence PCR detection method for detecting salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium based on a Taqman probe method, which is characterized by comprising the following steps: step 1, preparing a DNA template based on bacteria to be detected; step 2, preparing a specific primer probe group; step 3, performing fluorescent quantitative PCR amplification reaction, establishing a reaction system by adopting the specific primer probe set obtained in the step 2 based on the DNA template obtained in the step 1, and performing fluorescent quantitative PCR by adopting the reaction system under the condition of preset reaction parameters; and 4, judging whether salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium appear or not according to the fluorescent quantitative PCR amplification curve.
The quadruple fluorescence PCR detection method provided by the invention also has the following characteristics: the PCR template in the step 1 is DNA of bacteria to be detected, and the preparation method comprises the following steps: inoculating a strain of the bacteria to be detected to an LB liquid culture medium, culturing at 37 ℃ until OD600 is 1.0 to obtain a bacterial liquid, and extracting the genomic DNA of the bacterial liquid according to the instruction in the rhizobacteria genomic DNA extraction kit.
The quadruple fluorescence PCR detection method provided by the invention also has the following characteristics: in the preparation of the specific primer probe set, the concentration of each primer probe used in the primer set was 10. mu.M, and a diluent of each primer used was stored at-20 ℃ and then used.
The quadruple fluorescence PCR detection method provided by the invention also has the following characteristics: the condition of the predetermined fluorescence quantitative PCR reaction parameter is pre-denaturation at 95 ℃ for 30 s; 5s at 95 ℃, 40s at 57 ℃ and 40 cycles.
The quadruple fluorescence PCR detection method provided by the invention also has the following characteristics: the specific process of step 4 is to observe whether a characteristic S curve exists, judge the Salmonella enteritidis when a CY5 signal is detected and the characteristic S curve is presented, judge the Salmonella gallinarum or Salmonella pullorum when an FAM signal is detected and the characteristic S curve is presented, judge the Salmonella gallinarum when a JOE signal is detected and the characteristic S curve is presented, and judge the Salmonella typhimurium when a CY3 signal is detected and the characteristic S curve is presented.
The invention also provides an application of the primer group in preparing a quadruple fluorescent PCR detection kit for detecting salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium.
Advantageous effects
The quadruple fluorescent PCR primer probe group for detecting salmonella enteritidis, salmonella pullorum, salmonella typhimurium and salmonella typhimurium, the quadruple fluorescent PCR detection kit comprising the primer group and the quadruple fluorescent PCR detection method using the primer group, provided by the invention, have the advantages of sensitivity, specificity, rapidness and stability for detecting salmonella enteritidis, salmonella pullorum, salmonella typhimurium and salmonella typhimurium by using the quadruple fluorescent PCR detection kit of the primer probe group or the quadruple fluorescent PCR detection kit comprising the primer probe group, because the primer group has strong specificity and high sensitivity and simultaneously the primer group has good repeatability, meanwhile, the primer group can also be applied to the preparation of a quadruple fluorescent PCR detection kit for detecting salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium.
The kit can be used for rapidly identifying salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium, can be used as an auxiliary method for the traditional serological typing of the salmonella, and provides a novel method which is simple, rapid and good in repeatability for monitoring and laboratory diagnosis of the salmonella enteritidis, the salmonella pullorum, the salmonella gallinarum and the salmonella typhimurium in samples such as farms, animal products and feeds.
Drawings
FIG. 1 shows the results of the primer specificity evaluation in the single fluorescent quantitative PCR assay of evaluation example 1; wherein, A: when a CY5 signal is detected and a characteristic S curve is presented, the salmonella enteritidis is judged; b: when the FAM signal is detected and the characteristic S curve is presented, the salmonella gallinarum or the salmonella pullorum is judged; c: judging the salmonella gallinarum when the JOE signal is detected and the characteristic S curve is presented; d: salmonella typhimurium is judged when the CY3 signal is detected and a characteristic S-curve is presented.
FIG. 2 is a graph showing the results of the sensitivity evaluation of the quadruple fluorescent quantitative PCR method of evaluation example 2, in which Salmonella enteritidis A, Salmonella typhimurium/pullorum, Salmonella gallinarum C, and Salmonella typhimurium D
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 quadruple fluorescent PCR detection method
In this embodiment, primer probe sets for quadruple fluorescence PCR detection of salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium are used for detecting salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium, and the method specifically includes the following steps:
step 1, preparing PCR template based on bacteria to be detected
Inoculating a bacterial strain of the bacteria to be detected to an LB liquid culture medium, culturing overnight at the temperature of 37 ℃ to obtain bacterial liquid, and adding 2 mu L of the bacterial liquid serving as a template into a reaction system.
Step 2, preparing specific primer probe group
Preparing a primer probe group for quadruple fluorescent PCR detection of salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium: wherein the concentration of each primer group is 10pM, the diluent of the primer with the concentration is stored at the temperature of minus 20 ℃ for standby, repeated freeze thawing is avoided,
the primer probe set comprises the following components:
a primer pair of lysgD-F, lygD-R and a probe of lysgD-P for specifically amplifying the lysgD gene of the salmonella enteritidis; specifically amplifying a primer pair SGP-F, SGP-R and a probe SGP-P of the speC gene of the salmonella gallinarum/salmonella pullorum; a primer pair SG-F, SG-R and a probe SG-P for specifically amplifying the glgC gene of the salmonella gallinarum; a primer pair SAT-F, SAT-R and a probe SAT-P for specifically amplifying stm4495 gene of salmonella typhimurium,
the nucleotide sequences of the primers are respectively as follows:
lygD-F，5’-TCTGGGACGCCAAAAAGC-3’，(SEQ ID NO.1)
lygD-R，5’-TGACGGTAGATTGTGTCTCAAAGC-3’，(SEQ ID NO.2)
lygD-P，5’-TCAAACTTACTCAGGAGATCGCCGCTG-3’；(SEQ ID NO.3)
the reporter group of the probe lygD-P is CY5, and the quenching group is BHQ-2;
SGP-F，5’-GGATGTCCACGCTCATTTCTC-3’，(SEQ ID NO.4)
SGP-R，5’-TGAAAGCTGGCGTTACGGTTA-3’，(SEQ ID NO.5)
SGP-P，5’-CGTCAGGCCCACCGCCGACAG-3’；(SEQ ID NO.6)
a report group of the probe SGP-P is FAM, and a quenching group is BHQ-1;
SG-F，5’-CAGGCGATCATATCTACAAGCAGG-3’，(SEQ ID NO.7)
SG-R，5’-TCTTGTCGCTTTCATCGACCGC-3’，(SEQ ID NO.8)
SG-P，5’-ACTCGCGTATGTTTTGAAAAGGGC-3’；(SEQ ID NO.9)
the reporter group of the probe SG-P is JOE, and the quenching group is BHQ-1;
SAT-F，5’-GTTCAGCTCCGGTAAAGAGAA-3’，(SEQ ID NO.10)
SAT-R，5’-AGCAGCGGCACTACATATTC-3’，(SEQ ID NO.11)
SAT-P，5’-CGTTTGAGTGCCTGGTCTATCTGCA-3’(SEQ ID NO.12)
the reporter group of the probe SAT-P is CY3, and the quenching group is BHQ-2;
step 3, carrying out fluorescent quantitative PCR reaction
Based on the PCR template obtained in step 1, samplingUsing the specific primer probe set in step 2, according to TaKaRa Premix Ex TaqTM(Probe qPCR) protocol A single fluorescent quantitative PCR reaction system was set up: 2 XPremix Ex Taq (Probe qPCR) was added to each PCR tube in a volume of 10. mu.L, each of the primer pair of lysD-F and lysD-R was 0.4. mu.L, the Probe of lysD-P was 0.8. mu.L, each of the primer pair of SGP-F and SGP-R was 0.4. mu.L, the Probe of SGP-P was 0.8. mu.L, each of the primer pair of SG-F and SG-R was 0.4. mu.L, the Probe of SG-P was 0.8. mu.L, each of the primer pair of SAT-F and SAT-R was 0.4. mu.L, and the Probe of SAT-P was 0.8. mu.L, respectively. The bacterial liquid to be detected is 2 mu L of PCR template, and the water is supplemented to 20 mu L.
The reaction system is adopted to carry out the following steps under the condition of preset fluorescent quantitative PCR reaction parameters: the condition of the predetermined fluorescence quantitative PCR reaction parameter is pre-denaturation at 95 ℃ for 30 s; 5s at 95 ℃, 40s at 57 ℃ and 40 cycles.
Step 4, judging method
And observing whether the fluorescent quantitative PCR amplification has a characteristic S curve or not, judging as salmonella enteritidis when a CY5 signal is detected and the characteristic S curve is presented, judging as salmonella gallinarum or salmonella pullorum when an FAM signal is detected and the characteristic S curve is presented, judging as salmonella gallinarum when a JOE signal is detected and the characteristic S curve is presented, and judging as salmonella typhimurium when a CY3 signal is detected and the characteristic S curve is presented.
Example 2 detection with quadruple fluorescent PCR detection kit
This example, consistent with the procedure of example 1, except that the reaction system was established directly using the quadruple fluorescent PCR detection kit including the primer set mentioned in example 1.
The quadruple fluorescent quantitative PCR detection kit comprises the following components: wherein, the volume of 2 XPromix Ex Taq (Probe qPCR) is 10. mu.L, the volumes of the primer pair of lysD-F and lysD-R are 0.2. mu.L each, the volume of the Probe of lysD-P is 0.1. mu.L, the volumes of the primer pair of SGP-F and SGP-R are 0.1. mu.L each, the volume of the Probe of SGP-P is 0.1. mu.L, the volumes of the primer pair of SG-F and SG-R are 0.2. mu.L each, the volume of the Probe of SG-P is 0.1. mu.L, the volumes of the primer pair of SAT-F and SAT-R are 0.4. mu.L each, and the volume of the Probe of SAT-P is 0.8. mu.L in the primer set.
Evaluation example 1 evaluation of specificity
This example uses the single fluorescent quantitative PCR method of example 1 as an example to specifically detect the primer sets of examples 1 and 2:
selecting genomes of different serotypes of salmonella and other bacteria as templates to perform single fluorescent quantitative PCR.
FIG. 1 shows the results of the test for evaluating specificity in evaluation example 1.
As shown in FIG. 1, the results show that a characteristic S-curve can appear with Salmonella enteritidis, Salmonella pullorum, Salmonella gallinarum, and Salmonella enteritidis as templates, respectively; and the control samples of other bacteria such as salmonella choleraesuis, salmonella newbauer, salmonella heidelbergii, escherichia coli, vibrio parahaemolyticus, staphylococcus aureus, proteus, shigella, pseudomonas aeruginosa, listeria monocytogenes and the like are amplified, and the results are negative (the result is not shown). The primer sets in the embodiment 1 and the embodiment 2 have stronger specificity, and the quadruple fluorescent quantitative PCR detection method or the detection kit established by the primer sets can accurately, quickly, stably and specifically distinguish the salmonella enteritidis, the salmonella pullorum, the salmonella gallinarum and the salmonella enteritidis from other bacteria.
Evaluation example 2 evaluation of sensitivity
In this example, the sensitivity and the minimum detection limit of the detection kit were evaluated by taking the quadruple fluorescent quantitative PCR detection kit of example 2 as an example:
step 1, preparation of Positive bacterial template
Respectively inoculating salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium to LB liquid culture medium, inoculating the strains of the bacteria to be detected to the LB liquid culture medium, culturing at 37 ℃ until OD600 is 1.0 to obtain bacterial liquids, wherein the corresponding bacterial numbers after dilution by multiple times are respectively 2.5 multiplied by 107CFU、2.5×106CFU、2.5×105CFU、2.5×104CFU、2.5×103CFU、2.5×102CFU、2.5×101CFU, 2.5CFU, then according to the heaven root bacteriumThe instructions in the genome DNA extraction kit extract the genome DNA.
Step 2, sensitivity measurement
And taking the template subjected to gradient dilution for quadruple fluorescent quantitative PCR according to the operation instruction of the quadruple fluorescent quantitative PCR detection kit, and determining the sensitivity of the primer probe group to salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella enteritidis according to the test result. And after the reaction is finished, analyzing the relation between the Ct value and the number of bacteria according to an amplification curve, and determining the lowest detection limit. FIG. 2 shows the results of the quadruple fluorescent quantitative PCR sensitivity evaluation in the evaluation examples. As shown in FIG. 2, the results are analyzed as shown in Table 1, and the sensitivity of the detection gradient according to the determined quadruple fluorescent quantitative PCR reaction system is 2.5 CFU. When the positive template is less than 2.5CFU, no fluorescence can be detected. Then, the above template was used for ordinary PCR detection, and the results showed that the sensitivity of the ordinary PCR method was 2.5X 103CFU。
TABLE 1 Experimental results for quadruple fluorescent quantitative PCR sensitivity evaluation
(+: positive-: negative)
The primer sets in the embodiment 1 and the embodiment 2 are strong in sensitivity, and the established quadruple fluorescent quantitative PCR detection method or the quadruple fluorescent quantitative PCR detection kit can be used for sensitively distinguishing salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium from other bacteria.
Evaluation example 3 evaluation of reproducibility
In this example, the quadruple fluorescent quantitative PCR detection kit of example 2 is used as an example to evaluate the repeatability of the quadruple fluorescent quantitative PCR detection kit for salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium prepared in the following steps:
in this embodiment, the positive templates are detected under different time and different operation conditions (including, by the operator), and the detection results are shown in table 2.
TABLE 2 repeatability test results
| At different times | Replacement of operating personnel | Change laboratory | |
| Salmonella enteritidis | + | + | + |
| Salmonella pullorum | + | + | + |
| Salmonella gallinarum | + | + | + |
| Salmonella typhimurium | + | + | + |
(+: positive-: negative)
As shown in Table 2, according to the detection results, the primer sets in the examples 1 and 2 have strong repeatability, and the established quadruple fluorescent quantitative PCR detection method or the quadruple fluorescent quantitative PCR detection kit can be used for distinguishing salmonella enteritidis, salmonella pullorum, salmonella gallinarum, salmonella typhimurium from other bacteria with good repeatability.
Example 3 application of multiplex fluorescent quantitative PCR detection kit
By adopting the kit in the embodiment 2, 60 salmonella isolated from chicken farms can be detected, and salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium can be detected quickly and accurately. The method comprises the following specific steps:
step 1, separation of Salmonella
Collecting dead chicken samples from chicken farms in Jiangsu, Anhui and the like, inoculating the dead chicken samples to an SS plate in an aseptic manner, and separating and identifying 60 salmonella strains.
Step 2, fluorescent quantitative PCR method detection
Preparing the bacterial genome DNA of the salmonella isolate according to the method of the fluorescent quantitative PCR detection kit in the embodiment 2, then carrying out fluorescent quantitative PCR detection, observing whether the fluorescent quantitative PCR amplification has a characteristic S curve, and judging the result. And identifying 21 strains of salmonella enteritidis, 8 strains of salmonella pullorum, 5 strains of salmonella gallinarum and 26 strains of salmonella typhimurium.
Step 3, traditional serotype identification of salmonella
The serotype identification of 60 salmonella strains separated in the test is carried out by referring to the prior art and national standards, and the result shows that the 60 salmonella strains commonly comprise 21 salmonella enteritidis strains, 8 salmonella pullorum strains, 5 salmonella gallinarum strains and 26 salmonella typhimurium strains. The result shows that the identification result of the multiple fluorescence PCR method established by the invention is completely consistent with the identification result of the traditional serotype.
In this embodiment, the conventional serotype identification method is adopted to screen out salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium from 60 strains of salmonella, and at least 2 days are required. The PCR detection kit of embodiment 2 of the invention can accurately screen out Salmonella enteritidis, Salmonella pullorum, Salmonella typhimurium and Salmonella typhimurium from 60 strains of Salmonella only in 2-3 hours, and the fluorescent quantitative PCR identification result is completely coincident with the serotype identification result, with the accuracy rate of 100%.
Effects and effects of the embodiments
The traditional salmonella serotype identification method is usually carried out by taking a somatic antigen, a flagellum antigen and a capsular antigen of bacteria as the basis of serotyping by a seroagglutination method. And the salmonella has more than 2500 serotypes, so hundreds of single-factor serums need to be prepared, the time and the labor are wasted, and human errors exist in result judgment. However, in the present study, primers and probes are respectively designed for the lysgD gene of Salmonella enteritidis, the speC gene of Salmonella gallinarum and Salmonella pullorum, the glgC gene of Salmonella gallinarum, and the stm4495 gene of Salmonella typhimurium, and a method for detecting Salmonella enteritidis, Salmonella pullorum, Salmonella gallinarum, and Salmonella typhimurium by multiplex fluorescence PCR based on a TaqMan probe is established. The method has the advantages of simple operation, strong anti-interference performance, strong specificity, high sensitivity and good stability. In addition, the fluorescence PCR method does not need to carry out open electrophoresis treatment on the sample, reduces the risk of contacting toxic substances, can also realize whole-process monitoring, shortens the detection time and has wide application value.
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 (7)
1. A fluorescent quantitative primer group for detecting salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium is characterized by comprising the following components: a primer pair of lysgD-F, lygD-R and a probe of lysgD-P for specifically amplifying the lysgD gene of the salmonella enteritidis; specifically amplifying a primer pair SGP-F, SGP-R and a probe SGP-P of the speC gene of the salmonella gallinarum and the salmonella pullorum; a primer pair SG-F, SG-R and a probe SG-P for specifically amplifying the glgC gene of the salmonella gallinarum; a primer pair SAT-F, SAT-R and a probe SAT-P for specifically amplifying stm4495 gene of salmonella typhimurium,
the nucleotide sequences of the primers are respectively as follows:
lygD-F，5’-TCTGGGACGCCAAAAAGC-3’，(SEQ ID NO.1)
lygD-R，5’-TGACGGTAGATTGTGTCTCAAAGC-3’，(SEQ ID NO.2)
lygD-P，5’-TCAAACTTACTCAGGAGATCGCCGCTG-3’；(SEQ ID NO.3)
the reporter group of the probe lygD-P is CY5, and the quenching group is BHQ-2;
SGP-F，5’-GGATGTCCACGCTCATTTCTC-3’，(SEQ ID NO.4)
SGP-R，5’-TGAAAGCTGGCGTTACGGTTA-3’，(SEQ ID NO.5)
SGP-P，5’-CGTCAGGCCCACCGCCGACAG-3’；(SEQ ID NO.6)
a report group of the probe SGP-P is FAM, and a quenching group is BHQ-1;
SG-F，5’-CAGGCGATCATATCTACAAGCAGG-3’，(SEQ ID NO.7)
SG-R，5’-TCTTGTCGCTTTCATCGACCGC-3’，(SEQ ID NO.8)
SG-P，5’-ACTCGCGTATGTTTTGAAAAGGGC-3’；(SEQ ID NO.9)
the reporter group of the probe SG-P is JOE, and the quenching group is BHQ-1;
SAT-F，5’-GTTCAGCTCCGGTAAAGAGAA-3’，(SEQ ID NO.10)
SAT-R，5’-AGCAGCGGCACTACATATTC-3’，(SEQ ID NO.11)
SAT-P, 5'-CGTTTGAGTGCCTGGTCTATCTGCA-3'; (SEQ ID NO.12) the reporter group of the probe SAT-P was CY3, and the quencher group was BHQ-2.
2. A quadruple fluorescent quantitative PCR detection kit for salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium is characterized by comprising the following components in parts by weight: 2 XPremix Ex Taq (Probe qPCR) Premix and primer set for establishing PCR reaction system.
3. The quadruple fluorescent quantitative PCR detection kit of claim 2, wherein the volume of 2 x Premix Ex Taq (Probe qPCR) is 10. mu.L, the volumes of the primer pair of lygD-F and lygD-R are each 0.2. mu.L, the volume of the Probe lygD-P is 0.1. mu.L, the volumes of the primer pair of SGP-F and SGP-R are each 0.1. mu.L, the volume of the Probe SGP-P is 0.1. mu.L, the volumes of the primer pair of SG-F and SG-R are each 0.2. mu.L, the volume of the Probe SG-P is 0.1. mu.L, the volumes of the primer pair of SAT-F and SAT-R are each 0.4. mu.L, and the volume of the Probe SAT-P is 0.8. mu.L; the sequences of the primer pair and the probe are as described in claim 1.
4. A non-diagnostic purpose quadruple fluorescence PCR detection method for detecting salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium based on a Taqman probe method is characterized by comprising the following steps:
step 1, preparing a DNA template based on bacteria to be detected;
step 2, preparing a specific primer probe group;
step 3, performing fluorescent quantitative PCR amplification reaction, establishing a reaction system by adopting the specific primer probe set obtained in the step 2 based on the DNA template obtained in the step 1, and performing fluorescent quantitative PCR by adopting the reaction system under the condition of preset reaction parameters;
and 4, judging whether salmonella enteritidis, salmonella pullorum, salmonella gallinarum and salmonella typhimurium appear or not according to the fluorescent quantitative PCR amplification curve.
5. The quadruple fluorescent PCR detection method for non-diagnostic purposes as claimed in claim 4, wherein the specific primer probe set is the fluorescent quantitative primer set as claimed in claim 1.
6. The quadruple fluorescent PCR detection method for non-diagnostic purposes as claimed in claim 4 or 5, wherein the PCR template in step 1 is the DNA of the bacteria to be detected, and the preparation method comprises the following steps: inoculating a strain of bacteria to be detected to an LB liquid culture medium, culturing at 37 ℃ until OD600 is 1.0 to obtain a bacterial liquid, and extracting genomic DNA of the bacterial liquid according to the instruction in the bacterial genomic DNA extraction kit;
when the specific primer probe group is prepared, the concentration of each primer probe used in the primer group is 10 mu M, and the diluent of each used primer is stored at the temperature of-20 ℃ and then is used;
the condition of the predetermined fluorescence quantitative PCR reaction parameter is pre-denaturation at 95 ℃ for 30 s; 5s at 95 ℃, 40s at 57 ℃ and 40 times of circulation;
the specific process of step 4 is to observe whether a characteristic S curve exists, judge the Salmonella enteritidis when a CY5 signal is detected and the characteristic S curve is presented, judge the Salmonella gallinarum or Salmonella pullorum when an FAM signal is detected and the characteristic S curve is presented, judge the Salmonella gallinarum when a JOE signal is detected and the characteristic S curve is presented, and judge the Salmonella typhimurium when a CY3 signal is detected and the characteristic S curve is presented.
7. Use of the fluorescent quantitative primer set of claim 1 or the quadruple fluorescent quantitative PCR detection kit of claim 2 in preparation of a reagent for detecting Salmonella enteritidis, Salmonella pullorum, Salmonella gallinarum, and Salmonella typhimurium.
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