CN114752594B - Multiplex real-time fluorescent quantitative PCR primer and probe combination for detecting salmonella and cryptosporidium - Google Patents
Multiplex real-time fluorescent quantitative PCR primer and probe combination for detecting salmonella and cryptosporidium Download PDFInfo
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Abstract
The invention discloses a multiplex real-time fluorescent quantitative PCR primer and probe combination, which consists of two groups of primers and probes respectively designed for salmonella InvA genes and Cryptosporidium 18SrRNA genes, and also discloses a kit containing the primer and probe combination and a detection method. The method realizes double real-time fluorescent quantitative PCR detection of salmonella and cryptosporidium, is rapid, accurate, high in specificity and sensitivity, and can simultaneously realize qualitative and accurate quantitative detection of salmonella and cryptosporidium.
Description
Technical Field
The invention belongs to the field of molecular detection, relates to a primer and probe combination for detecting salmonella and cryptosporidium, and further relates to application of the primer and probe combination.
Technical Field
Salmonella can cause calf paratyphoid, the disease widely exists at home and abroad, and the trend of increasing year by year in China is presented. At present, large-scale outbreaks of salmonellosis are reported in various pastures such as Qinghai province and Gansu province in China, and infected cattle and cattle with bacteria can be used as main transmission sources of the salmonellosis and can become long-term carriers of the salmonellosis. When the constitution of the cattle is reduced and the resistance is weakened, the cattle can be ill, and the bacteria are discharged by the cattle with bacteria through urination, defecation, abortion and other ways, so that the surrounding environment is polluted. After the calf is infected with salmonella, the calf is mostly presented with enteritis and poisoning symptoms, the calf is raised in body temperature and accompanied with foul yellow diarrhea, even mucous blood feces appear, acute cases die from septicemia within several days, and huge economic losses are caused for the cattle raising industry.
Cryptosporidium (Cryptosporidium) is a parasitic protozoan that causes diarrhea in humans and animals, mainly Cryptosporidium parvum, and Cryptosporidium caused by Cryptosporidium parvum is a zoonotic disease. Cryptosporidium possesses a wide living area, can affect more than about two hundred forty mammals including human beings, can mutually infect through various channels such as water, food, air and the like, and mainly causes digestive system and respiratory diseases of young mammals and causes production performance reduction and even death of the young mammals. After the calves are infected by the cryptosporidium, clinical symptoms such as high fever, vomiting and diarrhea can appear, the calves are accompanied with water pasty malodorous excrement, the disease is repeated, the immune function of the calves is greatly damaged, the calves are difficult to grow, even progressive lethal diarrhea appears, the growth and development of the calves are seriously influenced, and even the calves die.
Clinically, mixed infection of multiple pathogens often occurs, great loss is caused to the cattle industry, and an effective gold standard is to carry out differential diagnosis of the pathogens, which is also a key place for preventing and controlling the infection. At present, the research on multiple detection of the pathogen causing bovine mixed infection in China is relatively less, serological detection and pathogen separation and identification are the most main diagnostic means at present, but the separation and identification of the pathogen and serological detection methods are time-consuming and labor-consuming, and most importantly, the method is incapable of carrying out mixed infection of multiple pathogens. Therefore, constructing a laboratory examination protocol that runs rapidly, simply, with high sensitivity and specificity is of great critical importance for early clinical judgment, epidemiological studies and final suppression of its spread.
The real-time fluorescent quantitative PCR technology can not only be used for qualitatively identifying pathogenic bacteria, but also be used for simply, rapidly, sensitively and accurately quantifying the DNA or RNA of the pathogenic bacteria, and can also be used for dynamically researching the revival or continuous infection of potential pathogenic bacteria in the whole course of disease. The research is based on a real-time fluorescent quantitative PCR technology, and establishes a dual real-time fluorescent quantitative PCR method capable of simultaneously detecting salmonella and cryptosporidium so as to provide technical support for detecting etiology of clinical bovine pathogens in China.
Disclosure of Invention
The invention aims to provide a multiplex real-time fluorescent quantitative PCR primer and probe combination for detecting two bovine pathogens, namely salmonella and cryptosporidium.
The above purpose is achieved by the following technical scheme:
a multiplex real-time fluorescent quantitative PCR primer and probe combination consists of two sets of primers and probes designed for salmonella InvA genes and cryptosporidium 18SrRNA genes, and the nucleotide sequences of the primers and probes in each set are as follows:
the nucleotide sequence of the upstream primer for detecting salmonella is shown as SED ID NO.1, the downstream primer is shown as SEQ ID NO.2, and the probe is shown as SEQ ID NO. 3; the nucleotide sequence of the upstream primer for detecting the cryptosporidium is shown as SED ID NO.4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.5, the nucleotide sequence of the probe is shown as SEQ ID NO.6, and the specific steps are as follows:
salmonella-F: 5'-ATTTCAATGGGAACTCTGCC-3' (SEQ ID No. 1)
salmonella-R: 5'-TCGCCTTTGCTGGTTTTAG-3' (SEQ ID No. 2)
salmonella-P: 5'-TTAAATTCCGTGAAGCAAAACGTAGCG-3' (SEQ ID No. 3)
Cryptosporidium-F: 5'-AATCAAAGTCTTTGGGTTCTGG-3' (SEQ ID No. 4)
Cryptosporidium-R: 5'-TGGTGAGTTTTCCCGTGTT-3' (SEQ ID No. 5)
Cryptosporidium-P: 5'-TTAAAGGAATTGACGGAAGGGCAC-3' (SEQ ID No. 6)
The 5 'end of the probe is connected with a fluorescent group, and the 3' end of the probe is connected with a fluorescence quenching group. The fluorescent group of SEQ ID NO.3 is selected from FAM, and the fluorescence quenching group is selected from BHQ1; the fluorescent group of SEQ ID NO.6 is selected from ROX, and the fluorescence quenching group is selected from BHQ2.
The primer can amplify a 134bp long sequence of an InvA gene region (GenBank accession number: MK 017940.1) of salmonella, the sequence is shown as SEQ ID No.7, and a 125bp long sequence of an 18SrRNA gene region (GenBank accession number: L16996.1) of Cryptosporidium, the sequence is shown as SEQ ID No. 8.
The primer and probe combination is applied to laboratory screening and identification of salmonella and cryptosporidium or clinical diagnosis of related diseases. Because the multiplex real-time fluorescent quantitative PCR primer and probe combination is provided, the simultaneous detection of two viruses can be realized by only carrying out one PCR amplification on a sample when identification or diagnosis is carried out.
The primer and probe combination can also be used for preparing a multiplex real-time fluorescent quantitative PCR detection kit for detecting salmonella and cryptosporidium.
A multiplex real-time fluorescent quantitative PCR detection kit for detecting salmonella and cryptosporidium comprises the primer and probe combination.
The invention further provides a method for detecting salmonella and cryptosporidium for non-diagnostic purposes, which comprises the steps of adding the primer and probe combination into a reaction system and performing multiplex real-time fluorescent quantitative PCR amplification.
Preferably, the reaction system is as follows: 2×T5Fast qPCR Mix 12.5. Mu.L; 1.1 mu L of each of the salmonella upstream and downstream primers and 0.9 mu L of each of the probes; 0.8 mu L of each of the upstream and downstream primers of Cryptosporidium and 1.1 mu L of each of the probes; 2. Mu.L of template DNA; the double distilled water was made up to 25 μl.
Preferably, the amplification reaction procedure is: 95 ℃ for 5min; annealing at 95℃for 10s and 60℃for 30s for 42 cycles, and automatically collecting fluorescence signals at the end of each cycle.
The beneficial effects of the invention are as follows:
according to the invention, a specific primer and a probe are designed according to the InvA gene region of salmonella and the 18SrRNA gene region of cryptosporidium, and a dual real-time fluorescence quantitative PCR method capable of simultaneously detecting salmonella and cryptosporidium is established, and the method is rapid, accurate, high in specificity and sensitivity, can simultaneously realize qualitative detection and accurate quantitative detection of salmonella and cryptosporidium, and has good development prospect and wide application space.
Drawings
Fig. 1: salmonella single-detection real-time fluorescence quantitative PCR sensitivity experimental result is sequentially 1 multiplied by 10 from left to right 7 ～1×10 2 Copy/. Mu.L.
Fig. 2: cryptosporidium single-detection real-time fluorescence quantitative PCR sensitivity experimental result is 1X 10 from left to right 7 ～1×10 2 Copy/. Mu.L.
Fig. 3: salmonella single-detection real-time fluorescence quantitative PCR standard curve.
Fig. 4: cryptosporidium single-check real-time fluorescent quantitative PCR standard curve.
Fig. 5: the salmonella sensitivity experimental result in the dual real-time fluorescence quantitative PCR is 1×10 from left to right 8 ～1×10 2 Copy/. Mu.L.
Fig. 6: cryptosporidium sensitivity experiment results in dual real-time fluorescence quantitative PCR (polymerase chain reaction) are sequentially 1 multiplied by 10 from left to right 8 ～1×10 2 Copy/. Mu.L.
Fig. 7: double real-time fluorescent quantitative PCR specificity experiment results. 1 is Cryptosporidium positive nucleic acid; 2 is salmonella positive nucleic acid; 3-12 are negative control and other viral nucleic acids, respectively.
Fig. 8: double real-time fluorescent quantitative PCR standard curve.
Fig. 9: the experimental result of sensitivity of salmonella in dual real-time fluorescence quantitative PCR in literature report is 1×10 from left to right 7 ～1×10 3 Copy/. Mu.L.
Fig. 10: cryptosporidium sensitivity experiment results in dual real-time fluorescence quantitative PCR in literature report are sequentially 1×10 from left to right 7 ～1×10 3 Copy/. Mu.L.
Detailed Description
Example 1 design of primers
All nucleotide sequences of the salmonella-InvA and Cryptosporidium-18 SrRNA gene regions were downloaded from GenBank, and compared and analyzed by MEGA software to select the gene sequence with the highest conservation degree (salmonella GenBank accession number: MK017940.1; cryptosporidium GenBank accession number: L16996.1) and the specific region thereof, and specific primers and probes were designed and screened (Table 1).
TABLE 1 primers and probes
Example 2 establishment and optimization of multiplex fluorescent quantitative PCR method
1. Establishment and optimization of single-detection real-time fluorescence quantitative PCR method for salmonella and cryptosporidium
1.1 Standard Positive plasmid preparation
Using the primers of Table 1, a sequence including the primer sequence of the 18SrRNA gene region of InvA and Cryptosporidium (Salmonella GenBank accession number: MK017940.1, cryptosporidium GenBank accession number: L16996.1) of Salmonella was inserted into the pMD19-T vector to construct a recombinant plasmid, which was designated pMD19-T-InvA and pMD19-T-18SrRNA, respectively, as a standard positive plasmid template and a positive control in the system construction.
1.2 establishment of Single-detection real-time fluorescence quantitative PCR method
Single factor variables were set, and primer concentration, probe concentration and annealing temperature were optimized for each single detection system using real-time fluorescent quantitative PCR according to the primers and probes of table 1. All optimization processes use standard positive plasmids as positive templates and double distilled water as negative templates. Along with the real-time fluorescence quantitative PCR reaction, the template is amplified, the fluorescence intensity is enhanced, the result can be analyzed through a fluorescence curve and a Ct value, a tube cover is not required to be opened in the whole process, the risk of aerosol pollution is reduced, and the optimal primer concentration, probe concentration and annealing temperature are selected according to the fluorescence signal intensity and the Ct value after the reaction is finished.
1.2.1 primer concentration optimization
A25.00. Mu.L reaction system was prepared, 8 gradients (0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3. Mu.L) were set for the amount of each pathogen upstream and downstream primer (10. Mu. Mol/L), the probe amount and annealing temperature were fixed, and the judgment was made based on the Ct value and fluorescence signal intensity.
1.2.2 probe concentration optimization
A25.00 mu L reaction system was prepared, 8 gradients (0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 and 1.3 mu L) were set for each pathogenic probe amount (10 mu mol/L), and the primer amounts were determined based on the 1.2.1 optimization results, the annealing temperature was fixed, and the Ct value and the fluorescence signal intensity were determined.
1.2.3 temperature optimization
A25.00. Mu.L reaction system was configured, and the amounts of each pathogenic primer and probe (10. Mu. Mol/L) were based on the optimized results of 1.2.1 and 1.2.2, and annealing temperatures were set to 56.5, 57, 57.5, 58, 58.5, 59, 59.5 and 8 annealing temperature gradients at 60 ℃. And judging according to the Ct value and the fluorescence signal intensity.
The results after optimization are shown in table 2.
Table 2 optimization results of real-time fluorescent quantitative PCR reaction system for single detection
In summary, the optimal system for determining the real-time fluorescent quantitative PCR reaction for each single sample is shown in Table 3.
Table 3 real-time fluorescent quantitative PCR final reaction system for each single sample
1.2.4 sensitivity analysis of real-time fluorescent quantitative PCR System for Single detection
In order to determine the detection limit (limit of detection, LOD) of each single-detection real-time fluorescent quantitative PCR system, we performed single-detection real-time fluorescent quantitative PCR reaction on each pathogen, using 10-fold serial dilution of standard plasmid, selecting1×10 0 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L, 1X 10 2 And (3) taking the copy/. Mu.L standard positive plasmid as a template, repeating each concentration for 20 times, and carrying out a sensitivity experiment according to the optimized result of a real-time fluorescence quantitative PCR system of each pathogen single-detection, wherein the lowest concentration meeting the positive detection rate of more than or equal to 90% is considered to be reliable LOD.
According to the configuration of 25.00 mu L of reaction system in Table 3, respectively carrying out real-time fluorescence quantitative PCR experiments on each system according to the temperature optimization result, wherein the procedure of each single-detection real-time fluorescence quantitative PCR amplification reaction is as follows: 95 ℃ for 5min;95℃10s, 30s of TM (Salmonella 60℃Cryptosporidium 60 ℃) for 42 cycles. The fluorescent signals are automatically collected at the end of each cycle, and the result shows that the detection limit of a single detection system of salmonella and cryptosporidium reaches 1 multiplied by 10 2 Copy/. Mu.L as shown in FIGS. 1-2 and Table 4.
TABLE 4 analytical sensitivity of real-time quantitative PCR reaction systems for each single assay
1.2.5 construction of a Standard Curve of a real-time fluorescent quantitative PCR System for Single detection
To verify the validity and reliability of each single-check real-time fluorescent quantitative PCR system. We respectively carry out real-time fluorescence quantitative PCR reaction on each single detection system, and the selection application range is 1×10 7 Copy/. Mu.L to 1X 10 0 A10-fold serial dilution of the standard positive plasmid at copy/. Mu.L was used as template, each concentration was tested 3 times and a negative control was set. And drawing a standard curve according to the optimization result of each single-sample system.
According to the configuration of 25.00 mu L of reaction system in Table 3, respectively carrying out real-time fluorescence quantitative PCR experiments on each single-detection system according to the temperature optimization result, wherein the procedures of each single-detection real-time fluorescence quantitative PCR amplification reaction are as follows: 95 ℃ for 5min;95℃10s, 30s of TM (Salmonella 60℃Cryptosporidium 60 ℃) for 42 cycles in total; fluorescence signals were automatically collected at the end of each cycle, and the results (fig. 3-4) showed: correlation coefficient R of salmonella and Cryptosporidium 2 The amplification efficiencies E are respectively 100.7 and 98.0 and are respectively 0.987 and 0.997, and the amplification efficiencies E are respectively between 90% and 110%, so that the linear relation is good; regression equations for Salmonella and Cryptosporidium are y, respectively 1 ＝-3.306x 1 +42.587 and y 2 ＝-3.370x 2 +44.053 shows that there is a good linear relationship between initial template concentration and threshold (Ct) value.
2. Establishment of dual real-time fluorescent quantitative PCR method
2.1 optimization of Dual real-time fluorescent quantitative PCR reaction conditions
25.00 mu L of reaction system is prepared, standard positive plasmids of salmonella and cryptosporidium are mixed in equal proportion to be used as templates, and 5 gradients are set for the use amount (10 mu mol/L) of the upstream and downstream primers of each pathogen by taking the optimization result of a single detection system as an intermediate value, for example: cryptosporidium sets (0.7, 0.8, 0.9, 1.0, 1.1 μl) five gradients; setting 5 gradients by taking the optimized result of a single detection system as an intermediate value (10 mu mol/L) of the consumption of each pathogenic probe; 8 annealing temperature gradients were set: 56.5, 57, 57.5, 58, 58.5, 59, 59.5 and 60 ℃. And (5) optimizing the double real-time fluorescent quantitative PCR reaction conditions. The amplification procedure was 95℃for 5min;95℃for 10s and a TM value of 30s for 42 cycles. Fluorescence signals are automatically collected at the end of each cycle. The results are shown in Table 5.
TABLE 5 optimization results of Dual real-time fluorescent quantitative PCR reaction System
In summary, the optimal system for determining the dual real-time fluorescent quantitative PCR reaction is shown in Table 6.
Table 6 Dual real-time fluorescent quantitative PCR final reaction system
2.2. Sensitivity analysis of dual real-time fluorescent quantitative PCR reaction system
To determine dual real-timeThe detection Limit (LOD) of the fluorescent quantitative PCR system on each pathogen is carried out by using a dual real-time fluorescent quantitative PCR system to respectively carry out sensitivity experiments on each pathogen, 10 times serial diluted standard positive plasmids are used, and 1X 10 is selected 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 3 The copy/. Mu.L of standard positive plasmid was used as a template, each concentration was repeated 20 times, and the lowest concentration satisfying the positive detection rate of 90% or more was considered as reliable LOD, and a sensitivity experiment was performed based on the results of the optimization of the dual system.
A 25.00 mu L reaction system is configured according to a table 6, and a real-time fluorescence quantitative PCR experiment is carried out on a double system according to a temperature optimization result, wherein the double real-time fluorescence quantitative PCR amplification reaction comprises the following procedures: 95 ℃ for 5min;95℃for 10s and TM60℃for 30s, for a total of 42 cycles. The fluorescence signal is automatically collected at the end of each cycle, and the result shows that the detection limit of the dual system on salmonella and cryptosporidium reaches 1 multiplied by 10 2 Copy/. Mu.L as shown in FIGS. 5-6 and Table 7.
TABLE 7 analytical sensitivity of Dual real-time quantitative PCR reaction System
2.3 specific analysis of Dual real-time fluorescent quantitative PCR reaction System
In order to determine the specificity of the dual real-time fluorescent quantitative PCR method, false positives caused by other pathogens are eliminated, DNA/cDNA of common calf diarrhea pathogens and other common pathogens including BVDV, BRV, BCV, escherichia coli F17, K99, F41, IBR1 and BPIV, BRSV, HM are used as templates, and salmonella and cryptosporidium nucleic acid are used as positive controls. And detecting by using the optimized double system, and evaluating the specificity. As shown in FIG. 7, only the positive control shows a typical amplification curve, and other pathogens are not amplified, so that the method has good analysis specificity and can be used for specific detection of salmonella and cryptosporidium.
2.4 construction of double real-time fluorescence quantitative PCR System Standard Curve
To verify dual real-time fluorescenceThe effectiveness and reliability of the photoperiod PCR reaction system. We choose to use a range from 1X 10 7 Copy/. Mu.L to 1X 10 0 Copies/. Mu.L of 10-fold serial dilutions of standard positive plasmid were used as templates and mixed in equal ratios, each concentration was tested 3 times and negative controls were set. And drawing a standard curve according to the optimization result of the dual system.
A 25.00 mu L reaction system is configured according to a table 6, and a real-time fluorescence quantitative PCR experiment is carried out on a double system according to a temperature optimization result, wherein the double real-time fluorescence quantitative PCR amplification reaction comprises the following procedures: 95 ℃ for 5min;95℃for 10s and TM60℃for 30s, for a total of 42 cycles. Automatically collecting fluorescent signals at the end of each cycle; the results (fig. 8) show: correlation coefficient R of salmonella and Cryptosporidium 2 0.997 and 0.900 respectively, and the amplification efficiencies E are 96.9 and 90.7 respectively, are 90-110%, and have good linear relations; regression equations for Salmonella and Cryptosporidium are y, respectively 1 ＝-3.397x 1 +38.630 and y 2 ＝-3.567x 2 +41.277 shows that there is a good linear relationship between initial template concentration and threshold (Ct) value.
2.2.5 repeatability experiments of double real-time fluorescent quantitative PCR reaction System
To determine the reproducibility of this dual real-time fluorescent quantitative PCR method, we selected 1X 10 3 Copy/. Mu.L, 1X 10 4 Copy/. Mu.L, 1X 10 5 Copy/. Mu.L of standard positive plasmid for each pathogen in 10-fold serial dilutions were mixed in equal proportions as templates and repeated experiments in and between groups were performed for Salmonella and Cryptosporidium, respectively, within a week, 3 times per reaction. The Coefficient of Variation (CV) of the Ct value of the sample at each concentration in the experiment was calculated to evaluate the reproducibility thereof. The results are shown in Table 8: the intra-and inter-group Coefficient of Variation (CVs) for each pathogen in the dual combination was less than 3%. The double real-time fluorescence quantitative PCR reaction system established by the research has good repeatability and high stability.
Table 8 results of repeatability experiments
Example 3 evaluation of Dual real-time fluorescent quantitative PCR System on clinical sample detection
Using the dual real-time fluorescent quantitative PCR system established by the present study and the general PCR detection method (table 9) obtained by referring to literature data, clinical sample detection and coincidence rate analysis were performed on 96 DNA samples stored in the laboratory, respectively. The results of each common PCR detection are verified by sequencing of Wohan qingke biotechnology Co., ltd; the determination standard of the dual real-time fluorescent quantitative PCR detection method is as follows:
1) The Ct value of the detected sample is less than 35, and positive is judged when a typical amplification curve appears;
2) The Ct value is greater than 35 and a review is required when a typical amplification curve occurs. The rechecking result is judged to be positive when the rechecking result is above, otherwise, the rechecking result is judged to be negative;
3) And when no Ct value exists and no amplification curve exists, the negative judgment is made.
The results are shown in Table 10: the detection rate of salmonella is 13/96 (13.54%), 11/96 (11.45%) and the coincidence rate is 92%; the detection rate of Cryptosporidium is 13/96 (13.54%), 10/96 (10.41%) and the coincidence rate is 87%.
TABLE 9 primer sequences for control PCR
TABLE 10 clinical sample test results
Example 4 sensitivity comparison with real-time fluorescent quantitative PCR method in literature report
In order to determine the superiority of the dual real-time fluorescent quantitative PCR method established in the study, reference is made to the literature to obtain the primer and probe sequences (Table 11) of the single-detection real-time fluorescent quantitative PCR method of each pathogen, and the primer and probe sequences are combined into a corresponding dual system according to the usage amount of the primer and the probe in the report of the literature. Sensitivity experiments were performed on this dual system, which was compared with the dual system established in this study.
Table 11 primer and probe sequences for real-time fluorescent quantitative PCR reported in the literature
4.1 initial experiments of sensitivity of Dual real-time fluorescent quantitative PCR reaction System reported in literature
To determine the limit of detection (LOD) of the dual real-time fluorescent quantitative PCR system for each pathogen, 1X 10 was selected using corresponding 10-fold serial dilutions of standard positive plasmids 7 Copy/. Mu.L to 1X 10 0 The initial experiment of sensitivity was performed on this dual system with copy/. Mu.L of standard positive plasmid as template.
The method comprises the steps of configuring a dual system and setting a reaction program according to the content reported in the literature, wherein the program of the dual real-time fluorescence quantitative PCR amplification reaction is as follows: 95 ℃ for 5min;95℃10s, TM60℃30s, 42 cycles total. The fluorescence signal was automatically collected at the end of each cycle, and the results of the initial experiments showed that the dual system had a detection limit of 1×10 for salmonella and cryptosporidium 3 Copy/. Mu.L, as shown in FIGS. 9-10.
4.2 Secondary experiments of sensitivity of Dual real-time fluorescent quantitative PCR reaction System reported in literature
Based on the results of the initial experiments, 10-fold serial dilutions of standard positive plasmids were used to select 1X 10 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 3 A secondary experiment of sensitivity was performed on the double system with copy/. Mu.L of standard positive plasmid as a template, and each concentration was repeated 20 times, and the lowest concentration satisfying the positive detection rate of 90% or more was considered to be a reliable sensitivity Limit (LOD).
The method comprises the steps of configuring a dual system and setting a reaction program according to the content reported in the literature, wherein the program of the dual real-time fluorescence quantitative PCR amplification reaction is as follows: 95 ℃ for 5min;95℃10s, TM60℃30s, 42 cycles total. At the end of each cycleThe fluorescence signal is automatically collected, and the result of the secondary experiment shows that the detection limit of the dual system on salmonella and cryptosporidium is 1 multiplied by 10 3 Copy/. Mu.L, as shown in Table 12.
Table 12 analytical sensitivity of the Dual real-time quantitative PCR reaction System reported in the literature
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Sequence listing
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Claims (9)
1. The multiplex real-time fluorescent quantitative PCR primer and probe combination is characterized by comprising two groups of primers and probes designed for salmonella InvA genes and cryptosporidium 18SrRNA genes respectively, wherein the nucleotide sequences of the primers and probes in each group are as follows:
a first group: the upstream primer sequence is shown as SEDIDNO 1, the downstream primer sequence is shown as SEQIDNO 2, and the probe sequence is shown as SEQIDNO 3;
second group: the upstream primer sequence is shown as SEQ ID NO.4, the downstream primer sequence is shown as SEQ ID NO.5, and the probe sequence is shown as SEQ ID NO. 6.
2. The multiplex real-time fluorescent quantitative PCR primer and probe combination of claim 1, wherein: the 5 'end of the probe is connected with a fluorescent group, and the 3' end of the probe is connected with a fluorescence quenching group.
3. The multiplex real-time fluorescent quantitative PCR primer and probe combination of claim 2, wherein the fluorescent groups of the first set of probes are FAM and the fluorescent quenching groups are BHQ1; the fluorescent group of the second group of probes is ROX, and the fluorescence quenching group is BHQ2.
4. Use of a multiplex real-time fluorescent quantitative PCR primer and probe combination according to any one of claims 1-3 for the detection of bovine pathogens, both salmonella and cryptosporidium, for non-diagnostic purposes.
5. Use of a multiplex real-time fluorescent quantitative PCR primer and probe combination according to any one of claims 1-3 for the preparation of a kit for the detection of bovine pathogens, salmonella and cryptosporidium.
6. A kit for detecting bovine pathogens, which comprises the multiplex real-time fluorescent quantitative PCR primer and probe combination of any one of claims 1-3, wherein the bovine pathogens are salmonella and cryptosporidium.
7. A method for detecting bovine pathogens, not diagnostic, which are salmonella and cryptosporidium, characterized in that: comprising the step of adding the primer and probe combination of any one of claims 1 to 3 to a reaction system and performing multiplex real-time fluorescent quantitative PCR amplification.
8. The method of claim 7, wherein the reaction system is as follows: 2×T5Fast qPCR Mix 12.5. Mu.L; 1.1 mu L of each of the first set of upstream and downstream primers and 0.9 mu L of each of the probes; the second set of upstream and downstream primers were each 0.8. Mu.L and the probe was 1.1. Mu.L; 2. Mu.L of template DNA; the double distilled water was made up to 25 μl.
9. The method of claim 7, wherein the amplification reaction procedure is: 95 ℃ for 5min;95℃for 10s,60℃for 30s, for a total of 42 cycles.
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| CN103074450A (en) * | 2013-01-25 | 2013-05-01 | 海尔施生物医药股份有限公司 | Kit for synchronously detecting thirty diarrhea pathogens and detection method of kit |
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| CN108796106A (en) * | 2018-06-29 | 2018-11-13 | 吉林大学 | Giardia bovis, Cryptosporidium multiple PCR detection kit and its method |
| CN114350829A (en) * | 2022-03-01 | 2022-04-15 | 内蒙古农业大学 | Primer-probe combination for simultaneously detecting enterotoxigenic escherichia coli and salmonella, kit and application |
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| CN104662167A (en) * | 2012-06-27 | 2015-05-27 | 莫比蒂亚戈公司 | Method for determining the presence of diarrhoea causing pathogens |
| CN103074450A (en) * | 2013-01-25 | 2013-05-01 | 海尔施生物医药股份有限公司 | Kit for synchronously detecting thirty diarrhea pathogens and detection method of kit |
| CN107090518A (en) * | 2017-04-05 | 2017-08-25 | 苏州协云基因科技有限公司 | The multiple RT PCR Polymorphism chip inspecting reagent units of the related pathogen of diarrhoea |
| CN108796106A (en) * | 2018-06-29 | 2018-11-13 | 吉林大学 | Giardia bovis, Cryptosporidium multiple PCR detection kit and its method |
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