CN110878366A - Nucleic acid composition, detection kit for intestinal pathogenic bacteria and use method of detection kit - Google Patents
Nucleic acid composition, detection kit for intestinal pathogenic bacteria and use method of detection kit Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C12Q2600/16—Primer sets for multiplex assays
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention relates to a nucleic acid composition, a detection kit for intestinal pathogenic bacteria and a use method thereof. The nucleic acid composition comprises a detection primer pair, wherein the detection primer pair comprises at least two of the following primer pairs: salmonella primer pair, staphylococcus aureus primer pair, clostridium difficile primer pair, shigella primer pair, klebsiella pneumoniae primer pair and pseudomonas aeruginosa primer pair. The nucleic acid composition can simultaneously detect at least two intestinal pathogenic bacteria at one time, the specificity of the primer pair is good, the interference between the primer pair is low, and the detection sensitivity can reach 100 copies/mu L.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a nucleic acid composition, a detection kit for enteropathogenic bacteria and a use method thereof.
Background
The balance of intestinal flora is closely related to human health, intestinal pathogenic bacteria seriously threaten the human health, and meanwhile, infectious diseases caused by the intestinal pathogenic bacteria are main factors endangering public health safety. Common enteropathogenic bacteria include Salmonella (Salmonella), Shigella (Shigella), Staphylococcus aureus (Staphyloccocuerurus), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Klebsiella pneumoniae (Klebsiella pneumoniae), Clostridium difficile (Clostridium difficile), Vibrio cholerae (Vibrio cholerae), and Escherichia coli (Escherichia coli).
Salmonella is a common zoonosis pathogen, which can cause typhoid fever, gastroenteritis, food poisoning, septicemia and the like, and in the event of food poisoning, the salmonella causes a number of cases listed as pre-cogongrass. Shigella, one of the major food-borne pathogens, can cause acute enteritis, and in severe cases, shock, cerebral edema, and respiratory failure. Staphylococcus aureus is commonly present in environment and food, has seasonal distribution, is easy to infect in spring and summer, and can cause a series of diseases such as staphylococcus aureus enteritis. Staphylococcus aureus can produce a large amount of toxins and can develop resistance to the body. Even at low concentrations, staphylococcus aureus can be a health hazard for humans and animals, such as acute pneumonia, sepsis, toxic shock, etc. Klebsiella pneumoniae is an enterobacteriaceae family that causes mainly pneumonia, respiratory infections, enteritis, diarrhea, and even sepsis in humans and various animals. Pseudomonas aeruginosa is widely distributed in the skin, respiratory tract, intestinal tract and the like of living organisms, and can cause skin diseases, pneumonia, intestinal diseases, meningitis, septicemia and the like after infection. Although pseudomonas aeruginosa is not used as a conventional index item for food microorganism test in daily food detection, pseudomonas aeruginosa has been identified as food-borne and water-borne pathogenic bacteria. Difficile is an intestinal conditional pathogen which is difficult to separate and culture, and long-term use of antibiotics can cause imbalance of intestinal flora, so that drug-resistant difficile can propagate in a large amount to cause related diarrhea and enteritis.
At present, the intestinal pathogenic bacteria have wide epidemic range of infectious diseases, high seasonal incidence and certain infectivity. Therefore, the establishment of an early rapid detection method has great significance for preventing and controlling the infection of the intestinal pathogenic bacteria.
The detection method of the intestinal pathogenic bacteria comprises a culture method and a molecular biological detection method. The culture method requires culturing the bacteria for a long time, for one or several days. The molecular biological detection method includes nucleic acid hybridization method, gene chip technology, polymerase chain reaction technology, etc. However, the traditional detection method is usually specific detection for one pathogen, the detection process of the mixture of multiple pathogens is complicated, and the detection is usually respectively specific to different pathogens and takes long time; when various primers of various pathogenic bacteria are mixed and simultaneously detected, the various primers of the various pathogenic bacteria are easy to interfere with each other, the specificity is poor, and the sensitivity is not high.
Disclosure of Invention
Based on the above, there is a need for a nucleic acid composition for detecting enteropathogenic bacteria, which is easy to detect, has good specificity and high sensitivity.
In addition, the kit for detecting the multiple intestinal pathogens is simple and convenient to detect, good in specificity and high in sensitivity, and the using method of the kit for detecting the multiple intestinal pathogens is simple and convenient.
A nucleic acid composition comprising a pair of detection primers comprising at least two of the following pairs of primers:
the primer pair of salmonella with sequences shown as SEQ ID No.1 and SEQ ID No.2, the primer pair of staphylococcus aureus with sequences shown as SEQ ID No.4 and SEQ ID No.5, the primer pair of clostridium difficile with sequences shown as SEQ ID No.7 and SEQ ID No.8, the primer pair of shigella with sequences shown as SEQ ID No.10 and SEQ ID No.11, the primer pair of klebsiella pneumoniae with sequences shown as SEQ ID No.13 and SEQ ID No.14, and the primer pair of pseudomonas aeruginosa with sequences shown as SEQ ID No.16 and SEQ ID No. 17.
The nucleic acid composition comprises at least two of a salmonella primer pair, a staphylococcus aureus primer pair, a clostridium difficile primer pair, a shigella primer pair, a klebsiella pneumoniae primer pair and a pseudomonas aeruginosa primer pair, can simultaneously detect at least two intestinal pathogens at one time, and has the advantages of good specificity of the primer pairs, low interference between the primer pairs and detection sensitivity reaching 100 copies/mu L.
In one embodiment, the kit further comprises a detection probe corresponding to the detection primer pair, and the detection probe corresponding to the detection primer pair comprises:
a salmonella probe with a sequence shown in SEQ ID No.3, a staphylococcus aureus probe with a sequence shown in SEQ ID No.6, a clostridium difficile probe with a sequence shown in SEQ ID No.9, a shigella probe with a sequence shown in SEQ ID No.12, a klebsiella pneumoniae probe with a sequence shown in SEQ ID No.15 and a pseudomonas aeruginosa probe with a sequence shown in SEQ ID No. 18;
and the detection probe corresponding to the detection primer pair is connected with a fluorescent group.
In one embodiment, the detection primer pair comprises the salmonella primer pair, the staphylococcus aureus primer pair, the clostridium difficile primer pair, the shigella primer pair, the klebsiella pneumoniae primer pair, and the pseudomonas aeruginosa primer pair; the detection primer pairs are divided into a plurality of groups, and the fluorescent groups connected to the detection probes corresponding to different detection primer pairs in the same group are different.
In one embodiment, the detection primers are split into two groups; the first group comprises the salmonella primer pair, the staphylococcus aureus primer pair and the clostridium difficile primer pair, and the second group comprises the shigella primer pair, the klebsiella pneumoniae primer pair and the pseudomonas aeruginosa primer pair.
In one embodiment, the fluorescent group linked to the detection probe corresponding to each detection primer pair in each set is selected from one of FAM, HEX, VIC, CY5, ROX, Texsa Red and Quasar 705.
In one embodiment, the fluorophores on the corresponding detection probes within the first set are ROX, HEX, and FAM, respectively; the fluorophores on the corresponding detection probes in the second group are ROX, HEX and FAM respectively.
In one embodiment, the nucleic acid composition further comprises an internal reference primer pair and an internal reference probe, wherein the sequences of the internal reference primer pair and the internal reference probe are shown as SEQ ID No.19 and SEQ ID No.20, and the sequence of the internal reference probe or the complementary sequence of the internal reference probe is shown as SEQ ID No. 21; the internal reference probe is connected with a fluorescent group different from the detection probe.
A kit for detecting enteric pathogenic bacteria comprises the nucleic acid composition.
In one embodiment, the kit further comprises at least one of a PCR reaction buffer, a nucleic acid extraction reagent, a PCR stabilizer and a PCR enhancer.
The use method of the detection kit for the enteropathogenic bacteria comprises the following steps:
extracting nucleic acid of a sample to be detected to obtain a nucleic acid sample;
adding a detection primer pair and a corresponding detection probe in the detection kit for the intestinal pathogenic bacteria by taking the nucleic acid sample as a template to perform real-time fluorescence PCR amplification reaction; and
and detecting the fluorescent signal in the real-time fluorescent PCR amplification reaction process to obtain a detection result.
Drawings
FIGS. 1 to 12 are amplification curves of the amplification reaction of salmonella, staphylococcus aureus, clostridium difficile, shigella, klebsiella pneumoniae, pseudomonas aeruginosa plasmid standards respectively used as templates to be tested by using the group A and group B fluorescent quantitative PCR systems in step (3);
FIGS. 13 to 14 are a immunofluorescence quantitative PCR amplification curve and an amplification standard curve of Salmonella, Staphylococcus aureus and Clostridium difficile, respectively;
FIGS. 15 to 16 are the PCR amplification curve and the standard amplification curve of Shigella, Klebsiella pneumoniae and Pseudomonas aeruginosa, respectively.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Some embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
One embodiment of the invention provides a nucleic acid composition, which can detect at least two intestinal pathogenic bacteria in a sample to be detected at one time, and has good specificity and higher detection sensitivity; the nucleic acid composition can be used for preparing a detection kit for detecting intestinal pathogenic bacteria.
In one embodiment, the sample to be tested is a human pharyngeal swab, a respiratory tract washing solution, a respiratory tract aspiration solution or other respiratory tract secretion sample.
In one embodiment, the nucleic acid composition comprises a pair of detection primers comprising at least two of the following pairs of primers: the primer pair of salmonella with sequences shown as SEQ ID No.1 and SEQ ID No.2, the primer pair of staphylococcus aureus with sequences shown as SEQ ID No.4 and SEQ ID No.5, the primer pair of clostridium difficile with sequences shown as SEQ ID No.7 and SEQ ID No.8, the primer pair of shigella with sequences shown as SEQ ID No.10 and SEQ ID No.11, the primer pair of klebsiella pneumoniae with sequences shown as SEQ ID No.13 and SEQ ID No.14, and the primer pair of pseudomonas aeruginosa with sequences shown as SEQ ID No.16 and SEQ ID No. 17.
Specifically, the sequence shown as SEQ ID No.1 is: 5'-CCGCAACCTACGACTCATACA-3' are provided. The sequence shown in SEQ ID No.2 is: 5'-TCGGGGCGTAATTGATCCAT-3' are provided. The sequence shown as SEQ ID No.4 is: 5'-ACCACATGCCTCTAATAATGCAAG-3' are provided. The sequence shown as SEQ ID No.5 is: 5'-CTCCAAATATCGCTAATGCACCGA-3' are provided. The sequence shown as SEQ ID No.7 is: 5'-TTTCAAGCCACCAATAAAGAACTTG-3' are provided. The sequence shown as SEQ ID No.8 is: 5'-ACACCTGTTTGTAACACTCCATT-3' are provided. The sequence shown as SEQ ID No.10 is: 5'-TGAACTTCGACTGGACCGTT-3' are provided. The sequence shown as SEQ ID No.11 is: 5'-AACAGGCAGTTCAGGTAGGT-3' are provided. The sequence shown as SEQ ID No.13 is: 5'-GCGAACCAGAGAGAGACCAA-3' are provided. The sequence shown as SEQ ID No.14 is: 5'-TCAGGAAATTGCCGACCTGA-3' are provided. The sequence shown as SEQ ID No.16 is: 5'-TACTTGCGGCTGGCTTTTTC-3' are provided. The sequence shown as SEQ ID No.17 is: 5'-CCTATCGCAAGGCTGACGA-3' are provided.
In one embodiment, the nucleic acid composition further comprises a detection probe corresponding to the detection primer pair, wherein the detection probe also comprises a fluorophore attached thereto.
Further, the detection probes corresponding to the detection primer pairs comprise a salmonella probe with a sequence shown in SEQ ID No.3, a staphylococcus aureus probe with a sequence shown in SEQ ID No.6, a clostridium difficile probe with a sequence shown in SEQ ID No.9, a shigella probe with a sequence shown in SEQ ID No.12, a klebsiella pneumoniae probe with a sequence shown in SEQ ID No.15 and a pseudomonas aeruginosa probe with a sequence shown in SEQ ID No. 18. The two ends of the detection probe corresponding to the detection primer pair are respectively connected with a fluorescent group and a quenching group. Preferably, the fluorescent group is located at the 5 'end of the detection probe and the quencher group is located at the 3' end of the detection probe.
Specifically, the sequence shown as SEQ ID No.3 is: 5'-TGGCCTGAGCGTGCTGCCGGT-3' are provided. The sequence shown as SEQ ID No.6 is: 5'-AAAGGTGGCATGGCCAAAGTATTGTTACCA-3' are provided. The sequence shown as SEQ ID No.9 is: 5'-CTCCGACTGAAGCAGCTCCACCA-3' are provided. The sequence shown as SEQ ID No.12 is: 5'-ACCTGACAACTTACCAGCTCAGATAACGCTG-3' are provided. The sequence shown as SEQ ID No.15 is: 5'-TTCCACGACTTCGGTACCGGCGTTCTGAA-3' are provided. The sequence shown as SEQ ID No.18 is: 5'-ACGAGGCTAACGAGCGTGCCCTGC-3' are provided.
In one embodiment, the fluorophore attached to the detection probe is selected from the group consisting of FAM, HEX, VIC, CY5, ROX, Texsa Red, and Quasar 705. Preferably, the fluorescent group connected to the detection probe is selected from one of FAM, HEX, CY5 and ROX. The fluorescent group is not limited to the above-mentioned fluorescent group, and other fluorescent groups may be used.
In one embodiment, the detection primer pair comprises a salmonella primer pair with sequences shown as SEQ ID No.1 and SEQ ID No.2, a staphylococcus aureus primer pair with sequences shown as SEQ ID No.4 and SEQ ID No.5, a clostridium difficile primer pair with sequences shown as SEQ ID No.7 and SEQ ID No.8, a shigella primer pair with sequences shown as SEQ ID No.10 and SEQ ID No.11, a klebsiella pneumoniae primer pair with sequences shown as SEQ ID No.13 and SEQ ID No.14, and a pseudomonas aeruginosa primer pair with sequences shown as SEQ ID No.16 and SEQ ID No. 17.
Furthermore, the detection primer pairs are divided into a plurality of groups, and the fluorescent groups connected to the detection probes corresponding to different detection primer pairs in the same group are different.
Further, the detection primers are divided into two groups; the first group comprises the salmonella primer pair, the staphylococcus aureus primer pair and the clostridium difficile primer pair, and the second group comprises the shigella primer pair, the klebsiella pneumoniae primer pair and the pseudomonas aeruginosa primer pair. The fluorescent group connected to the detection probe in each group is selected from one of FAM, HEX, VIC, CY5, ROX, Texsa Red and Quasar 705. According to the grouping, the mutual interference among the primer probes of each group can be avoided, and the detection result is easy to read.
Of course, in some embodiments, the detection primer pairs may also be a set, as long as the fluorescent groups attached to the detection probes corresponding to different detection primer pairs are different, so as to be able to distinguish different detection primer pairs.
In one embodiment, the nucleic acid composition further comprises an internal reference primer pair and an internal reference probe, wherein the sequences of the internal reference primer pair and the internal reference probe are shown as SEQ ID No.19 and SEQ ID No.20, and the sequence of the internal reference probe or the complementary sequence thereof is shown as SEQ ID No. 21; the internal reference probe is connected with a fluorescent group different from the detection probe.
In one embodiment, the fluorophores on the corresponding detection probes within the first set are ROX, HEX, and FAM, respectively; the fluorophores on the corresponding detection probes in the second group are ROX, HEX and FAM respectively. The fluorescent genes on the internal reference probes corresponding to the internal reference primer pairs in each group are ROX.
The nucleic acid composition avoids mutual interference of a plurality of detection primer pairs and corresponding detection probes through ingenious design of the detection primer pairs and the detection probes, and can simultaneously detect at least two intestinal pathogenic bacteria at one time. And proved that the nucleic acid composition has good specificity, strong anti-interference capability and high detection result accuracy when used for detection, and the sensitivity reaches 100 copies/mu L.
The nucleic acid composition can be used for simultaneously detecting at least two intestinal pathogenic bacteria, has high detection sensitivity and strong anti-interference capability, and can be used for preparing a detection kit for the intestinal pathogenic bacteria.
The invention also provides a detection kit for the intestinal pathogenic bacteria, which comprises the nucleic acid composition.
In one embodiment, the kit further comprises at least one of a PCR reaction buffer, a nucleic acid extraction reagent, a PCR stabilizer and a PCR enhancer. Specifically, the nucleic acid extraction reagent comprises a lysis solution. The PCR reaction buffer solution comprises Tris-HCl, ammonium sulfate, potassium chloride, Tween 20, magnesium sulfate, Taq DNA polymerase, dNTPs and BSA.
In one embodiment, the detection kit for enteropathogenic bacteria further comprises a positive control and a negative control. Furthermore, the positive control is a plasmid containing specific fragments of various intestinal pathogenic bacteria, and the negative control is human genome DNA.
The detection kit for the intestinal pathogenic bacteria comprises the nucleic acid composition, can detect at least two intestinal pathogenic bacteria at one time, and has the advantages of high detection sensitivity, good specificity and strong anti-interference capability.
The embodiment of the invention also provides a using method of the detection kit for the intestinal pathogenic bacteria, which comprises the following steps:
s110, extracting nucleic acid of a sample to be detected to obtain a nucleic acid sample.
Specifically, the nucleic acid sample is DNA extracted from a clinical sample. The clinical samples are nasal swabs, pharyngeal swabs or secretions and the like.
S130, taking a nucleic acid sample as a template, adding the detection primer pair and the corresponding detection probe in the nucleic acid composition, and carrying out real-time fluorescence PCR amplification reaction.
Specifically, a nucleic acid sample is mixed with a PCR reaction buffer solution, a detection primer pair in the nucleic acid composition and a corresponding detection probe to obtain a reaction solution. Then, the reaction solution is subjected to real-time fluorescent PCR amplification reaction.
Further, mixing the PCR reaction buffer solution, the detection primer pair in the nucleic acid composition and the corresponding detection probe to obtain a pre-reaction solution, and then mixing the pre-reaction solution and the nucleic acid sample to obtain a reaction solution. Wherein, the DNA of the nucleic acid sample to be detected in the reaction solution is 0.5 ng-500 ng; the final concentration of the upstream primer and the downstream primer of each detection primer pair in the reaction solution is 100 nM-500 nM; the final concentration of the detection probe corresponding to each detection primer pair in the reaction solution is 100 nM-300 nM. Wherein the reaction solution also comprises an internal reference primer pair and an internal reference probe. The final concentration of the internal reference primer pair is 100 nM-500 nM, and the final concentration of the internal reference probe is 100 nM-300 nM. In this embodiment, the PCR reaction buffer is 25 XTaqmanqPCR Master Mix.
In one embodiment, the nucleic acid samples are divided into a plurality of groups, and the detection primer pairs in the nucleic acid composition, the corresponding detection probes and the PCR reaction buffer are mixed with the nucleic acid samples of each group respectively to obtain a plurality of groups of reaction solutions.
Specifically, the amplification conditions of the real-time fluorescent PCR amplification reaction are as follows: 90-98 ℃ for 1-10 min; 90-98 ℃, 5-30 s, 55-65 ℃, 10-60 s, and 30-45 cycles. In one embodiment, the amplification conditions of the real-time fluorescent PCR amplification reaction are: 10min at 95 ℃ for 1 cycle; 95 ℃, 5s,60 ℃, 40s, 40 cycles.
In one embodiment, the positive control and the negative control are provided with an internal reference for each reaction tube. Diluting the standard substance to 1 × 10 with sterile deionized water7copies/μL～1×102copies/μL。
S130, detecting a fluorescent signal in the real-time fluorescent PCR amplification reaction process to obtain a detection result.
And (3) analyzing a detection result: when the Ct value (the cycle number of reaction when each fluorescence signal in the reaction tube reaches a set threshold) of the specific probe is less than or equal to 35, the detection result is positive corresponding to the pathogenic bacteria; when the Ct value of the specific probe is 40, the detection result is negative to the corresponding pathogenic bacteria; and when the Ct value is between 35 and 40, retesting is needed, and the negative result is that the Ct value is more than 37. And according to the obtained standard curve, quantitative calculation can be carried out on the sample to be detected.
The use method of the detection kit for the intestinal pathogenic bacteria is simple and convenient and quick.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.
Example 1
(1) Materials: entrusting Jinzhi biotechnology limited Suzhou to synthesize plasmid standards of salmonella, staphylococcus aureus, clostridium difficile, shigella, klebsiella pneumoniae and pseudomonas aeruginosa; the detection primer pair and detection probe of Suzhou Jinzhi Biotechnology Limited are entrusted. The specific sequences of the plasmid standard, the detection primer pair and the detection probe are shown in Table 1. Wherein: the 5 'end of the salmonella probe is connected with ROX, and the 3' end of the salmonella probe is connected with BHQ 1; the 5 'end of the staphylococcus aureus probe is connected with HEX, and the 3' end of the staphylococcus aureus probe is connected with BHQ 1; the 5 'end of the clostridium difficile probe is connected with FAM, and the 3' end of the clostridium difficile probe is connected with BHQ 1; the 5 'end of the Klebsiella pneumoniae probe is connected with ROX, and the 3' end of the Klebsiella pneumoniae probe is connected with BHQ 1; the 5 'end of the pseudomonas aeruginosa probe is connected with HEX, and the 3' end of the pseudomonas aeruginosa probe is connected with BHQ 1; the 5 'end of the shigella probe is connected with FAM, and the 3' end of the shigella probe is connected with BHQ 1; the 5 'end of the internal reference probe is connected with CY5, and the 3' end is connected with BHQ 2.
TABLE 1
(2) The synthesized plasmid standards were quantified using the Qubit3.0 and the Qubit dsDNA assay kit and converted to copies/. mu.l.
(3) The single-fluorescence quantitative PCR amplification reaction system corresponding to salmonella, staphylococcus aureus, clostridium difficile, shigella, klebsiella pneumoniae and pseudomonas aeruginosa is as follows:
the A group of fluorescent quantitative PCR amplification reaction system is as follows:
The B group of fluorescent quantitative PCR amplification reaction system is as follows:
The reaction parameter settings of the fluorescent quantitative PCR reaction systems of the group A and the group B are as follows: the reaction is started at 95 ℃ for 10min, and then the fluorescence signal detection is carried out for 40 cycles, wherein the reaction conditions of each cycle are 95 ℃ for 5s and 60 ℃ for 40 s.
(4) And (3) specific detection: the same dilution (1X 10)7Copied/mu L) of salmonella plasmid standard, staphylococcus aureus plasmid standard, clostridium difficile plasmid standard, shigella plasmid standard, klebsiella pneumoniae plasmid standard and pseudomonas aeruginosa plasmid standard respectively serve as templates to be detected, and the A group and B group fluorescent quantitative PCR systems in the step (3) are used for reaction so as to detect the specificity of the primer-probe, wherein the results are shown in figures 1-12.
In FIGS. 1 and 2, 2. mu.L of 1X 10 was taken7And (3) taking the copied/mu L salmonella plasmid standard as a template to be detected, and respectively preparing a group A fluorescence quantitative PCR amplification reaction system and a group B fluorescence quantitative PCR amplification reaction system in the step (3), and then carrying out amplification reaction. As can be seen from FIG. 1, the Salmonella plasmid standard only reacted with its specific primer-probe, but did not perform non-specific amplification with other probes in the group A reaction system, and the reference amplification curve was normal in the group A reaction system. As can be seen from FIG. 1, sandThe phylum bacterium plasmid standard substance and all probes in the group B reaction system do not carry out non-specific amplification, and the internal reference amplification curve in the group B reaction system is normal.
In FIGS. 3 and 4, 2. mu.L of 1X 10 was taken7Taking the copied/mu L staphylococcus aureus plasmid standard as templates to be detected, and respectively obtaining the result of the amplification reaction of the group A fluorescence quantitative PCR amplification reaction system and the group B fluorescence quantitative PCR amplification reaction system in the step (3). As can be seen from FIG. 3, the Staphylococcus aureus plasmid standard reacted only with its specific primer-probe, but did not undergo non-specific amplification with the other probes in the group A reaction system, and the reference amplification curve was normal in the group A reaction system. As can be seen from FIG. 4, the Staphylococcus aureus plasmid standard and all the probes in the group B reaction system did not undergo non-specific amplification, and the reference amplification curve was normal in the group B reaction system.
In FIGS. 5 and 6, 2. mu.L of 1X 10 was taken7And (3) taking the copied/mu L clostridium difficile plasmid standard product as a template to be detected, and respectively obtaining the result of the amplification reaction of the A group of fluorescence quantitative PCR amplification reaction system and the B group of fluorescence quantitative PCR amplification reaction system in the step (3). As can be seen from FIG. 5, the Clostridium difficile plasmid standard reacted only with its specific primer-probe, but did not undergo non-specific amplification with the other probes in the group A reaction system, and the reference amplification curve was normal in the group A reaction system. As can be seen from FIG. 6, the C.difficile plasmid standard and all the probes in the group B reaction system did not undergo non-specific amplification, and the reference amplification curve was normal in the group B reaction system.
In FIGS. 7 and 8, 2. mu.L of 1X 10 was taken7Taking the copied/mu L shigella plasmid standard product as a template to be detected, and respectively preparing a group A fluorescent quantitative PCR amplification reaction system and a group B fluorescent quantitative PCR amplification reaction system in the step (3) and then carrying out an amplification reaction. As can be seen from FIG. 7, the Shigella plasmid standard and all probes in the group A reaction system did not undergo non-specific amplification, and the reference amplification curve was normal in the group A reaction system. As can be seen from FIG. 8, the shigella plasmid standard only reacts with its specific primer-probe, but does not nonspecifically react with other probes in the group B reaction systemAnd (3) performing anisotropic amplification, wherein the internal reference amplification curve is normal in the group B reaction system.
In FIGS. 9 and 10, 2. mu.L of 1X 10 was taken7And (3) taking the copied/mu L Klebsiella pneumoniae plasmid standard substance as a template to be detected, and respectively preparing a group A fluorescence quantitative PCR amplification reaction system and a group B fluorescence quantitative PCR amplification reaction system in the step (3), and then carrying out amplification reaction. As can be seen from FIG. 9, all the probes in the reaction systems of Klebsiella pneumoniae and group A were not non-specifically amplified, and the reference amplification curve was normal in the reaction system of group A. As can be seen from fig. 10, klebsiella pneumoniae reacted only with its specific primer-probe, but did not non-specifically amplify with other probes in the group B reaction system, and the reference amplification curve was normal in the group B reaction system.
In FIGS. 11 and 12, 2. mu.L of 1X 10 was taken7And (3) taking the copied/mu L pseudomonas aeruginosa plasmid standard as a template to be detected, and respectively obtaining the result of the amplification reaction of the A group of fluorescence quantitative PCR amplification reaction system and the B group of fluorescence quantitative PCR amplification reaction system in the step (3). As can be seen from FIG. 11, the Pseudomonas aeruginosa plasmid standard and all the probes in the group A reaction system did not undergo non-specific amplification, and the reference amplification curve was normal in the group A reaction system. As can be seen from FIG. 12, the Pseudomonas aeruginosa plasmid standard only reacted with its specific primer-probe, but did not perform non-specific amplification with other probes in the group B reaction system, and the reference amplification curve was normal in the group B reaction system.
The results show that the 6 groups of primers and probes designed in the embodiment have good specificity, the amplification of the internal reference is normal, and the internal reference and the intestinal pathogenic bacteria have no cross interference. Can be used for subsequent multiplex fluorescent quantitative PCR reaction.
Example 2
(1) Preparing a multiple fluorescent quantitative PCR amplification reaction system:
the A group of multiplex fluorescent quantitative PCR amplification reaction system is as follows:
The group B multiplex fluorescent quantitative PCR amplification reaction system is as follows:
In the system, 3 templates corresponding to the primer-probe respectively refer to a shigella plasmid standard, a klebsiella pneumoniae plasmid standard and a pseudomonas aeruginosa plasmid standard.
The reaction parameter settings of the multiple fluorescent quantitative PCR reaction systems of the group A and the group B are as follows: the reaction is started at 95 ℃ for 10min, and then the fluorescence signal detection is carried out for 40 cycles, wherein the reaction conditions of each cycle are 95 ℃ for 5s and 60 ℃ for 40 s.
(2) And (3) sensitivity detection: respectively carrying out continuous gradient dilution on the salmonella plasmid standard, the staphylococcus aureus plasmid standard, the clostridium difficile plasmid standard, the shigella plasmid standard, the klebsiella pneumoniae plasmid standard and the pseudomonas aeruginosa plasmid standard to 1 multiplied by 107Copy/. mu.L-1X 102Copies/. mu.L. And (2) applying the continuously diluted salmonella plasmid standard, staphylococcus aureus plasmid standard and clostridium difficile plasmid standard to the group A fluorescent quantitative PCR amplification reaction system in the step (1) for amplification reaction, and applying the continuously diluted shigella plasmid standard, klebsiella pneumoniae plasmid standard and pseudomonas aeruginosa plasmid standard to the group B fluorescent quantitative PCR system in the step (1) for amplification reaction. The results are shown in FIGS. 13 to 16.
FIG. 13 is a re-fluorescence quantitative PCR amplification curve for Salmonella plasmid standards, Staphylococcus aureus plasmid standards, and Clostridium difficile plasmid standards; FIG. 14 is a multiple fluorescence quantitative PCR amplification standard curve for Salmonella plasmid standards, Staphylococcus aureus plasmid standards, and Clostridium difficile plasmid standards. As can be seen from fig. 13 and 14, in the multiplex fluorescence quantitative PCR system, the Ct values detected by the salmonella plasmid standard, the staphylococcus aureus plasmid standard and the clostridium difficile plasmid standard are relatively close to each other at the same dilution.
FIG. 15 is a multiple fluorescence quantitative PCR amplification curve of Shigella plasmid standards, Klebsiella pneumoniae plasmid standards, and Pseudomonas aeruginosa plasmid standards; FIG. 16 is a multiple fluorescence quantitative PCR amplification standard curve of Shigella plasmid standard, Klebsiella pneumoniae plasmid standard and Pseudomonas aeruginosa plasmid standard. As can be seen from FIGS. 15 and 16, Ct values detected by the Shigella plasmid standard, the Klebsiella pneumoniae plasmid standard and the Pseudomonas aeruginosa plasmid standard are relatively close to each other at the same dilution in the multiplex fluorescence quantitative PCR system.
(6) Comparing Ct values of single and multiple fluorescence quantitative PCR results of the salmonella plasmid standard, the staphylococcus aureus plasmid standard and the clostridium difficile plasmid standard; comparing the Ct values of the single weight and the multiple fluorescence quantitative PCR result of the shigella plasmid standard, the klebsiella pneumoniae plasmid standard and the pseudomonas aeruginosa plasmid standard; the results are shown in tables 2 and 3.
TABLE 2
TABLE 2
Therefore, multiple fluorescence quantitative PCR amplification results of salmonella, staphylococcus aureus, clostridium difficile, shigella, klebsiella pneumoniae and pseudomonas aeruginosa show that the standard curve obtained by the reaction is perfect, the detection linear range is wide, three pathogenic bacteria in the same reaction system can be simultaneously quantified and detected within the concentration range of 6 orders of magnitude, and the detection sensitivity can reach 1 multiplied by 102Copies/. mu.L.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Anthenian Biotechnology (Shenzhen) Limited
Shenzhen Qinghua university institute
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Claims (10)
1. A nucleic acid composition comprising a pair of detection primers, said pair of detection primers comprising at least two of the following pairs of primers:
the primer pair of salmonella with sequences shown as SEQ ID No.1 and SEQ ID No.2, the primer pair of staphylococcus aureus with sequences shown as SEQ ID No.4 and SEQ ID No.5, the primer pair of clostridium difficile with sequences shown as SEQ ID No.7 and SEQ ID No.8, the primer pair of shigella with sequences shown as SEQ ID No.10 and SEQ ID No.11, the primer pair of klebsiella pneumoniae with sequences shown as SEQ ID No.13 and SEQ ID No.14, and the primer pair of pseudomonas aeruginosa with sequences shown as SEQ ID No.16 and SEQ ID No. 17.
2. The nucleic acid composition of claim 1, further comprising a detection probe corresponding to the detection primer pair, the detection probe corresponding to the detection primer pair comprising:
a salmonella probe with a sequence shown in SEQ ID No.3, a staphylococcus aureus probe with a sequence shown in SEQ ID No.6, a clostridium difficile probe with a sequence shown in SEQ ID No.9, a shigella probe with a sequence shown in SEQ ID No.12, a klebsiella pneumoniae probe with a sequence shown in SEQ ID No.15 and a pseudomonas aeruginosa probe with a sequence shown in SEQ ID No. 18;
and the detection probe corresponding to the detection primer pair is connected with a fluorescent group.
3. The nucleic acid composition of claim 2, wherein the detection primer pair comprises the salmonella primer pair, the staphylococcus aureus primer pair, the clostridium difficile primer pair, the shigella primer pair, the klebsiella pneumoniae primer pair, and the pseudomonas aeruginosa primer pair; the detection primer pairs are divided into a plurality of groups, and the fluorescent groups connected to the detection probes corresponding to different detection primer pairs in the same group are different.
4. The nucleic acid composition of claim 3, wherein the detection primers pair into two groups; the first group comprises the salmonella primer pair, the staphylococcus aureus primer pair and the clostridium difficile primer pair, and the second group comprises the shigella primer pair, the klebsiella pneumoniae primer pair and the pseudomonas aeruginosa primer pair.
5. The nucleic acid composition of claim 3, wherein the fluorescent group attached to the detection probe corresponding to each detection primer pair in each set is selected from one of FAM, HEX, VIC, CY5, ROX, Texsa Red, and Quasar 705.
6. The nucleic acid composition of claim 5, wherein the fluorophores on the corresponding detection probes in the first set are ROX, HEX, and FAM, respectively; the fluorophores on the corresponding detection probes in the second group are ROX, HEX and FAM respectively.
7. The nucleic acid composition of any one of claims 2 to 6, further comprising an internal reference primer pair and an internal reference probe, wherein the sequences of the internal reference primer pair and the internal reference probe are shown as SEQ ID No.19 and SEQ ID No.20, and the sequence of the internal reference probe or the complementary sequence thereof is shown as SEQ ID No. 21; the internal reference probe is connected with a fluorescent group different from the detection probe.
8. A kit for detecting enteropathogenic bacteria, comprising the nucleic acid composition of any one of claims 1 to 7.
9. The detection kit for enteropathogenic bacteria according to claim 8, further comprising at least one of a PCR reaction buffer, a nucleic acid extraction reagent, a PCR stabilizer and a PCR enhancer.
10. The use method of the detection kit for the enteropathogenic bacteria is characterized by comprising the following steps:
extracting nucleic acid of a sample to be detected to obtain a nucleic acid sample;
adding a detection primer pair and a corresponding detection probe in the detection kit for the enteropathogenic bacteria according to any one of claims 8 to 9 by taking the nucleic acid sample as a template to perform a real-time fluorescent PCR amplification reaction; and
and detecting the fluorescent signal in the real-time fluorescent PCR amplification reaction process to obtain a detection result.
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