CN105331710A - Nucleic acid isothermal amplification detection kit for Salmonella and detection method - Google Patents
Nucleic acid isothermal amplification detection kit for Salmonella and detection method Download PDFInfo
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Abstract
The invention discloses a nucleic acid isothermal amplification detection kit for Salmonella and a detection method. The detection kit comprises a DNA extraction reagent, an isothermal amplification reaction liquid, a positive control and a negative control. The detection kit is high in specificity and high in sensitivity; the temperature of all nucleic acid isothermal amplification reactions is consistent, only 35 minutes are required for isothermal amplification, 1-2 minutes are required for a detection result of a nucleic acid test strip, only about 1 hour is required for the whole detection process from sample receiving to result acquisition, only one isothermal instrument is required in the whole reaction process, and the detection kit is particularly suitable for on-site and rapid detection and elimination of the Salmonella in food.
Description
(1) technical field
The present invention relates to a kind of constant-temperature amplification Fast Detection Technique for Salmonellas nucleic acid, be applicable to the qualitative detection to Salmonellas.
(2) background technology
Salmonellas is extensively present in nature, and the serotype had been found that so far just has more than 2500 kinds, and China has found 216.The serotype relevant with human diseases mainly concentrates on A ~ E group, comprise: salmonella typhi, first, second, moscow' paratyphi C, Salmonella typhimurium, Salmonella choleraesuls, Salmonella enteritidis, Salmonella anatis, Salmonella newport etc., wherein common with Salmonella typhimurium, Salmonella enteritidis and Salmonella choleraesuls.Salmonellal food poisoning accounts for first place, the common bacteria of Yi Shi China bacterial food poisoning Japan, America and Europe.In China hinterland, having 70% ~ 80% bacterial food poisoning to be by salmonellal, is class foodborne bacterial pathogens human and animal's health being had to high risks.
Traditional detection many employings microbial culture of Salmonellas, the method such as biochemistry and serological identification, this class methods complex operation, waste time and energy, required reagent is various, Financial cost is high.Along with immunology and molecular biological development, establish now immunological detection method based on antigen as ELISA method, and the detection technique based on PCR.ELISA operation is comparatively loaded down with trivial details, time-consuming longer; PCR detection method is quick, and sensitive, accuracy is higher, but needs certain equipment and technical requirements, and reagent expense is more expensive, is difficult to popularize.Extensively can carry out the rapid detection of Salmonella enteritidis to realize basic unit, such bacterial food poisoning of effective prevention and control, set up practical, easy, Salmonella enteritidis detection method is very necessary fast and accurately.
In recent years, LAMP method receives publicity, the method with specific nucleic acid be target under isothermal conditions by there being the archaeal dna polymerase effect of strand displacement enzymic activity to increase, have that specificity is high, susceptibility be strong, and easy feature with low cost fast.In numerous areas widespread uses such as microorganism detection.The technology of the present invention have developed constant-temperature amplification Salmonellas nucleic acid, and the colour developing of bind nucleic acid test strip carrys out the detection method of result of determination, and whole reaction process does not open reaction tubes lid, test strip flow detection process is airtight, has stopped nucleic acid amplification product and has polluted extraneous possibility.Research shows that this salmonella constant temperature amplification-nucleic acid test strip detection method has the advantages such as high specificity, susceptibility is high, simple to instrument requirements, detection time is short, use so be useful in very much inspection body of basic unit, to diagnose with bedside clinically at the Site Detection of public health emergency simultaneously and also there is good application prospect.
(3) summary of the invention
The object of the invention is to provide a kind of constant-temperature amplification detection kit that is accurate, sensitive, detection Salmonellas nucleic acid rapidly and detection method thereof.
The technical solution used in the present invention is:
The invention provides the nucleic acid isothermal amplification detection kit of a kind of Salmonellas, described test kit comprises following composition:
(1) the DNA extraction reagent (BacterialGenomicDNAExtractionKit (article No. DV810A) of preferred Takara company;
(2) isothermal amplification reactions liquid, comprising: primer, reverse peripheral primer, two probes, two intersections amplimer, 1 × Thermolbuffer, MgSO just to the periphery
4, dNTPs solution, BstDNA polysaccharase and aseptic double-distilled water; Wherein:
Described peripheral primer is respectively:
Described primer just is to the periphery: 5 '-AACAGTGCTCGTTTACGACC-3 ' (SEQIDNO.2);
Reverse peripheral primer is: 5 '-CTGATCGATAATGCCAGACG-3 ' (SEQIDNO.3);
The sequence of described two probes is respectively:
Forward 5 ' holds Biotin label probe: 5 '-Biotin-AGGTAGGTAATGGAATGACGA-3 ' (SEQIDNO.4);
Reverse 5 ' end Fitc label probe 5 '-Fitc-CGTTCTACATTGACAGAATCCTCAG-3 ' (SEQIDNO.5);
Described two amplification cross primers are respectively:
Amplification forward primer:
5′-GCGATCAGGAAATCAACCAGATAGAATGGTGATGATCATTTCTATGTTC-3′(SEQIDNO.6)；
Amplification reverse primer:
5′-CGTACTGGCGATATTGGTGTTTATATTAACAGTACCGCAGGAAAC-3′(SEQIDNO.7)；
(3) positive control: the DNA plasmid containing Salmonellas invA gene fragment;
(4) negative control: aseptic double-distilled water.
Further, 1 × Thermolbuffer of the present invention consists of: (the NH of KCl, 10mM of Tris-HCl, 10mM of 20mM
4)
2sO
4, 2mM MgSO
4and mass concentration is the TritonX-100 of 0.1%, pH7.5.
Further, positive control of the present invention is the fragment of the Salmonellas invA gene order containing long 238bp, the nucleotide sequence following (SEQIDNO.1) of described fragment:
AACAGTGCTCGTTTACGACCTGAATTACTGATTCTGGTACTAATGGTGATGATCATTTCTATGTTCGTCATTCCATTACCTACCTATCTGGTTGATTTCCTGATCGCACTGAATATCGTACTGGCGATATTGGTGTTTATGGGGTCGTTCTACATTGACAGAATCCTCAGTTTTTCAACGTTTCCTGCGGTACTGTTAATTACCACGCTCTTTCGTCTGGCATTATCGATCAGTACCA
Further, positive control of the present invention is prepared as follows:
To comprise the Salmonellas invA gene fragment of amplification region for template, carry out pcr amplification by two peripheral primers and obtain goal gene; PCR purification kit (Promega) is utilized to carry out purifying to pcr amplification product; Amplified production after purifying is built the complete plasmid sequence containing goal gene by T-easy plasmid transfection test kit (Promega), with spectrophotometer, A is surveyed to the plasmid of institute's extracting
280quantitatively also 10 times of dilutions ,-20 DEG C of preservations.
Further, isothermal amplification reactions liquid of the present invention consists of: two peripheral primer 10pmol separately, two probe 20pmol separately, two cross primer 40pmol separately, 1 × Thermolbuffer, MgSO
4for 6mmol, dNTPs solution each 0.4mmol, BstDNA polysaccharase 8U, aseptic double-distilled water composition complements to 25 μ l.
The present invention also provides the detection method of the constant-temperature amplification detection kit of a kind of described Salmonellas, and described detection method is:
A) from sample to be detected, DNA is extracted with DNA extraction reagent;
B) step a) being extracted the DNA obtained joins in the PCR pipe that isothermal amplification reactions liquid is housed as template, amplified reaction 35 minutes at 65 DEG C; Standard positive template (i.e. positive control) adds in positive control PCR pipe, standard negative template (i.e. negative control) adds in negative control PCR pipe, described standard positive template is the plasmid containing Salmonellas invA gene fragment, and described standard negative template is aseptic double-distilled water;
C) reacted PCR pipe is placed in the anti-pollution proofing unit of nucleic acid test strip (Hangzhou You Sida biotech company No. 3 devices) and detects, sentence read result after 2 minutes, when the detection line of test strip is positive, containing Salmonellas nucleic acid in interpret sample.This test strip is included on the liner with non-setting adhesive and is provided with sample pad, coloured particle binding substances pad and absorbent filter pad in order, each part mentioned above partly overlaps at adjacent, tunica fibrosa is provided with detection line and nature controlling line, coloured particle wherein on coloured particle binding substances pad has anti-Fitc antibody bag quilt, detection line there is avidin bag quilt, nature controlling line has anti-Fitc antibody, coloured particle is selected from colloid gold particle, latex particle.
In test kit provided by the invention, there are two peripheral primers, two cross primers and two detection probes.6 oligonucleotide sequences in this test kit rely on the highly active strand displacement characteristic of BstDNA polysaccharase, make strand displacement DNA synthesize continuous self-amplification cycles.In the constant-temperature amplification detection kit of Salmonellas nucleic acid provided by the invention, different reaction conditionss is optimized, as the concentration of primer and probe, Mg
2+concentration, the optimization of temperature of reaction etc., and the present invention is combined with nucleic acid detection test strip detection system, establish the method for Salmonellas nucleic acid constant-temperature amplification qualitative detection.The sensitivity of this test kit can detect 10 copies in each reaction system, can meet the requirement of rapid detection Salmonellas nucleic acid.
The present invention compared with prior art, has the following advantages and effect:
(1) specificity of the nucleic acid isothermal amplification detection kit of Salmonellas of the present invention is good, highly sensitive, and step is simple, repeatable high;
(2) speed of response is fast, and single sample, from sample process to completing detection, only needs 1 hours;
(3) do not need to open PCR pipe lid in whole amplification and testing process, decrease the chance that amplified production pollutes; Whole reaction process does not need complicated instrument.
(4) accompanying drawing explanation
Fig. 1 is nucleic acid detection test strip principle schematic.
Fig. 2 is that test kit of the present invention is for detecting the specific outcome of Salmonellas, in figure, 1-13 represents salmonella typhi CMCC50071 respectively, Salmonella typhimurium CMCC50115, shigella flexneri CMCC51572, pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC27853, streptococcus aureus ATCC25923, colon bacillus ATCC25922, enterobacter cloacae ATCC13047, Vibrio parahemolyticus ATCC17802, campylobacter jejuni ATCC33291, single increasing listeria spp ATCC19111, clostridium difficile ATCC9689, positive reference substance, the experimental result of negative controls.
Fig. 3 be test kit of the present invention for detecting the sensitivity of Salmonellas, in figure, 1-6 represents 10 respectively
4copy/microlitre, 10
3copy/microlitre, 10
2copy/microlitre, 10
1copy/microlitre, 10
0the experimental result of copy/microlitre, negative controls.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
The composition of embodiment 1 test kit of the present invention and preparation
A) the DNA extraction reagent (BacterialGenomicDNAExtractionKit (article No. DV810A) of preferred Takara company;
B) Salmonellas nucleic acid constant-temperature amplification reaction solution: two peripheral primers (10pmol), two probes (20pmol) and two cross primers (40pmol), 1 × Thermolbuffer, MgSO
4(6mmol), dNTPs solution (each 0.4mmol), BstDNA polysaccharase (8U) and aseptic double-distilled water composition, it is 25 μ l that total reaction liquid amasss;
Primer is just to the periphery: 5 '-AACAGTGCTCGTTTACGACC-3 ';
Reverse peripheral primer is: 5 '-CTGATCGATAATGCCAGACG-3 ';
Forward 5 ' holds Biotin label probe: 5 '-Biotin-AGGTAGGTAATGGAATGACGA-3;
Reverse 5 ' end Fitc label probe: 5 '-Fitc-CGTTCTACATTGACAGAATCCTCAG-3 ';
Amplification forward primer:
5′-GCGATCAGGAAATCAACCAGATAGAATGGTGATGATCATTTCTATGTTC-3′；
Amplification reverse primer:
5′-CGTACTGGCGATATTGGTGTTTATATTAACAGTACCGCAGGAAAC-3′；
1 described × Thermolbuffer consists of: (the NH of KCl, 10mM of Tris-HCl, 10mM of 20mM
4)
2sO
4, 2mM MgSO
4and mass concentration is the TritonX-100 of 0.1%, pH7.5.
All primers and probe are by the synthesis of Shanghai raw work biological company limited.
C) positive control: positive control is the Salmonellas invA gene order containing long 238bp, and sequence is as follows:
AACAGTGCTCGTTTACGACCTGAATTACTGATTCTGGTACTAATGGTGATGATCAT TTCTATGTTCGTCATTCCATTACCTACCTATCTGGTTGATTTCCTGATCGCACTGA ATATCGTACTGGCGATATTGGTGTTTATGGGGTCGTTCTACATTGACAGAATCCTC AGTTTTTCAACGTTTCCTGCGGTACTGTTAATTACCACGCTCTTTCGTCTGGCATT ATCGATCAGTACCA positive control is prepared as follows:
To comprise the Salmonellas invA gene fragment of amplification region for template, carry out pcr amplification by two peripheral primers and obtain goal gene; PCR purification kit is utilized to carry out purifying to pcr amplification product; Amplified production after purifying is built the complete plasmid sequence containing goal gene by T-easy plasmid transfection test kit, with spectrophotometer, A is surveyed to the plasmid of institute's extracting
280quantitatively and be diluted to 20 copy/μ l ,-20 DEG C of preservations.
D) negative control: aseptic double-distilled water.
Embodiment 2 test kit of the present invention detects the concrete grammar of Salmonellas nucleic acid
A) from sample to be detected, DNA is extracted with DNA extraction liquid in salmonella constant temperature amplification detection kit.
B) get specimen dna as template, join in the PCR pipe that salmonella constant temperature amplification reaction solution is housed, wherein specimen dna 5 μ l, reaction solution 20 μ l, 65 DEG C of amplified reactions 35 minutes; Positive template (Salmonellas invA gene, SEQIDNO.1) and negative template (distilled water) is added respectively in the positive and negative control PCR pipe.
C) reacted PCR pipe is placed in the anti-pollution proofing unit of nucleic acid test strip (Hangzhou You Sida biotech company No. 3 devices) and detects, sentence read result after 2 minutes.When containing Salmonellas nucleic acid in sample, the detection line of test strip is positive (C, T line is redness).
Repeatedly repeat experiment 3 times, detected result there was no significant difference, the detected result between the different batches that this test kit is described has comparability, has good repeatability.Above-described embodiment illustrates, detects reproducible, and only needs 1 hours just can complete to the detection of sample, greatly shorten detection time with test kit of the present invention.
Embodiment 3 test kit of the present invention detects the specificity of Salmonellas
Salmonella typhi CMCC50071 is detected according to the method for embodiment 2, Salmonella typhimurium CMCC50115, shigella flexneri CMCC51572, pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC27853, streptococcus aureus ATCC25923, colon bacillus ATCC25922, enterobacter cloacae ATCC13047, Vibrio parahemolyticus ATCC17802, campylobacter jejuni ATCC33291, single increasing listeria spp ATCC19111, clostridium difficile ATCC9689 and Salmeterol fluticasone propionate test kit positive reference substance, result display test kit of the present invention detects Salmonellas nucleic acid and has very strong specificity, except salmonella typhi CMCC50071, Salmonella typhimurium CMCC50115 and test kit positive reference substance, other bacterial strains detect and are feminine gender.As shown in Figure 2, in figure, 1-13 represents salmonella typhi CMCC50071, Salmonella typhimurium CMCC50115, shigella flexneri CMCC51572, pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC27853, streptococcus aureus ATCC25923, colon bacillus ATCC25922, enterobacter cloacae ATCC13047, Vibrio parahemolyticus ATCC17802, campylobacter jejuni ATCC33291, single experimental result increasing listeria spp ATCC19111, clostridium difficile ATCC9689, positive reference substance, negative controls respectively.
Embodiment 4 test kit of the present invention detects the sensitivity of Salmonellas nucleic acid
Carry out quantitatively to the plasmid DNA containing Salmonellas invA gene fragment, being diluted to concentration is respectively 10
4copy/microlitre, 10
3copy/microlitre, 10
2copy/microlitre, 10
1copy/microlitre, 10
0copy/microlitre, adopts method described in embodiment 2 to determine that test kit of the present invention is for detecting the sensitivity of Salmonellas nucleic acid.As shown in Figure 3, in figure, 1-6 represents 10 to result respectively
4copy/microlitre, 10
3copy/microlitre, 10
2copy/microlitre, 10
1copy/microlitre, 10
0the experimental result of copy/microlitre, negative controls, can find that this test kit can detect 10 copies in each reaction system, have very high sensitivity, can meet the requirement of rapid detection Salmonellas.
Claims (6)
1. a Salmonellas nucleic acid isothermal amplification detection kit, is characterized in that described test kit comprises following composition:
(1) DNA extraction reagent;
(2) isothermal amplification reactions liquid, comprising: primer, reverse peripheral primer, two probes, two increase cross primer, 1 × ThermolBuffer, MgSO just to the periphery
4, dNTPs solution, BstDNA polysaccharase and aseptic double-distilled water;
Described primer just is to the periphery: 5 '-AACAGTGCTCGTTTACGACC-3 ';
Reverse peripheral primer is: 5 '-CTGATCGATAATGCCAGACG-3 ';
The sequence of described two probes is respectively:
Forward 5 ' holds Biotin label probe: 5 '-Biotin-AGGTAGGTAATGGAATGACGA-3 ';
Reverse 5 ' end Fitc label probe: 5 '-Fitc-CGTTCTACATTGACAGAATCCTCAG-3 ';
Described two amplification cross primers are respectively:
Amplification forward primer:
5′-GCGATCAGGAAATCAACCAGATAGAATGGTGATGATCATTTCTATGTTC-3′；
Amplification reverse primer:
5′-CGTACTGGCGATATTGGTGTTTATATTAACAGTACCGCAGGAAAC-3′；
(3) positive control: the DNA plasmid containing Salmonellas invA gene;
(4) negative control: aseptic double-distilled water.
2. Salmonellas nucleic acid isothermal amplification detection kit as claimed in claim 1, is characterized in that described 1 × ThermolBuffer consists of: (the NH of KCl, 10mM of Tris-HCl, 10mM of 20mM
4)
2sO
4, 2mM MgSO
4and mass concentration is the TritonX-100 of 0.1%, pH7.5.
3. Salmonellas nucleic acid isothermal amplification detection kit as claimed in claim 1, it is characterized in that described positive control is the fragment of the Salmonellas invA gene order containing long 238bp, the nucleotides sequence of described fragment is classified as shown in SEQIDNO.1.
4. Salmonellas nucleic acid isothermal amplification detection kit as claimed in claim 1, is characterized in that described positive control is prepared as follows:
To comprise the Salmonellas invA gene fragment of amplification region for template, carry out pcr amplification by two peripheral primers and obtain goal gene; PCR purification kit is utilized to carry out purifying to pcr amplification product; Amplified production after purifying is built the complete plasmid sequence containing goal gene by T-easy plasmid transfection test kit, with spectrophotometer to the plasmid of institute's extracting quantitatively and be diluted to 20 copy/μ l, acquisition positive control ,-20 DEG C of preservations.
5. Salmonellas nucleic acid isothermal amplification detection kit as claimed in claim 1, it is characterized in that described isothermal amplification reactions liquid consists of: two peripheral primer 10pmol separately, two probe 20pmol separately, two cross primer 40pmol separately, 1 × Thermolbuffer, MgSO
4for 6mmol, dNTPs solution each 0.4mmol, BstDNA polysaccharase 8U, aseptic double-distilled water composition complements to 25 μ l.
6. Salmonellas nucleic acid isothermal amplification detection kit described in claim 1 a detection method, it is characterized in that described detection method is:
A) from sample to be detected, DNA is extracted with DNA extraction reagent;
B) step a) is extracted the DNA that obtains as template, join in the PCR pipe that isothermal amplification reactions liquid is housed, standard positive template adds in positive control PCR pipe, standard negative template adds in negative control PCR pipe, described standard positive template is the plasmid containing Salmonellas invA gene fragment, described standard negative template is aseptic double-distilled water, amplified reaction 35 minutes at 65 DEG C;
C) reacted PCR pipe is placed in the anti-pollution proofing unit of nucleic acid test strip detects, sentence read result after 2 minutes, when the detection line of test strip is positive, containing Salmonellas invA gene DNA nucleic acid in interpret sample.
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Cited By (7)
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| CN105755124A (en) * | 2016-03-22 | 2016-07-13 | 济南大学 | Method for detecting salmonella with fluorescence method on basis of enzymatic remediation isothermal cycle amplification |
| CN106636387A (en) * | 2016-12-14 | 2017-05-10 | 天津科技大学 | Salmonella nucleic acid rapid detection kit, test strip and detection method |
| CN108060209A (en) * | 2017-12-26 | 2018-05-22 | 中科智测(天津)科技有限公司 | A kind of kit and detection method of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella |
| CN109402271A (en) * | 2017-08-14 | 2019-03-01 | 中国科学院微生物研究所 | For detecting the primer system and kit and their application and the method for detecting salmonella of salmonella |
| CN109652575A (en) * | 2019-03-05 | 2019-04-19 | 许绍坤 | A kind of nucleic acid rapid detection method for salmonella |
| CN110982914A (en) * | 2019-12-18 | 2020-04-10 | 江苏海洋大学 | Specific forward and reverse primers and probe for salmonella, detection kit and application of specific forward and reverse primers and probe |
| CN113462797A (en) * | 2021-07-02 | 2021-10-01 | 华南理工大学 | CPA detection primer, detection kit and detection method for salmonella |
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Cited By (9)
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| CN105755124A (en) * | 2016-03-22 | 2016-07-13 | 济南大学 | Method for detecting salmonella with fluorescence method on basis of enzymatic remediation isothermal cycle amplification |
| CN105755124B (en) * | 2016-03-22 | 2019-02-05 | 济南大学 | A kind of method that enzyme repairs isothermal circulation amplification Fluorometric assay salmonella |
| CN106636387A (en) * | 2016-12-14 | 2017-05-10 | 天津科技大学 | Salmonella nucleic acid rapid detection kit, test strip and detection method |
| CN106636387B (en) * | 2016-12-14 | 2020-10-27 | 天津科技大学 | Kit, test paper and detection method for rapid detection of salmonella nucleic acid |
| CN109402271A (en) * | 2017-08-14 | 2019-03-01 | 中国科学院微生物研究所 | For detecting the primer system and kit and their application and the method for detecting salmonella of salmonella |
| CN108060209A (en) * | 2017-12-26 | 2018-05-22 | 中科智测(天津)科技有限公司 | A kind of kit and detection method of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella |
| CN109652575A (en) * | 2019-03-05 | 2019-04-19 | 许绍坤 | A kind of nucleic acid rapid detection method for salmonella |
| CN110982914A (en) * | 2019-12-18 | 2020-04-10 | 江苏海洋大学 | Specific forward and reverse primers and probe for salmonella, detection kit and application of specific forward and reverse primers and probe |
| CN113462797A (en) * | 2021-07-02 | 2021-10-01 | 华南理工大学 | CPA detection primer, detection kit and detection method for salmonella |
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