CN116356049A - Kit for detecting clostridium difficile - Google Patents
Kit for detecting clostridium difficile Download PDFInfo
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Abstract
The invention discloses a detection kit of clostridium difficile based on the LAMP technology. LAMP primers were designed based on the specific conserved sequences of Clostridium difficile, each set of primers contained 6 oligonucleotides. For clostridium difficile detection, a white precipitate can be observed by naked eyes; the isothermal nucleic acid amplification analyzer can also be used for detecting and data processing analysis of fluorescent signals emitted after the amplified products are combined with the nucleic acid dye, so as to form an amplification curve, and the detection result is judged according to the Tt value. The invention provides a new technical platform for clostridium difficile detection, and is suitable for popularization and application in basic units, on-site monitoring and bedside detection.
Description
Technical Field
The invention belongs to application of a molecular biological detection method represented by isothermal amplification in clostridium difficile detection, in particular to a loop-mediated isothermal amplification detection method and a kit for clostridium difficile.
Background
Complex and relatively stable flora is planted in human gastrointestinal tract, the species is about 500, and the number is 10 14 About one. Dysregulation of intestinal flora, reduced numbers of probiotics, increased pathogenic bacteria can destroy the intestinal barrier, cause inflammatory reactions, and increase the incidence of intestinal infectious diseases, the most common of which is clostridium difficile (Clostridium difficile, CD). Clostridium difficile is widely distributed in nature and has flagella, a gram-positive anaerobic bacillus with spores, exposed to the skyThe clostridium difficile in the atmosphere can die quickly, and spores can survive for months in the external environment, are resistant to drying, heat and various disinfectants, and are the only anaerobic bacteria which can cause nosocomial infection.
Clostridium difficile is a strict intestinal anaerobe, a major causative factor responsible for hospital diarrhea, antibiotic-associated diarrhea, colon and pseudomembranous enteritis, and is associated with the toxic product A, B toxin produced thereby. Clostridium difficile is not a resident bacterium in the intestinal tract, with adult bacterial rates of only 5% and neonates and infants up to 90%. Research shows that the high risk factor of clostridium difficile infection and the diseases caused by the clostridium difficile are common in clinic. Clinically, the content of clostridium difficile in the intestinal faeces of the children is detected, the correlation between the clostridium difficile and the diseases is clear, and experimental data and clinical basis are provided for preventing and treating the related diseases caused by the clostridium difficile.
In recent years, clostridium difficile has an accelerated spreading trend in europe and america, and is valued locally, but has not been popular in large areas in China, but is paid attention to. The detection method of clostridium difficile generally comprises anaerobic culture method, enzyme immune method, traditional PCR, tissue culture cytotoxin test and the like.
The anaerobic culture method needs expensive anaerobic culture equipment, has strict inoculation requirements, and is difficult to popularize and develop; although the enzyme immune method is convenient and quick, the enzyme immune method lacks sensitivity and has lower positive detection rate; after the traditional PCR amplification is finished, complicated steps such as electrophoresis, development and the like are needed to be carried out on the product; although the tissue culture cytotoxin assay is considered a gold standard for detection of clostridium difficile from faeces, it requires at least a incubation period of 24 to 48 hours and only toxin B can be detected. Thus, there is a need for a simple, rapid, specific, comprehensive assay for early diagnosis of clostridium difficile infection.
In recent years, molecular biology methods have been continuously applied to rapid detection of digestive tract pathogens, and laboratory diagnosis methods of digestive tract pathogens have been greatly advanced, and nucleic acid detection has become a development direction of laboratory diagnosis of digestive tract pathogens. Compared with the traditional laboratory detection method, the molecular diagnosis technology has incomparable detection speed, specificity and sensitivity, and has become a new standard of laboratory diagnosis.
The loop-mediated isothermal amplification method has the advantages of high detection sensitivity, visual result judgment, no need of expensive equipment such as a fluorescent quantitative PCR instrument, and the like, and has wide application prospect.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification technology, has the advantages of rapidness, simplicity, economy, sensitivity and the like, and is widely applied to the field of rapid nucleic acid detection. The LAMP principle is to design 3 pairs of primers (F3, B3, FIP [ F1c+F2], BIP [ B2+B1c ], LF and LB) aiming at 6 regions on a target gene, and perform nucleic acid amplification under isothermal conditions in a short time (30 min) under the action of strand displacement DNA polymerase. The amplified product and the nucleic acid dye are combined to emit fluorescent signals, the fluorescent signals can be captured by an instrument and processed and analyzed to form an amplification curve, and the detection result is judged according to the Tt value. Compared with the traditional PCR, the LAMP has the characteristics of simple operation, high sensitivity, strong specificity, simple result judgment, low cost and the like. LAMP detection sensitivity is at least 2 orders of magnitude higher than that of ordinary PCR. In addition, isothermal amplification only needs a isothermal amplification and fluorescence detection device, the requirement on equipment is simple, the operation process is short, and the isothermal amplification can be completed within 1 hour. The detection of the LAMP reaction result does not need gel electrophoresis like ordinary PCR, and only needs to observe turbid products by naked eyes or detect fluorescent curves by an instrument, thereby being an efficient, simple, convenient and quick detection method.
Therefore, the development of the loop-mediated isothermal amplification kit for clostridium difficile has important social significance for rapidly locking disease infectious sources, accurately diagnosing and taking medicines and preventing the spread of input epidemic diseases.
Disclosure of Invention
The invention aims at: providing a method for clostridium difficile nucleic acid detection; another object is to provide a kit for use in the method.
1. LAMP detection special primer for clostridium difficile
According to the clostridium difficile genome specific conserved sequence, the online primer design software Primer Explorer V is applied, and after uploading the target sequence, a plurality of groups of primer sequences can be obtained preliminarily.
The LAMP primer is obtained by screening the LAMP primer mainly comprising the stability of the terminal ends of the primer, the GC content, the distance between the primers and the secondary structure according to the key factors of the LAMP primer design. Specifically, in order to make F1c and B1c more easily bent during the reaction, a double stem-loop structure can be formed, and the primers F1c and B1c should have a Tm value of about 5℃higher than those of the other primers. To improve the annealing efficiency of the nucleotides with the template, the free energy delta G value of the six bases at the extreme end of each primer is less than or equal to-4 Kcal/mol, the delta G value of the 3 '-end of F3/B3, F2/B2 and LF/LB is less than or equal to-4 Kcal/mol, and the delta G value of the 5' -end of F1c and B1c is less than or equal to-4 Kcal/mol. The GC content of the primer is set to be 40% -60%. For the distance between the primers, the distance from the 5 '-end of F2 to the 5' -end of B2 is 120-180 bp, the distance from the 5 '-end of F2 to the 3' -end of F1c, namely the stem-loop segment is 40-60 bp, and the distance from the 5 '-end of F2 to the 3' -end of F3 is 0-20 bp. Finally, special attention must be paid to the inability to form secondary structures between the primers. The final primers were obtained by screening according to the above design principle as follows.
F3: 5’-GTGGGGAGCAAACAGGATT-3’；
B3: 5’-TCTTCGCGTTGCTTCGAATT-3’；
FIP: 5’-CGTTAGCTGCGGCACCGAAGAGTCCACGCTGTAAACGATG-3’；
BIP: 5’-CCTGGGAAGTACGCTCGCAAACATGCTCCGCTACTTGTG-3’；
LF: 5’-GGTAACCCCCGACACCTAGTAC-3’；
LB: 5’-CTCAAAGGAATTGACGGGGAC-3’。
2. LAMP detection method for clostridium difficile
The LAMP detection method of clostridium difficile provided by the invention mainly comprises the following steps:
[1] nucleic acid extraction: the nucleic acid extraction reagent of the magnetic bead method produced by Shanghai Co.Ltd is used for extracting the nucleic acid of the sample to be detected.
[2] LAMP amplification
The extracted nucleic acid to be detected is used as a template and amplified under the mediation of a special primer. Wherein the LAMP reaction system (30. Mu.L) comprises: genomic nucleic acid of the sample 12. Mu.L, 20mM Tris-HCl (pH 8.8), 10mM KCl,10mM MgSO 4 ，10mM (NH 4 ) 2 SO 4 0.1% Tween20,1M Betaine,1 XSYBR GREEN,1.5mM dNTP each,9U Bst DNA polymerase, primer amounts were: 6pmol F3 and B3, 48pmol FIP and BIP,12pmol LF and LB. The reaction tube is placed at a constant temperature of 54-68 ℃ for 15-45 min, preferably 63 ℃ for 30min.
[3] Result determination
1) Visual observation of
After the reaction was completed, whether the product was cloudy or not was visually observed. Compared with a negative control, a sample with obvious turbidity is positive, and a positive product is centrifuged to form a precipitate at the bottom of the test tube; if there is no turbidity, it is a negative specimen.
2) Fluorescence detection
And detecting and processing and analyzing fluorescent signals emitted after the amplified products are combined with the nucleic acid dye by using a constant-temperature nucleic acid amplification analyzer to form an amplification curve, and judging a detection result according to the Tt value.
3. LAMP detection kit for clostridium difficile
The LAMP detection kit for clostridium difficile provided by the invention comprises the special primer, the main reagent and the reaction parameters for LAMP detection, and has the following advantages:
[1] high specificity
The recognition of 6 specific regions of the target sequence by the 6 primers ensures the high specificity of LAMP amplification, namely LAMP can search out the corresponding target sequence from a gene specimen differing by only one nucleotide for amplification;
[2] efficient amplification
Sensitivity was about 100-fold higher than that of the conventional PCR;
[3] easy and convenient to operate
The result can be judged only by placing the sample to be detected (target nucleic acid) and the detection reagent in a constant-temperature water bath kettle at about 63 ℃ for about 30 min;
[4] visual result
The results can be detected by visual inspection or by a isothermal nucleic acid amplification analyzer. The method can simply, conveniently and rapidly (the reaction is carried out for 30min at the constant temperature of 63 ℃) under the isothermal condition, and can efficiently and specifically detect clostridium difficile. The method does not need complex instruments, provides a new technical platform for clostridium difficile detection, is particularly suitable for crowd screening, has wide market prospect and large economic and social benefits, and is suitable for large-scale popularization and application.
Detailed Description
The present invention will now be described with reference to specific embodiments and specific operation procedures on the premise of the technical proposal of the present invention, but the scope of the present invention is not limited to the following examples. The methods used in the examples described below are conventional methods unless otherwise specified.
Example 1 primer design for LAMP detection of Clostridium difficile
And (3) obtaining a clostridium difficile genome specificity conserved sequence by utilizing NCBI database retrieval, and preliminarily obtaining a plurality of groups of primer sequences after uploading target sequences by applying on-line primer design software PrimerExplorer V5. The LAMP primer is obtained by screening the LAMP primer mainly comprising the stability of the terminal ends of the primer, the GC content, the distance between the primers and the secondary structure according to the key factors of the LAMP primer design.
Example 2 establishment of LAMP detection method for Clostridium difficile
The primers obtained in example 1 for LAMP detection of Clostridium difficile were used to carry out LAMP detection of nucleic acid extract from nasopharyngeal swab samples, and the specific procedures were as follows:
[1] reaction system
Nucleic acid extraction was performed on the sample to be tested using a magnetic bead method nucleic acid extraction reagent (nucleic acid extraction and purification reagent) produced by samsung diagnostics technologies (Shanghai), and isothermal amplification was performed under the guidance of the LAMP-specific primers obtained in example 1, using the extracted product as a template. Wherein, LAMP reverseThe reaction system (30. Mu.L) comprises: genomic nucleic acid of the sample 12. Mu.L, 20mM Tris-HCl (pH 8.8), 10mM KCl,10mM MgSO 4 ，10mM (NH 4 ) 2 SO 4 0.1% Tween20,1M Betaine,1 XSYBR GREEN,1.5mM dNTP each,9U Bst DNA polymerase, primer amounts were: 6pmol F3 and B3, 48pmol FIP and BIP,12pmol LF and LB.
[2] Result determination
And detecting a fluorescence curve by using a constant temperature amplification instrument, wherein the fluorescence curve indicates that clostridium difficile exists in the sample to be detected (positive), and the non-amplification curve indicates that clostridium difficile exists in the sample to be detected (negative).
Example 3 LAMP detection kit for preparing Clostridium difficile
The LAMP reaction mixture, i.e., LAMP reaction primers (1.2 uM F3 and B3;9.6uM FIP and BIP;2.4uM LF and LB), LAMP reaction enzyme (3U/. Mu. L Bst DNA polymerase), reaction buffer (60 mM Tris. HCl (pH 8.8); 30mM KCl;30mM MgSO) 4 ；30mM (NH 4 ) 2 SO 4 The method comprises the steps of carrying out a first treatment on the surface of the 0.3% Tween20;3M Betaine;4.5mM dNTP each, 3 XSYBR Green), negative control and positive control (plasmid dilution containing Clostridium difficile genome specific conserved sequence) were packaged together to obtain a LAMP detection kit for Clostridium difficile.
Claims (4)
1. Loop-mediated isothermal amplification (Loop-Mediated Isothermal Amplification, LAMP) detection target for clostridium difficile (Clostridium difficile, CD): the special primer for rapidly detecting clostridium difficile is designed according to a specific conserved target sequence of a clostridium difficile genome and is used for qualitatively detecting clostridium difficile in a sample to be detected; each set of primers contained 6 oligonucleotide sequences, namely 2 outer primers (F3, B3), 2 inner primers (FIP, BIP) and 2 loop primers (LF, LB).
2. The LAMP detection method of clostridium difficile mainly comprises the following steps: performing LAMP amplification under the guidance of the special primer according to claim 1 by using genomic nucleic acid of an object to be detected as a template; the amplified product and the nucleic acid dye are combined to emit fluorescent signals, the fluorescent signals can be captured by an instrument and processed and analyzed to form an amplification curve, and the detection result is judged according to the Tt value.
3. The detection method according to claim 2, wherein: nucleic acid extraction is carried out on a sample to be detected by using a magnetic bead method nucleic acid extraction reagent (a nucleic acid extraction and purification reagent) produced by Shanghai Corp of complex star diagnosis technology; the LAMP reaction system comprises: genomic nucleic acid of the sample 12. Mu.L, 20mM Tris-HCl (pH 8.8), 10mM KCl,10mM MgSO 4 ，10mM (NH 4 ) 2 SO 4 0.1% Tween20,1M Betaine,1 XSYBR GREEN,1.5mM dNTP each,9U Bst DNA polymerase, primer amounts were: 6pmol F3 and B3, 48pmol FIP and BIP,12pmol LF and LB.
4. The detection method according to claim 2, characterized in that the LAMP amplification conditions are: the reaction tube is placed at a constant temperature of 54-68 ℃ for 15-45 min, preferably 63 ℃ for 30min.
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