CN115961060A - Primer probe group, kit, method and application for isothermal amplification detection of mycobacterium tuberculosis based on nanogold - Google Patents
Primer probe group, kit, method and application for isothermal amplification detection of mycobacterium tuberculosis based on nanogold Download PDFInfo
- Publication number
- CN115961060A CN115961060A CN202211008195.5A CN202211008195A CN115961060A CN 115961060 A CN115961060 A CN 115961060A CN 202211008195 A CN202211008195 A CN 202211008195A CN 115961060 A CN115961060 A CN 115961060A
- Authority
- CN
- China
- Prior art keywords
- mycobacterium tuberculosis
- probe
- amplification
- seq
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of biological detection, in particular to a primer probe group, a kit, a method and application for isothermal amplification detection of mycobacterium tuberculosis based on nanogold. The primer probe group comprises an outer primer pair F3 and B3, an inner primer pair FIP and BIP, a loop primer pair LF and LB and a gold nano particle probe; the gold nanoparticle probe is a probe molecule which is obtained by modifying a section of antisense oligonucleotide chain complementary with an amplification target on gold nanoparticles with a certain particle size. The method has more visual detection result and lower price, and can meet the requirement of rapid, high-sensitivity and visual detection of the mycobacterium tuberculosis on site in relatively laggard basic regions and underdeveloped countries.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer probe group, a kit, a method and application for isothermal amplification detection of mycobacterium tuberculosis based on nanogold.
Background
Delayed diagnosis of tuberculosis and drug-resistant tuberculosis are great threats to global control of tuberculosis diseases, and early diagnosis of active tuberculosis, especially detection of mycobacterium tuberculosis, is a necessary prerequisite for effective treatment and public health intervention [1] 。
At present, laboratory diagnosis mainly depends on smear staphyloscopy, mycobacterium tuberculosis culture and other traditional laboratory test methods. The smear staining microscopy method has the defects of low positive rate, complex operation, high technical requirement and the like, and the culture of the mycobacterium tuberculosis also has the defects of high pollution rate, long detection time and the like, so that nearly half of tuberculosis cases in the world cannot be confirmed by laboratories, and the diagnosis is delayed [1] . Therefore, in the world-wide struggle with tuberculosis, finding a simple, fast and efficient diagnostic method remains a critical issue. The PCR method based on molecular technology solves the problems to a certain extent [2，3] Although this method has improved sensitivity and specificity, the whole detection process still requires 5-6 hours, and the method requires a skilled technician, a specific experimental area, and precise temperature control. In addition, methods such as nested PCR and real-time PCR have been applied to rapid detection of Mycobacterium tuberculosis [4，5] Although the sensitivity is improved, the detection process is complex, expensive instruments and equipment are required, and the requirements of simplicity and rapidness cannot be met.
In 2006, baptista et al applied gold nanoparticles for the first time to detect Mycobacterium tuberculosis. The assay involves exploring the differential stability of gold nanoparticle probes in the presence of different DNA targets. The gold nanoparticle probe is combined with the complementary DNA target spot, so that the aggregation of the gold nanoparticles is hindered, the solution keeps red or pink, and the missing DNA target spot or non-complementary/mismatched solution does not hinder the aggregation of the gold nanoparticle probe, so that the visible color is changed from red to blue or purple. The method uses oligonucleotide functionalized gold nanoparticle probe with sulfhydryl modification for clinical samplesThe identification of the mycobacterium tuberculosis in the method and the method for carrying out targeted amplification by combining PCR show high sensitivity. However, PCR analysis requires expertise and highly complex environment, and is too expensive to diagnose [6] . These methods cannot combine the convenience of use and economy at the same time, and thus cannot fully meet the clinical requirements of tuberculosis laboratory diagnosis.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal amplification technique for nucleic acids [7] The principle is as follows: 4 primers are designed aiming at four regions on a target gene, and the other two primers, namely 'loop primers', are used for accelerating isothermal amplification reaction, under the action of strand displacement type DNA polymerase and under the constant temperature condition of 60-65 ℃, a large amount of target genes can be synthesized within 15-60 min, so that the reaction speed is greatly improved. The LAMP technology can only react under the condition that four specific independent regions are simultaneously recognized, so that the method has high specificity. In addition, the whole reaction is carried out under the constant temperature condition, the detection can be carried out without complex instruments and equipment, and the reaction is finished within 1 hour, so that the experimental time is greatly saved. Therefore, the LAMP method has played a great role in the detection of various bacteria [8，9] . At present, when the loop-mediated isothermal amplification technology is used for detecting mycobacterium tuberculosis, a real-time fluorescence quantitative PCR instrument is required to read a fluorescence signal, and the real-time fluorescence quantitative PCR instrument is expensive and needs to be operated by a professional, so that manpower and material resources are consumed.
The colloidal gold method for detecting the mycobacterium tuberculosis is used for detecting anti-mycobacterium tuberculosis IgG and IgM antibodies in human serum, is also called nano gold, has negative charges in a weak alkali environment, can form firm combination with positive charge groups of protein molecules, and does not influence the biological characteristics of the protein because the combination is electrostatic combination. Colloidal gold labeling is essentially a coating process in which a polymer such as a protein is adsorbed on the surface of colloidal gold particles. The adsorption mechanism is that the negative charges on the surface of the colloidal gold particles are firmly combined with the positive charge groups of the protein due to electrostatic adsorption. The immuno-gold labeling technique mainly utilizes the characteristic of gold particles with high electron density, and when the labels are aggregated in a large amount at corresponding ligands, red or pink spots can be seen by naked eyes, so that the immuno-gold labeling technique is used in a qualitative rapid immunoassay method. The colloidal gold method is used for detecting the anti-mycobacterium tuberculosis IgG and IgM antibodies in human serum, has low sensitivity, is difficult to detect and easy to miss detection when the content of the anti-mycobacterium tuberculosis IgG and IgM antibodies in the human serum is low, and cannot detect early infection.
Early diagnosis of patients in primary tuberculosis prevention and treatment is a key link of prevention and treatment, and accurate, rapid and simple experimental detection technology is needed to play a role in early finding and accurately diagnosing tuberculosis, particularly widely finding infectious pulmonary tuberculosis patients [10] . Therefore, the research is based on the nanogold combined with the loop-mediated isothermal amplification method, and a method for detecting the mycobacterium tuberculosis with high sensitivity, visualization and rapidness is developed by utilizing the advantages of the nanogold combined with the loop-mediated isothermal amplification method, and can be used for basic community investigation and detection.
Reference documents
[1] Molecular diagnostic progress of mycobacterium tuberculosis [ J ] western medicine, 2019,34 (08): 839-843.
[2]Deshpande,P.S.,et al.Evaluation of the IS6110 PCR assay for therapid diagnosis of tuberculous meningitis.Cerebrospinal Fluid Res.2007,4:10。
[3]Nagdev,K.J.,et al.Comparative evaluation of a PCR assay with an in-house ELISA method for diagnosis of tuberculous meningitis.Med.Sci.Monit.2010,16:CR289–CR295。
[4]Takahashi,T.,et al.Nested polymerase chain reaction for assessing the clinical course of tuberculous meningitis.Neurology.2005,64:1789–1793。
[5]Takahashi,T.,et al.Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA:clinical application for diagnosis of tuberculous meningitis.J.Clin.Microbiol.2008,46:1698–1707。
[6]P.V.Baptista,M.Koziol-Montewka,J.Paluch-Oles,G.Doria,R.Franco,Goldnanoparticle-probe-based assay for rapid and direct detection of Mycobacterium tuberculosis DNA in clinical samples,Clinical Chemistry,Vol.52,2006,pp.1433-1434。
[7]Notomi,T.,H.Okayama,H.Masubuchi,T.Yonekawa,K.Watanabe,N.Amino,and T.Hase.Loop-mediated isothermal amplification of DNA.Nucleic Acids Res.2000,28:E63。
[8]Kuboki,N.,Inoue,N.,Sakurai,T.,Cello,F.D.,Grab,D.J.,Suzuki,H.,Sugimoto,C.&Igarashi,I.Loop-mediated isothermal amplification for detection of African trypanosomes.J Clin Microbiol.2003,41:5517–5524。
[9]Parida,M.,Inoue,G.P.S.,Hasebe,F.&Morita,K.Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus.J Clin Microbiol.2004,42:257–263。
[10] Zhang hui, lihui, wang Yu, ma birch, huang Xiao Ying.2 nucleic acid amplification fluorescence methods are used for detecting mycobacterium tuberculosis in clinical sputum specimens [ J ]. China journal of sanitary inspection, 2022,32 (03): 301-303.
Disclosure of Invention
The invention provides a primer probe set, a kit, a method and application for detecting mycobacterium tuberculosis by isothermal amplification based on nanogold, the detection result is more visual, the price is lower, the on-site rapid, high-sensitivity and visual detection of mycobacterium tuberculosis in relatively laggard basic regions and under-developed countries can be met, and the problems in the prior art are solved.
One of the technical schemes adopted by the invention is as follows:
a primer probe group for detecting mycobacterium tuberculosis through isothermal amplification based on nanogold comprises an outer primer pair F3 and B3, an inner primer pair FIP and BIP, a loop primer pair LF and LB and a gold nanoparticle probe;
the sequences of the outer primer pair F3 and B3 are shown as SEQ ID No.1 and SEQ ID No. 2;
the sequences of the inner primer pair FIP and BIP are shown as SEQ ID No.3 and SEQ ID No. 4;
the sequences of the loop primer pair LF and LB are shown in SEQ ID No.5 and SEQ ID No. 6;
the gold nanoparticle probe is a probe molecule with a section of antisense oligonucleotide chain which is complementary with an amplification target and is modified on a gold nanoparticle with a certain particle size.
Furthermore, the size of the gold nanoparticles used by the gold nanoparticle probe is 20nm, and the probe sequence is 5'-SH-TCTGTCCGGGACCACT-3'.
Further, the preparation of the gold nanoparticle probe comprises the following steps:
(1) Preparing gold nanoparticles with the diameter of 20nm into a gold nanoparticle solution for later use;
(2) The probe sequence is as follows: 5'-SH-TCTGTCCGGGACCACT-3' as shown in SEQ ID No. 7;
(3) Synthesis of gold nanoparticle probes
And (3) incubating the sulfhydryl-modified oligonucleotide chain obtained in the step (2) and the gold nanoparticle solution obtained in the step (1), and according to the amount concentration of the substance SH-DNA: and (4) mixing and modifying AuNPs = 150.
Further, the operation of preparing the gold nanoparticle solution in the step (1) is as follows:
adding clean stirring magnetons and 142.5mL of ultrapure water into a three-neck round-bottom flask, adding 1.5mL of 1% chloroauric acid, and simultaneously heating to slightly boil; then, 6mL of 1% sodium citrate is quickly added, stirring is continuously carried out, slight boiling is kept, the solution is changed into blue immediately, heating and stirring are continuously carried out, the solution is changed into purple from blue, finally, the solution is changed into red, after the solution is stable and does not change any more, heating and stirring are continuously carried out for 10min, the solution is cooled to room temperature, the maximum absorption peak is measured at 520nM, the calculated concentration is 1nM, and the solution is transferred to a centrifugal tube and is wrapped by tin foil paper to be protected from light and stored at 4 ℃.
The second technical scheme adopted by the invention is as follows:
a kit for detecting mycobacterium tuberculosis based on nanogold isothermal amplification comprises the primer probe group.
Further, the kit for detecting mycobacterium tuberculosis through isothermal amplification based on nanogold also comprises Buffer, dNTP and MgSO 4 BstDNA polymerase.
The third technical scheme adopted by the invention is as follows:
a method for detecting mycobacterium tuberculosis by isothermal amplification based on nanogold comprises the following operation steps:
(1) Construction of an amplification System
An amplification system comprising the primer probe set of claim 1 or 2, and Buffer, dNTP, mgSO 4 BstDNA polymerase, water and template;
(2) Placing the amplification system in a thermostat water bath at 65 ℃, reacting for 40min, taking out, adding a certain volume of 6M/L NaCl solution, and observing color change within 10 min; the positive result is pink, and the negative result is transparent.
Further, in the step (2), the amounts of the amplification system and the NaCl solution used were 25. Mu.L each.
The fourth technical scheme adopted by the invention is as follows:
the primer probe set or the kit is applied to the detection of the mycobacterium tuberculosis.
Further, the detection sensitivity for detecting Mycobacterium tuberculosis was 5 copy/. Mu.L.
According to the technical scheme, different color changes can be generated by combining the aggregation and dispersion states of the nanogold, so that the principle of detecting various pathogenic bacteria by naked eye colorimetry is realized, firstly, a novel nucleic acid isothermal amplification technology, namely a loop-mediated isothermal amplification technology, is utilized, 4 primers are designed aiming at four regions on a target gene, the other two primers, namely loop primers, are used for accelerating isothermal amplification reaction, under the action of strand displacement type DNA polymerase and under the constant temperature condition of 65 ℃, a large amount of target genes can be synthesized within 40min, and the efficiency is extremely high. Therefore, aiming at the mycobacterium tuberculosis IS6110 gene sequence, the accession number in NCBI repository IS X52471.1, the nucleotide sequence for designing the primer IS selected to be 1 to 1000, the 1000 nucleotide bases are added into PrimerExplorer V5 software, 3 pairs of primers suitable for the current loop-mediated isothermal amplification system are designed, and the amplification system IS constructed to amplify the detection signal. And then, modifying a section of antisense oligonucleotide chain which is complementary with the amplification target on the gold nanoparticles, and detecting the antisense oligonucleotide chain as a probe molecule. Adding the gold nanoparticle probe into an amplification system for reaction, and adding excessive salt after amplification is finished. In a system without LAMP amplification products, because a large amount of salt neutralizes all negative charges on the modified antisense oligonucleotide chain and the nano-gold, a large amount of gold nano-particles losing electrostatic repulsive force are aggregated, and the color of the aggregated gold nano-particles is changed into blue gray. In a system with LAMP amplification products, the nano gold particles can be aggregated under high salt concentration, but the gold nanoparticle probe can be combined with a target, and the LAMP amplification products are mixed products of DNA with different numbers of stem loops, so that a complex network structure can be formed, a steric hindrance effect exists, aggregation of the gold nanoparticles is hindered, and the gold nanoparticles are still in a dispersed state and are pink. Therefore, the positive result is pink, and the negative result is transparent.
The invention has the beneficial effects that:
(1) The detection accuracy of the mycobacterium tuberculosis is high, and the specificity is strong: the invention carries out target screening and primer design aiming at the mycobacterium tuberculosis IS6110 gene, has the advantages of high accuracy and strong specificity, has the sensitivity of 5 copy/mu L, and can realize accurate detection of the mycobacterium tuberculosis.
(2) And (3) rapid detection: the detection method of the invention has the advantages that the amplification is carried out at the constant temperature of 65 ℃ without temperature rise and fall and circulation, so the required time is greatly shortened, the amplification only needs 40min, the detection only needs 10min, and the total time is 50min.
(3) And (4) visualizing the result: the addition of the gold nanoparticle probe can realize the visual diagnosis of the detection result.
(4) The equipment is simple, and the cost is low: the nucleic acid amplification process can be completed by only one constant-temperature water bath, and the detection cost is greatly reduced.
The constructed naked-eye colorimetric method for detecting the mycobacterium tuberculosis by combining nanogold with loop-mediated isothermal amplification has the advantages that the detection result is more visual, the price is lower, the detection is quick, the on-site quick, high-sensitivity and visual detection of the mycobacterium tuberculosis in relatively laggard basic regions and under-developed countries can be met, the important function is played in the clinical diagnosis of the mycobacterium tuberculosis, the early diagnosis, the early blocking and the early treatment are facilitated, the medical resource burden caused by the diagnosis of the mycobacterium tuberculosis is reduced, and the application prospect is wide.
Drawings
FIG. 1 is a schematic diagram of the experimental principle of the present invention;
FIG. 2 is a schematic diagram showing the amplification effect of the first set of primers screened according to the present invention;
FIG. 3 is a schematic diagram illustrating the amplification effect of the second set of primers screened according to the present invention;
FIG. 4 is a schematic diagram showing the amplification effect of the third set of primers screened according to the present invention;
FIG. 5 is a schematic diagram of the color rendering effect of the present invention.
Detailed Description
In order to clearly explain the technical features of the present invention, the present invention will be explained in detail by the following embodiments with reference to the attached drawings.
By taking IS6110 gene specific to Mycobacterium tuberculosis as a target sequence (the sequence information IS shown in the following table 1), because the length of an amplification sequence cannot be too long and cannot exceed 300bp, partial fragments of the IS6110 gene are selected and amplified finally in the experiment, and the length IS 199bp (the specific sequence IS the underlined part).
TABLE 1
1. Primer design
Because the GC content of the target sequence area is rich, the GC content of the primer is controlled to be 50-60 percent, the Tm value of the primer is controlled to be 60-65 ℃, and the optimal primer size and the distance between primer binding regions are selected; the Tm values of F2 and B2 are slightly higher for F1c and B1c so as to form a ring structure immediately upon release of the single-stranded template; in order to improve the stability of the primer, besides avoiding the occurrence of secondary structures, the dG content in 6 bases at the 5 'end of F1c (or B1 c) and the 3' end of F2 and F3 (or B2 and B3) is made to be less than-4 Kcal/mol.
Three sets of primers are obtained by screening according to the design principle, and the primer information is as follows:
sequence information of the first set of primers:
F3：5’-CGCCGCCAACTACGGT-3’
B3：5’-CGGCGCTGGACGAGAT-3’
FIP：5’-CATCTGGCCACCTCGATGCttttGGTGCCCGCAAAGTG-3’
BIP：5’-CGGCTGATGACCAAACTCGGttttGCTGTGGCCGGATCA-3’
LF：5’-TCACGGTTCAGGGTTAGCC-3’
LB：5’-CAAAGCCCGCAGGACCA-3’
second set of primer sequence information:
F3：5’-GCCCCGATGGTTTGCG-3’
B3：5’-GGGTTAGCCACACTTTGCG-3’
FIP：5’-TAGGTCGATGGGGCGATCGGttttGGGTGTCGAGTCGATCTGC-3’
BIP：5’-GCGCGATGGCGAACTCAAGGttttGGCACCGTAAACACCGTAG-3’
LF：5’-CAGCTCGGTCAGCTGTGT-3’
LB：5’-ACATCAGCCGCGTCCA-3’
sequence information of the third set of primers:
F3：5’-TCCACGCCGCCAACT-3’(SEQ ID No.1)
B3：5’-CGGCGCTGGACGAGAT-3’(SEQ ID No.2)
FIP：5’-GCATCTGGCCACCTCGATGCCttttCGGTGTTTACGGTGCCCG-3’(SEQ ID No.3)
BIP：5’-ACGGCTGATGACCAAACTCGGCttttGGCTGTGGCCGGATCA-3’(SEQ ID No.4)
LF：5’-CGGTTCAGGGTTAGCCACACTTTG-3’(SEQ ID No.5)
LB：5’-AAAGCCCGCAGGACCA-3’(SEQ ID No.6)
the following table shows an amplification system, the amplification effects of three sets of primers are screened by using ABI7500, and the set of primers with the best amplification effect is selected for subsequent color development reaction by checking an amplification fluorescence curve.
The amplification results of the first two sets of primers have false positive, and the sensitivity is relatively low. FIG. 2 is a schematic diagram showing the amplification effect of the first set of primers, and it can be seen from the graph that the peak is early and 10 3 copy/mL can not be detected completely, the sensitivity is lower, and the negative control group also has a peak indicating that false positive is generated; FIG. 3 is a schematic diagram showing the amplification effect of the second set of primers, 10 3 copy/mL can not be detected completely, the sensitivity is low, and false positive is generated; as shown in FIG. 4, the amplification effect of the third set of primers is shown schematically, and it is clear that the peak formation is early and 10 can be detected by using the third set of primers 3 The plasmid of copy/mL has higher sensitivity and no false positive phenomenon.
After the third set of primers is optimized, 1copy/uL plasmid can be detected, the sensitivity is high, and false positive is not generated.
2. Preparing gold nano particle solution, designing oligonucleotide chain probe sequence, and synthesizing gold nano particle probe
2.1 preparing gold nanoparticle solution:
the size of the gold nano-particle used in the invention is 20nm, and the preparation process is as follows:
adding clean stirring magnetons and 142.5mL of ultrapure water into a three-neck round-bottom flask, adding 1.5mL of 1% chloroauric acid, and simultaneously heating to slightly boil; then, adding 6mL of 1% sodium citrate rapidly, continuing to stir and keep slight boiling, changing the solution color to blue immediately, continuing to heat and stir, changing the solution color from blue to purple, finally changing to red, continuing to heat and stir for 10min after the solution color is stable and does not change any more, continuing to stir, cooling the solution to room temperature, transferring the solution to a centrifugal tube, wrapping the solution with tin foil paper in the dark, and storing at 4 ℃.
2.2 Probe sequence selection
Firstly, the probe sequence must avoid overlapping or complementary with the primer sequence so as to avoid influencing the amplification efficiency of the primer, the length is between 15 and 20bp, the GC percent is between 40 and 60 percent, the Tm value is between 50 and 60 ℃, and the probe sequence is designed according to the consideration: 5 '-SH-TCTGTCCGGGACCAC-3' (SEQ ID No. 7).
2.3 Synthesis of gold nanoparticle Probe
The thiol-modified oligonucleotide chain obtained in 2.2 was incubated with the gold nanoparticle solution prepared in 2.1, at an amount concentration of SH-DNA: auNPs =150, 1, modifying the thiol-modified oligonucleotide chain onto the gold nanoparticle, thereby preparing the gold nanoparticle probe, which can be used for subsequent amplification and color development experiments.
The specific steps of the synthesis of the gold nanoparticle probe are as follows:
a. firstly, SH-DNA is reduced, 1 mu L of TCEP (0.5M) is mixed with 50 mu L of LSH-DNA (the theoretical concentration provided by the company is 100 mu M), and the mixture is incubated for 6h at room temperature;
b. mu.L of 3M CH 3 Adding COONa into the mixed solution containing TCEP DNA, adding 200 μ L of glacial ethanol, incubating at-80 deg.C for 1h, centrifuging at 10 deg.C for 35min at 12000rpm, and removing supernatant; then, adding 500 μ L of glacial ethanol, vortexing, centrifuging at 12000rpm at 10 deg.C for 35min, removing most of supernatant, and standing the residual liquid at room temperature to volatilize ethanol;
c. adding 50 mu L of water for redissolving and mixing evenly, taking out 1 mu L of sample and 9 mu L of DNA concentration diluted by water, and determining the concentration after reduction to be 84 mu M; the remaining sample was used for modification;
d. 1mL of AuNPs (15nm, 2.5nM) was taken and 10. Mu. LBSPP (20 mg/mL) was added; the mixture is incubated overnight at room temperature in the dark or incubated for 1h at 50 ℃;
e. centrifuging (12 000rmp,25 deg.C, 12-15 min), removing supernatant, and removing excessive BSPP and sodium citrate; adding 1mL of water to redissolve the lower-layer substance AuNPs;
f. introducing SH-DNA and AuNPs according to the proportion of 150 (substance quantity ratio) and incubating for more than 6h at room temperature;
g. gradually adding 5M NaCl to the mixture to make the solution have a final concentration of 50mM;
h. incubating the mixture overnight; the mixture was then centrifuged 2 times, the first time (12000rpm, 25 ℃,30 min), the supernatant was discarded and 1mL of water was added to redissolve; centrifuging for the second time (12000rpm, 25 deg.C, 30 min), discarding supernatant, and adding 45 μ L water for redissolving; and redispersed in water at a concentration of 16.8nM for subsequent experiments.
3. The concentrations of the components in the nucleic acid amplification and color development system are as follows:
4. adjusting the optimal concentration of color-developing salt
In 25. Mu.L of the amplification and color development system, 25. Mu.L of 6M/L NaCl solution was added to obtain the best color development effect.
The experimental steps are as follows: the amplification color development system is put into a thermostat water bath kettle at 65 ℃ for reaction for 40min, after being taken out, 25 mu L of NaCl solution at 6M/L is added, color change can be observed within 10min, and the system can detect 5 copy/mu L of plasmid at present.
As shown in FIG. 5, the left side is a positive control group, the right side is a negative control group, the positive control group is a template obtained by diluting plasmids by 10 times and 5 times respectively, and then the amplification is carried out by adopting the amplification and color development system, the amplification result shows that the positive is pink purple, the negative is transparent, and the minimum 5copy/mL of plasmids can be detected.
The above-described embodiments should not be construed as limiting the scope of the invention, and any alternative modifications or alterations to the embodiments of the present invention will be apparent to those skilled in the art.
The present invention is not described in detail, but is known to those skilled in the art.
Claims (7)
1. A primer probe group for detecting mycobacterium tuberculosis by isothermal amplification based on nanogold is characterized by comprising an outer primer pair F3 and B3, an inner primer pair FIP and BIP, a loop primer pair LF and LB and a gold nanoparticle probe;
the sequences of the outer primer pair F3 and B3 are shown as SEQ ID No.1 and SEQ ID No. 2;
the sequences of the inner primer pair FIP and BIP are shown as SEQ ID No.3 and SEQ ID No. 4;
the sequences of the loop primer pair LF and LB are shown as SEQ ID No.5 and SEQ ID No. 6;
the gold nanoparticle probe is a probe molecule with a section of antisense oligonucleotide chain which is complementary with an amplification target and is modified on a gold nanoparticle with a certain particle size.
2. The primer probe group for detecting mycobacterium tuberculosis based on isothermal amplification of nanogold according to claim 1, wherein the gold nanoparticle probe is 20nm in size of gold nanoparticles; the probe sequence is 5'-SH-TCTGTCCGGGACCACT-3', and is shown in SEQ ID No. 7.
3. A kit for detecting Mycobacterium tuberculosis based on nano-gold isothermal amplification, comprising the primer probe set of claim 1 or 2.
4. The kit for isothermal amplification detection of mycobacterium tuberculosis based on nanogold according to claim 3, further comprising Buffer, dNTP, mgSO 4 BstDNA polymerase.
5. A method for detecting mycobacterium tuberculosis by isothermal amplification based on nanogold is characterized by comprising the following operation steps:
(1) Construction of an amplification System
An amplification system comprising a primer probe set according to claim 1 or 2, and Buffer, dNTP, mgSO 4 BstDNA polymerase, water and template;
(2) Putting the amplification system into a water bath kettle with the constant temperature of 65 ℃, reacting for 40min, taking out, adding 6M/L NaCl solution with a certain volume, and observing color change within 10 min; the positive result is pink, and the negative result is transparent.
6. Use of the primer probe set of claim 1 or 2, the kit of claim 3 or 4 for the detection of mycobacterium tuberculosis.
7. The use according to claim 6, wherein the detection sensitivity for detecting Mycobacterium tuberculosis is 5 copy/. Mu.L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211008195.5A CN115961060A (en) | 2022-08-22 | 2022-08-22 | Primer probe group, kit, method and application for isothermal amplification detection of mycobacterium tuberculosis based on nanogold |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211008195.5A CN115961060A (en) | 2022-08-22 | 2022-08-22 | Primer probe group, kit, method and application for isothermal amplification detection of mycobacterium tuberculosis based on nanogold |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115961060A true CN115961060A (en) | 2023-04-14 |
Family
ID=87351635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211008195.5A Pending CN115961060A (en) | 2022-08-22 | 2022-08-22 | Primer probe group, kit, method and application for isothermal amplification detection of mycobacterium tuberculosis based on nanogold |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115961060A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5851761A (en) * | 1989-02-22 | 1998-12-22 | Cogent Limited | Probes, kits and methods for the detection and differentiation of mycobacteria |
| CN101413031A (en) * | 2008-12-02 | 2009-04-22 | 博奥生物有限公司 | Method for identifying Mycobacterium tuberculosis and non-tuberculous mycobacteria, and special reagent kit therefor |
| CN101659999A (en) * | 2009-08-04 | 2010-03-03 | 天津朝海科技有限公司 | Method for detecting product of loop-mediated isothermal amplification |
| WO2012096646A1 (en) * | 2011-01-10 | 2012-07-19 | The American University In Cairo | Direct detection of unamplified hepatitis c virus rna using unmodified gold nanoparticles |
| CN103937884A (en) * | 2014-03-28 | 2014-07-23 | 中国检验检疫科学研究院 | Loop-mediated isothermal amplification kit for detecting mycobacterium tuberculosis and application method of kit |
| CN105274193A (en) * | 2014-08-12 | 2016-01-27 | 沈阳国际旅行卫生保健中心 | Mycobacterium tuberculosis constant temperature rapid detection kit through LAMP method and detection method |
| CN112011633A (en) * | 2020-10-14 | 2020-12-01 | 河南智泰生物科技有限公司 | Primer group, kit and method for LAMP (loop-mediated isothermal amplification) simultaneous detection of mycobacterium tuberculosis complex group and rpoB gene mutation |
| CN112126695A (en) * | 2020-09-04 | 2020-12-25 | 四川大学华西医院 | Primer composition for Mycobacterium tuberculosis complex group loop-mediated isothermal amplification detection and reaction system |
-
2022
- 2022-08-22 CN CN202211008195.5A patent/CN115961060A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5851761A (en) * | 1989-02-22 | 1998-12-22 | Cogent Limited | Probes, kits and methods for the detection and differentiation of mycobacteria |
| CN101413031A (en) * | 2008-12-02 | 2009-04-22 | 博奥生物有限公司 | Method for identifying Mycobacterium tuberculosis and non-tuberculous mycobacteria, and special reagent kit therefor |
| CN101659999A (en) * | 2009-08-04 | 2010-03-03 | 天津朝海科技有限公司 | Method for detecting product of loop-mediated isothermal amplification |
| WO2012096646A1 (en) * | 2011-01-10 | 2012-07-19 | The American University In Cairo | Direct detection of unamplified hepatitis c virus rna using unmodified gold nanoparticles |
| CN103937884A (en) * | 2014-03-28 | 2014-07-23 | 中国检验检疫科学研究院 | Loop-mediated isothermal amplification kit for detecting mycobacterium tuberculosis and application method of kit |
| CN105274193A (en) * | 2014-08-12 | 2016-01-27 | 沈阳国际旅行卫生保健中心 | Mycobacterium tuberculosis constant temperature rapid detection kit through LAMP method and detection method |
| CN112126695A (en) * | 2020-09-04 | 2020-12-25 | 四川大学华西医院 | Primer composition for Mycobacterium tuberculosis complex group loop-mediated isothermal amplification detection and reaction system |
| CN112011633A (en) * | 2020-10-14 | 2020-12-01 | 河南智泰生物科技有限公司 | Primer group, kit and method for LAMP (loop-mediated isothermal amplification) simultaneous detection of mycobacterium tuberculosis complex group and rpoB gene mutation |
Non-Patent Citations (3)
| Title |
|---|
| 匡红;孙薏;张聪;府伟灵;陈庆海;冯怀志;李硕;李玉红;: "结核分支杆菌扩增体系的建立、优化及评价", 四川医学, no. 05 * |
| 朱诗应, 杨毓华, 邵海枫: "PCR检测结核分枝杆菌复合群IS_(986)DNA重复序列", 医学研究生学报, no. 03 * |
| 陈庆海;边志衡;张雪;匡红;敖琳;府伟灵;: "结核分枝杆菌高特异性MB-PCR体系优化及荧光检测研究", 中华医院感染学杂志, no. 03 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Reslova et al. | xMAP technology: applications in detection of pathogens | |
| Wang et al. | Loop-mediated isothermal amplification label-based gold nanoparticles lateral flow biosensor for detection of Enterococcus faecalis and Staphylococcus aureus | |
| CN106399517B (en) | Nucleic acid detection technology combining multi-cross constant-temperature amplification with gold nano biosensing | |
| CN109913565B (en) | Kit, primer pair, probe and method for detecting vibrio parahaemolyticus | |
| CN113718045B (en) | DNA fragment, primer, probe and kit for detecting 4 kinds of Bordetella pertussis and specifically detecting Bordetella pertussis and application | |
| CN110499393A (en) | The real-time fluorescence quantitative PCR detection of hepatitis infectiosa canis virus, hepatitis infectiosa canis virus I type and hepatitis infectiosa canis virus II type and diagnostic primers and probe | |
| CN111549184A (en) | PCR fluorescence detection kit for respiratory adenovirus and application thereof | |
| Zhou et al. | Rapid detection of monkeypox virus by multiple cross displacement amplification combined with nanoparticle‐based biosensor platform | |
| CN114540526A (en) | Primer, probe and method for typing detection of five input plasmodium | |
| Nicholls et al. | Nucleic acid analysis by sandwich hybridization | |
| CN112094930B (en) | Streptococcus pneumoniae serum typing kit and typing method | |
| Erhardt et al. | Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence | |
| Mido et al. | Sensitive detection of live Escherichia coli by bacteriophage amplification-coupled immunoassay on the Luminex® MAGPIX instrument | |
| CN116814857A (en) | Cat parvovirus and kit thereof and fluorescent recombinase polymerase amplification method | |
| CN115961060A (en) | Primer probe group, kit, method and application for isothermal amplification detection of mycobacterium tuberculosis based on nanogold | |
| CN106636474A (en) | Primer group, kit and method for detecting six mouse viruses by multiple immunity fluorescence | |
| WO2010030243A1 (en) | Gene sequences for the detection of chikungunya and dengue fever | |
| CN110042175A (en) | Primer, probe, kit and application based on RPA technology detection Bean common mosaic virus | |
| CN113265479B (en) | Primer composition for detecting rickettsia morganii and application thereof | |
| Park et al. | Naked eye detection of an amplified gene using metal particle-based DNA transport within functionalized porous interfaces | |
| CN113512607A (en) | Primer and method for fluorescence quantitative PCR detection of prawn enterobacter hepatica | |
| WO2005030027A2 (en) | Salmonella detection identification | |
| CN114085926A (en) | Primer, probe, kit and detection method for SNP site polymorphism of ABCB1 gene C3435T | |
| CN108362876B (en) | Immunomagnetic bead chromatography test strip for detecting Cronobacter sakazakii and rapid detection method | |
| CN112553379A (en) | Method and kit for detecting respiratory infectious disease virus based on liquid-phase chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |