CN110551607A - mycobacterium tuberculosis drug-resistant gene multiple detection method based on RCA amplification - Google Patents
mycobacterium tuberculosis drug-resistant gene multiple detection method based on RCA amplification Download PDFInfo
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Abstract
The invention belongs to the technical field of gene detection, and discloses a Mycobacterium tuberculosis drug-resistant gene multiple detection method based on RCA amplification, wherein a base sequence required by detection is SEQ ID NO: 1 to SEQ ID NO: 28. the paper-based microfluidic analytical device includes: the device comprises a sample pad, a colloidal gold combination pad, a detection line, a control line, an NC membrane, a water absorption pad and a bottom plate; the bottom plate is sequentially provided with a sample pad, a colloidal gold combination pad, an NC membrane and a water absorption pad from left to right, and the surface of the NC membrane is marked with a detection line and a control line. The invention generates color reaction indication detection result by hybridizing a universal sequence probe modified by nano gold particles and a single base mutation probe with a large amount of tandem repeat sequences of RCA reaction products on the surface of a solid phase. A detection platform combining isothermal amplification with a paper-based color development sensing technology is constructed, and an experimental method for rapidly identifying and detecting the drug resistance of multiple tubercle bacilli is established.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a Mycobacterium tuberculosis drug-resistant gene multiple detection method based on RCA amplification.
Background
currently, the current state of the art commonly used in the industry is such that: the global tuberculosis report issued by the World Health Organization (WHO) shows that tuberculosis is one of the first ten causes of death in the world with air droplet transmission property, is a chronic infectious disease with serious health hazard, and is one of important prevention and control infectious diseases in China and even in the world. In tuberculosis, due to the increase of population number and mobility, delay of treatment, irregular use of antituberculosis drugs and the prevalence of immunodeficiency diseases such as AIDS, mycobacterium tuberculosis drug-resistant strains appear, and the formed multidrug-resistant tuberculosis (MDRTB) and extensive drug-resistant tuberculosis (XDRTB) are the difficulties in preventing and treating tuberculosis in the world at present. Such as tuberculosis, which is resistant to the most potent antitubercular drugs Isoniazid (INH) and Rifampicin (RFP). At present, the cure rate of common tuberculosis patients is more than 85 percent, and the cure rate of multi-drug resistance is only about 50 percent. However, only about 7% of patients with multidrug-resistant tuberculosis can be diagnosed. The traditional method for detecting the mycobacterium tuberculosis and the drug resistance thereof mainly depends on comprehensive analysis of a plurality of tests such as sputum smear, culture, drug sensitivity, PCR and the like, and has slow and fussy detection, low positive rate and poor specificity; however, in the current molecular biology technology based on drug-resistant gene detection, such as direct sequencing method, microarray gene chip method, etc., the traditional detection method usually needs expensive large-scale instruments and diagnostic reagents, and may also need to be combined with a plurality of experimental methods for comprehensive analysis to obtain a detection result, and technical personnel with relevant professional knowledge are needed to operate, so that the accurate diagnosis can not be carried out in the remote areas where the emergency situation of medical personnel is not diagnosed on site or resources are deficient. The common POCT (on-site rapid detection) method, such as Tuberculosis (TB) antibody detection card, is a TB qualitative detection card, and is suitable for detecting whole blood samples. The colloidal gold labeled anti-human immunoglobulin IgG monoclonal antibody is used for detecting tuberculosis specific antigen, and common people can also use the detection card to carry out self regular monitoring. However, the sample required for detection needs fresh whole blood, and the detection effect of the sample which is old, agglutinated or taken from other parts is not clear. And the specificity is poor, and if the antibody concentration in the whole blood is too low, a negative result cannot be obtained. So the limit of resource environment and detection method is too complex, which results in low diagnosis rate of the disease. The nucleic acid detection based on isothermal amplification can obviously improve the detection sensitivity due to the advantage of amplification, can accurately detect the drug-resistant tubercle bacillus, and is used for the medication guidance of early patients so as to improve the cure rate of the patients. Therefore, the novel method for detecting the drug-resistant genotype of the tubercle bacillus is established, is used for quickly detecting the drug-resistant tubercle bacillus and has important significance for early treatment of tuberculosis and control of transmission of the drug-resistant bacillus.
In summary, the problems of the prior art are as follows: the existing molecular biology technology based on drug-resistant gene detection is expensive, high in operation requirement and long in time consumption of the traditional detection method.
The difficulty and significance for solving the technical problems are as follows:
The amplification product is combined with the paper substrate microfluidic chip, and the paper substrate chip, namely the paper detection card, is simple to manufacture, rich in source, low in cost and easy to carry out a large amount of repeated experimental research and condition optimization. Under the condition that the diagnostic value is not influenced, the establishment of a novel rapid diagnostic method has very important significance for controlling the spread of diseases and early treatment. A novel POCT paper-based micro-fluidic chip for simply, sensitively and quickly identifying drug resistance of tubercle bacillus is constructed, and isothermal nucleic acid amplification and visual color development are integrated on the micro-fluidic chip. The detection processes of amplification, paper base color development and the like of a sample to be detected under a constant temperature condition are completed within 2-4 hours, temperature circulation is not needed, and complicated instrument equipment is not needed.
Disclosure of Invention
aiming at the problems in the prior art, the invention provides a multiple detection method of mycobacterium tuberculosis drug-resistant genes based on RCA amplification.
the invention is realized in such a way that a paper microfluid chip for nucleic acid detection is based on, the paper microfluid chip for nucleic acid detection is a nano-gold probe, and the sequence of the nano-gold probe is as follows: SEQ ID NO: 1 to SEQ ID NO: 28.
It is another object of the present invention to provide a paper-based microfluidic analysis device for nucleic acid detection comprising the same, comprising: the device comprises a sample pad, a colloidal gold combined pad, a detection Line (C Line), a control Line (T Line), an NC membrane, a water absorption pad and a bottom plate;
further, the sample pad, the colloidal gold combined pad, the NC membrane and the water absorption pad are mutually overlapped for 2mm, and the whole width is 4 mm.
Further, the sample pad, the colloidal gold combined pad and the NC membrane are respectively a polyester cellulose membrane or a glass cellulose membrane, a nitrocellulose membrane, absorbent paper and a bottom plate with sticky PVC.
Further, the multiple drug-resistant mutation site detection card is shown in fig. 4 and 5. The single manufactured detection cards shown in fig. 1 are recombined to form the detection card (r) of fig. 4: the detection card consists of 6 detection strips, but only needs to be dripped once to detect a sample, 6 different nano-gold probes are respectively fixed on 6 colloidal gold combination pads, and substances fixed at other positions are the same. No. 1: and (4) performing multiplex detection. Since 4 PLPs for identifying drug-resistant mutation sites all have the same universal sequence, the amplification products also have the same DNA, and the universal probe designed according to the gene sequence can indicate whether mutations occur in a detectable range, namely whether single-base mutations of 4 drug-resistant genes exist or not, but which mutation or mutations occur is unknown. No. 2: and (5) negative control. The probes on the conjugate pad do not bind complementarily to all of the product sequences, but are captured by the complementary sequences on the control line. No. 3-6: the detection of single-base mutation of KatG315, inhA-15, rpo531 and rpsL43 can verify the detection result of position 1, determine whether the mutation really occurs, and indicate which mutation occurs.
Further, the relevant DNA sequence: 1-6, and the colloidal gold bonding pad is modified with the nucleic acid sequence shown in SEQ ID NO: 11. SEQ ID NO: 16. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15; streptavidin is fixed on all control lines; the detection lines are respectively fixed with SEQ ID NO: 23. SEQ ID NO: 28. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27.
FIG. 5 shows a detection card: the multiplex detection is concentrated on a detection card, wherein a universal probe is modified on the colloidal gold bonding pad to capture all amplification products in the detection range; the detection lines are multiple, each line is 2mm away from the other line, each line indicates a point mutation, substances fixed at the point for capturing are related to amplification reaction primers, different capture substances are fixed on the T line according to different substances modified on the primers, detection of multiple drug-resistant mutation sites can be realized, and redundant universal probes modified by nano-gold flow to the control line fixed with complementary bases; when the amplification reaction solution is detected, under the condition that the color of the C line changes, the change of the number and the position of the T line can indicate the occurrence of the drug resistance mutation.
Further, the related DNA sequence was modified with the universal sequence SEQ ID NO: 11 on a colloidal gold conjugate pad, T 1 immobilized avidin, T 2 immobilized digoxin, indicating whether KatG315, inhA-15 single base mutation occurred or not, respectively, depending on primer modification, and C-line immobilized SEQ ID NO: 23 (all base sequences were in solution when used).
The invention also aims to provide application of the paper microfluidic chip for nucleic acid detection in drug-resistant gene detection of mycobacterium tuberculosis.
In summary, the advantages and positive effects of the invention are: the microfluidic chip is a device for sample preparation, biochemical reaction, result detection and analysis on a small chip with square centimeter as a unit. The important points are that the detection efficiency is improved, the loss is reduced, the device is miniaturized and high-throughput analysis can be carried out. Because the Paper material has rich sources, low price, recyclability, easy processing and easy chemical modification, the Paper material can be used as a substrate to replace materials such as silicon, glass, high polymer and the like, and can be combined with a nanogold immunochromatography technology to prepare a Paper-based microfluidic analytical device (mu PADs) with rapid diagnosis capability. Compared with the microfluidic chip in the general sense, the microfluidic chip has the advantages of low cost, simple preparation, no need of complex peripheral equipment, capability of performing disposable, low-price and portable analysis in the true sense, has attracted more and more attention, is generally regarded as one of the development trends of field real-time diagnosis in the future, and meets the requirements of Point-of-Care Test (POCT).
The rolling circle amplification technology (RCA) is a simple and efficient isothermal amplification technology, a padlock probes (PLPs) with sequences complementary to target DNA at two ends are designed, single base mutation of a target gene is specifically recognized, linear PLP is connected into a ring under the action of E.coli DNA ligase, a primer which is designed in advance is added, amplification can be carried out under an isothermal state, amplification products are long chains with thousands of repeated sequences complementary to the PLP, so the amplification method does not need temperature circulation, has high amplification efficiency and good specificity, RCA reaction cannot be carried out if the target DNA sequence and the PLP cannot be completely complementary, wherein 5 different regions are arranged on the designed PLP, namely detection arms T 1 and T 2 at two ends of a target gene are recognized, a universal sequence S (all the sequences are identical), a restriction endonuclease site R, a primer binding region G is designed after amplification is finished, two nanogold probes are designed, one amplified with AuP sequences, the AuP probes are connected with a universal gold probe modified with a gold sequence, a probe which is connected with a target DNA molecule, a probe which can be subjected to covalent detection, and the amplified, the amplified by a probe with a target DNA molecule, and a target DNA molecule is subjected to covalent detection reaction, and a probe with a probe which can be subjected to a covalent detection reaction, and a nanometer detection molecule which can be subjected to a change in a covalent detection reaction after the surface of a nanometer detection molecule is detected, so that the amplification reaction can be detected, the nanometer probe is detected, the nanometer gold probe is detected, the amplified, the nanometer gold probe is detected, the nanometer gold probe is connected with a nanometer gold molecule, the nanometer probe is connected with a nanometer gold molecule, the nanometer.
the invention generates color reaction indication detection result by hybridizing a universal sequence probe modified by nano gold particles and a single base mutation probe with a large amount of tandem repeat sequences of RCA reaction products on the surface of a solid phase. A detection platform combining isothermal amplification with a paper-based color development sensing technology is constructed, and an experimental method for rapidly identifying and detecting the drug resistance of multiple tubercle bacilli is established. The high selectivity of the RCA ligation process enables the method to have high specificity and realize the identification of single base mutation of a target gene. The whole process including the steps of connection, amplification and paper-based detection can be completed within a few hours. All detection steps are carried out at constant temperature without complex instruments and equipment, and the problem that the temperature of PCR reaction needs to be raised and lowered repeatedly is solved.
The experiment adopts an isothermal amplification method to replace the traditional PCR and combines a colloidal gold chromatography technology to detect the drug-resistant mycobacterium tuberculosis with single base mutation.
In order to evaluate the feasibility of the method, the invention firstly designs a DNA sequence segment which is completely consistent with the target gene TB mutant gene sequence, so as to evaluate the feasibility and the specificity of the method. In the presence of the target sequence, a significant T-line was produced on the test card, whereas the blank had no detectable signal on the T-line. The method can obviously distinguish the mutant genes. To further determine the specificity of the ligation reaction, a listeria hlyA DNA fragment (Negative control) with no homology to mycobacterium tuberculosis was used as a Negative control. No 4 RCA products of the tubercle bacillus can be combined with the nano-gold probe of the control bacterium, and the T line of the negative control detection card can not generate color reaction.
drawings
FIG. 1 is a schematic diagram of a paper-based microfluidic analytical device according to an embodiment of the present invention;
in the figure: 1. a sample pad; 2. a colloidal gold bonding pad; 3. detecting lines; 4. a control line; 5. NC film; 6. a water absorbent pad; 7. a base plate.
FIG. 2 is a flow chart of a method for rapidly detecting nucleic acid by isothermal amplification according to an embodiment of the present invention.
FIG. 3 is a schematic representation of RCA amplification provided by an embodiment of the invention.
Fig. 4 is a schematic diagram of a multiple test card 1 according to an embodiment of the present invention.
Fig. 5 is a schematic diagram of the multiple detection card 2 according to the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention constructs a novel POCT paper-based microfluidic analysis device for simply, sensitively and rapidly identifying drug resistance of tubercle bacillus, and integrates isothermal nucleic acid amplification and visual color development on the paper-based microfluidic analysis device. The detection processes of amplification, paper base color development and the like of the sample to be detected under the constant temperature condition are completed within 2-4 hours, and complex instrument equipment is not needed.
the following detailed description of the principles of the invention is provided in connection with the accompanying drawings.
As shown in fig. 1, an embodiment of the present invention provides a paper-based microfluidic analysis device including: the device comprises a sample pad 1, a colloidal gold combination pad 2, a detection line 3, a control line 4, an NC membrane 5, a water absorption pad 6 and a bottom plate 7.
The bottom plate 7 is sequentially provided with a sample pad 1, a colloidal gold combination pad 2, an NC membrane 5 and a water absorption pad 6 from left to right, and the surface of the NC membrane 5 is marked with a detection line 3 and a control line 4.
The sample pad 1, the colloidal gold combined pad 2, the NC membrane 5 and the water absorption pad 6 are mutually overlapped by about 2mm, and the whole width is 4 mm;
The sample pad 1, the colloidal gold combined pad 2, the NC membrane 5 and the water absorption pad 6 are respectively a polyester cellulose membrane (or a glass cellulose membrane), a glass cellulose membrane, a nitrocellulose membrane, water absorption paper and a bottom plate 7 with sticky PVC.
as shown in fig. 2, the method for rapidly detecting nucleic acid by isothermal amplification provided by the embodiment of the present invention comprises the following steps:
S201, mixing 100nM of linear PLP (muts) phosphorylated at the 5' end with 100nM of two target sequences (mutant and wild type) in a 20. mu.L ligation system comprising (30mM Tris-HClpH8.0, 4mM MgCl 2, 10mM (NH 4) 2 SO 4, 1.2mM EDTA, 0.1mM NAD, 0.005% BSA), denaturing at 95 ℃ for 5min and cooling to 4 ℃, then incubating at 37 ℃ for 30min, adding 5U E.coli DNA ligase and 0.05% BSA to the system, and reacting at 37 ℃ for 1h to ligate the circular template;
S202, adding the ligation product obtained in the previous step into a 40-L external-contact reaction system (67mM glycine-KOH pH9.5, 6.7mM MgCl 2, 1mM DTT), adding 10U of exonuclease I and exonuclease III to remove unclyclized linear PLP and redundant target DNA, reacting at 37 ℃ for 30min, and then reacting at 90 ℃ for 5mim to inactivate the enzyme;
S203: mu.L of circularized PLPs and 2. mu.L of amplification primers were placed in RCA Reaction system and incubated at 37 ℃ for 30min containing 10 × Reaction Buffer (330mM Tris-acetate pH 7.9, 100mM Mg-acetate, 660mM K-acetate, 10mM DTT, 1% (v/v) Tween 20), 1mM dNTPs, 0.2. mu.g/. mu.LBSA, 5% DMSO. 0.5U/. mu.L of Phi29 DNA polymerase was then added and the mixture was amplified at 37 ℃ for 1h and then left at 90 ℃ for 10min to inactivate the enzyme. Adding 4 reaction systems in proportion during multiple detection;
S204: taking 5 mu L of RCA product, mixing the product with 5 mu L of enzyme digestion reaction system (5U HhaI restriction endonuclease, 33mM Tris-acetate (pH 7.9), 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/mLBSA), carrying out enzyme digestion for 1h at 37 ℃, and treating for 20min at 80 ℃ to inactivate the enzyme;
S205: preparing 1% agarose gel electrophoresis, adding 10. mu.L of RCA product (enzyme digestion product) stained by SYBR Green II, carrying out electrophoresis at a voltage of 80V for 30min, and observing and recording gel images.
the pH test paper invented by England chemist Robert & Boyle in 17 th century is the earliest application of mu PADs, and is made up by using the characteristics of litmus which can be changed into red colour when it is met with acid and can be changed into blue colour when it is met with alkali. Since then, pregnancy test strips using immunochromatography technology began to appear, and the application and development of μ PADs were pushed to the peak. Modern diagnostic medicine has used such portable detection methods on a large scale, so the experiment adopts an isothermal amplification method to replace the traditional PCR, and combines a colloidal gold chromatography technology for detecting the drug-resistant mycobacterium tuberculosis with single base mutation.
To evaluate the feasibility of this approach, a DNA sequence fragment identical to the TB mutant gene sequence of the target gene was first designed to evaluate the feasibility and specificity of the approach. In the presence of the target sequence, a significant T-line was produced on the test card, whereas the blank had no detectable signal on the T-line. The method can obviously distinguish the mutant genes. To further determine the specificity of the ligation reaction, a Listeria hlyADNA fragment (Negative control) that is not homologous to Mycobacterium tuberculosis was used as a Negative control. None of the 4 RCA products of the tubercle bacillus could be combined with the capture probe of the control bacteria, so that the T-line of the negative control detection card would produce a color reaction.
The application of the principles of the present invention will now be described in further detail with reference to the accompanying drawings.
1. four clinical common antituberculous first-line drug resistance mutation sites are screened:
Isoniazid (INH) resistant KatG315(AGC → ACC), inhA-15(ACG → ATG);
Rifampicin (RFP) resistance rpo531(TCG → TTG);
Streptomycin (SM) resistant rpsL43(AAG → AGG).
2. Designing a desired padlock probe (PLP), capture probe (capture probe), etc. according to each mutation type
SEQ ID NO：1
TGGTGATCGCGTCCTGTATACGCTTCTTCGGTGCCCATGCGCTGCGACCTCAGCATCGACCTGTCTAGACCTCGATGCCGG
SEQ ID NO：2
TCTCGCCGCGGCCGGGTATACGCTTCTTCGGTGCCCATGCGCGACCACCTTGCGATCGGGTAGTCTACCGACAACCTATCA
SEQ ID NO：3
ACAGTCGGCGCTTGGTATACGCTTCTTCGGTGCCCATGCGCCAGCGGTAGACCACCTATCGGTCTACCCAGCGCCA
SEQ ID NO：4
TCGGAGTGGTGGTGTACGTATACGCTTCTTCGGTGCCCATGCGCGACCGGTATGCGACCTGGTAGTCTAGAGTTCGGCTTCC
SEQ ID NO：5
AGGTCGATGCTGAGGTCGCA
SEQ ID NO：6
TACCCGATCGCAAGGTGGTC
SEQ ID NO：7
CGATAGGTGGTCTACCGCTG
SEQ ID NO：8
TACCAGGTCGCATACCGGTC
SEQ ID NO：9
GGATAGGACATAATAAGCGA
SEQ ID NO：10
CGCTTCTTCGGTGCCCAT
SEQ ID NO：11
CGCTTCTTCGGTGCCCAT
SEQ ID NO：12
TCCTGCGCTAGTGGTGGACGTAGCTCCAG
SEQ ID NO：13
GGCCGGCGCCGCTCTACTATCCAACAGCC
SEQ ID NO：14
GTTCGCGGCTGACAACCGCGACCC
SEQ ID NO：15
CATGTGGTGGTGAGGCTCCTTCGGCTTGAG
SEQ ID NO：16
CGTAAGTCTCCGAGGTTGCCATCGATGATTTGAACTTCATC
SEQ ID NO：17
TGGCACCGGAACCGGTAAGGACGCGATCACCAGCGGCATCGAGGTCGTATGGACGAA
SEQ ID NO：18
TGGCACCGGAACCGGTAAGGACGCGATCACCACCGGCATCGAGGTCGTATGGACGAA
SEQ ID NO：19
TACCGATTTCGGCCCGGCCGCGGCGAGATGATAGGTTGTCGGGGTGACTG
SEQ ID NO：20
TCGGGGTTGACCCACAAGCGCCGACTGTTGGCGCTGGGGCCCGGCGGTCT
SEQ ID NO：21
GCACCCGCGTGTACACCACCACTCCGAGGAAGCCGAACTCGGCGCTTCGG
SEQ ID NO：22
TGTGTAACAACATGAAGATTGTAGGTCAGAACTCACCTGTTAGAAACTGTGAAGATCGCTTATTATGTCCTATCCTCAGC
SEQ ID NO：23
GCGAAGAAGCCACGGGTA
SEQ ID NO：24
AGGACGCGATCACCACCGGCATCGAGGTC
SEQ ID NO：25
CCGGCCGCGGCGAGATGATAGGTTGTCGG
SEQ ID NO：26
CAAGCGCCGACTGTTGGCGCTGGG
SEQ ID NO：27
GTACACCACCACTCCGAGGAAGCCGAACTC
SEQ ID NO：28
GCATTCAGAGGCTCCAACGGTAGCTACTAAACTTGAAGTAC
Padlockprobes (PLP) a linear single-stranded DNA probe with a detection arm. The sequences of oligonucleotides for identifying mutations of Isoniazid (INH) resistant KatG315, inhA-15, Rifampicin (RFP) resistant rpo531 and Streptomycin (SM) rpsL43 were determined from top to bottom, respectively. Oblique body region: the detection arms at both ends are multiplied by 2, and the boxed base is the mutation site. Hyphenated bold area: a primer binding region. Marking area: universal sequence, which is combined with the probe on the colloidal gold combined pad. GCGC: and (4) enzyme cutting sites.
"p": the 5' phosphate, reacts with the ligase.
Primer: the 5' end of the primer for starting the RCA reaction is modified with biotin and can be connected with a primer with avidin.
AuNPs probes: the 5' end is decorated with sulfydryl, can be combined with the oligonucleotide probe of the nano-gold, is responsible for being combined with the RCA amplification product and is positioned on the colloidal gold combination pad. katG315, inhA-15, rpoB531, rpsL43 are responsible for the recognition of the corresponding single-base mutations. The Universal primer can be combined with four mutation products, and can simultaneously recognize 4 single base mutations. MismatchProbe as a negative control failed to complementarily bind the four mutation sites.
Target sequences: a target sequence of interest. The sequence is intercepted from a related drug-resistant gene sequence of the tubercle bacillus, a lineation region is a PLP detection arm complementary combination region, and a mutation site is arranged in the middle. katG315(W) represents a normal, non-mutated partial gene segment of the isoniazid drug action sequence. Negative control, unrecognizable by PLP probe.
capture probes: and the capture probe positioned on the control line can be complementarily combined with the nanogold probe to capture the redundant AuNPs probes.
3. sequence specificity verification
3.1 in order to ensure the specificity of the reaction, the primer sequence in the middle section of PLP is compared with the whole genome of the target sequence to be detected (Mycobacterium tuberculosis H37RV) for homology and specificity analysis.
3.2 to ensure the hybridization efficiency, should try to make the specific hybridization region located in the probe loop, avoid the possibility of generating internal secondary structure sequence, according to the online analysis software http:// sfold. wadsworth. org/and http:// mfold. rna. albany. edu/obtain the circular template (circular Padlock probes) single-stranded secondary structure results and hybridization thermodynamic parameters (Ironic conditions: [ Na + ] ═ 0.1M, [ Mg2+ ] ═ 0.01M).
(1) The appropriate length of the detection arms at the two ends of the probe is 14-25 bp;
(2) The hairpin structure of the detection arms at the two ends is excluded and the GC content is less than 60 percent;
(3) the Tm values of the detection arms at both ends are respectively as follows: t1(49 ℃ C.), T2(37.3 ℃ C.). The Tm of the capture probe was 49.4 ℃.
(4) the connection temperature of the PLP is between the Tm values at two ends;
(5) The link sequence in the middle segment of PLP is independent of the target sequence to be detected, and comprises a universal primer sequence and a HhaI enzyme cutting site (GCG ^ C);
(6) The secondary structure of the PLP after the loop formation and the primer hybridization sequence are positioned at a circular position.
4. RCA amplification (see FIG. 3)
4.1 cyclization ligation of Linear PLP:
100nM of linear PLP (Muts), or Padlock probes (PLP), phosphorylated at the 5' end, were mixed with 100nM of each of the two Target sequences (mutant and wild type), Target sequences, in a 20. mu.L ligation system comprising (30mM Tris-HCl pH8.0, 4mM MgCl 2, 10mM (NH 4) 2 SO 4, 1.2mM EDTA, 0.1mM NAD, 0.005% BSA), denatured at 95 ℃ for 5min, cooled to 4 ℃ and then incubated at 37 ℃ for 30min, 5U E.coli DNaligase and 0.05% BSA were added to the system and reacted at 37 ℃ for 1h to form circular template ligations.
4.2 digestion reaction:
The ligation product of the above reaction was added to 40. mu.L of an exo-reaction system (67mM glycine-KOH pH9.5, 6.7mM MgCl 2, 1mM DTT), 10U of exouchase I and exouchase III were added to remove unclyclized linear PLP and excess target DNA, reacted at 37 ℃ for 30min, and then reacted at 90 ℃ for 5mM to inactivate the enzyme.
4.3RCA amplification reaction:
mu.L of circularized PLPs and 2. mu.L of amplification primers (Primer) were placed in RCA Reaction system and incubated at 37 ℃ for 30min containing 10 × Reaction Buffer (330mM Tris-acetate pH 7.9, 100mM Mg-acetate, 660mM K-acetate, 10mM DTT, 1% (v/v) Tween 20), 1mM dNTPs, 0.2. mu.g/. mu.LBSA, 5% DMSO. 0.5U/. mu.L of Phi29 DNA polymerase was then added and the mixture was amplified at 37 ℃ for 1h and then left at 90 ℃ for 10min to inactivate the enzyme. And 4 reaction systems are added in proportion during multiple detection.
4.4 enzyme digestion reaction:
mu.L of the RCA product was taken, mixed with 5. mu.L of a digestion reaction system (33mM Tris-acetate (pH 7.9), 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/mL BSA), and 5U of HhaI restriction enzyme was added, digested at 37 ℃ for 1 hour, and then treated at 80 ℃ for 20min to inactivate the enzyme.
4.5 electrophoresis of the cleavage products:
preparing 1% agarose gel electrophoresis, adding 10. mu.L of RCA product (enzyme digestion product) stained by SYBR Green II, carrying out electrophoresis at a voltage of 80V for 30min, and observing and recording gel images.
5. Marking of the nanogold probe and assembling of the paper-based microfluidic analysis device:
5.1 marking of the nano-gold probe:
3 4Adding 10 mu L of 100 mu mol/L sulfhydryl labeled DNA (1 mu mol/L) namely AuNPs probes into a centrifuge tube, adding 0.33 mu L of 500 mu mol/L acetate buffer solution (pH5.2) and 0.5 mu L of 10mmol/LTCEP into the centrifuge tube to activate sulfhydryl, incubating for 1 hour at room temperature in a dark place, adding 1mL of 10nmol/L nanogold (20nm-40nm) into another centrifuge tube, oscillating at a low speed, adding activated DNA, placing for more than 16 hours at room temperature in a dark place, gradually adding 10 mu L of 500mmol/L Tris acetate buffer solution (pH8.2) and 1mol/L NaCl to final concentrations of 5mmol/L respectively, mixing the mixture at 0.1mol/L, incubating for 48 hours at room temperature in a dark place at 4 ℃, centrifuging for 30 minutes at 3 times, washing with 500 mu L of 10mmol/L buffer solution (PBS), washing with pH 7.0.14000 r/L of PBS, rinsing with sucrose, adding 50% of 50 mu L of 50% of colloidal suspension, removing Na, 5% of heavy suspension, 5 mu L of 5% of PBS, 5% of Na, 84% heavy suspension, 5% of PEG, 5% heavy suspending agent, and 5% heavy suspending agent (20% of 5% of Na and 5% heavy suspending agent).
5.2 assembling and sample detecting of the single paper-based microfluidic analysis device:
Firstly, as shown in figure 1, a processed sample pad (15mm), a nanogold bonding area (6mm), an NC membrane (20mm), a water absorption pad (13mm) and a bottom plate are assembled in sequence, are overlapped by about 2mm, and have the whole width of 4 mm. The materials of each part are respectively a polyester cellulose membrane (or a glass cellulose membrane), a glass cellulose membrane, a nitrocellulose membrane, absorbent paper and a PVC bottom plate with viscosity. And secondly, respectively drawing a Control Line and a detection Line (distance is 2mm) on the NC membrane by using an XYZ three-dimensional film-drawing gold spraying instrument, dotting the marked nanogold probe solution on the membrane to a nanogold binding region, forming an avidin point membrane to a C Line (Control Line) which is the Control Line, and forming a Capture probe solution which is the Capture probe point membrane to a T Line (Test Line) detection Line. Drying at 37 ℃ for 1h after finishing film spotting, and then placing in a room temperature drying environment for later use. Dropping 20 mu L and 20 mu L hybridized buffer of the amplified product to be detected on a sample pad of the detection device or immersing the detection strip (card), namely the paper-based microfluidic analysis device, in the sample liquid (without touching the colloidal gold combined pad) for 30s, and flatly placing the detection card. 20 mu L of hybridization buffer can be dripped every 3 minutes, the color change appears on the detection card after 5-10min, if red bands appear on the C line and the T line simultaneously, the mutation of the target is proved, if the red bands appear on the T line only, the mutation is not realized, and if the color change does not appear on both bands, the detection card is invalid.
The detection principle is as follows: the PLP can specifically recognize a target sequence with single base mutation, only if the target sequence is completely complementary with the PLP, the linear PLP can be cyclized under the action of ligase to generate RCA reaction under the action of polymerase, and the product has a large number of repeated sequences complementary with the PLP, namely sequences complementarily combined with a universal probe, a primer sequence modified with biotin and sequences complementary with a complete detection arm with a mutation site. The colloidal gold combined pad of the detection card is provided with a nano-gold probe which can be complementarily combined with an amplification product. The C line is fixed with avidin, when the target gene generates related mutation to generate RCA reaction, the product combined with the nano-gold probe is captured on the line due to the fact that the biotin (modified at the 5' end of the primer) exists, because one avidin can be combined with four biotin, the compound of the two has extremely high affinity and stability, and the nano-gold probe combined with the product is gathered and develops color at the position. The redundant nanogold probe is captured by the complementary probe fixed on the T line. Therefore, the simultaneous color development of the C line and the T line represents a positive reaction, i.e., a drug-resistant mutation occurs in the detection range. The C line is negative reaction when being singly developed, and no mutation is generated.
5.3 multiple drug resistant sudden change point detection card
The method comprises the following steps:
as shown in FIG. 4, the test card consists of 6 test strips, but only one test sample needs to be dropped, 6 different nanogold probes are respectively fixed on 6 colloidal gold bonding pads, and the substances fixed at other positions are the same. No. 1: and (4) performing multiplex detection. Since 4 PLPs recognizing the drug-resistant mutation site have the same universal sequence, the amplification products thereof also have the same DNA, and designing a universal probe based on the gene sequence can indicate whether a mutation occurs in a detectable range, i.e., whether a single-base mutation of 4 drug-resistant genes (KatG315, inhA-15, rpo531, rpsL43) exists, but which kind or kinds of mutation occurred is unknown. No. 2: and (5) negative control. The probes on the conjugate pad do not bind complementarily to all of the product sequences, but are captured by the complementary sequences on the control line. No. 3-6: the detection of single-base mutation of KatG315, inhA-15, rpo531 and rpsL43 can verify the detection result of position 1, determine whether the mutation really occurs, and indicate which mutation occurs.
the second method comprises the following steps:
As shown in FIG. 5, the multiplex detection is focused on one detection card, in which a universal probe is modified on a colloidal gold conjugate pad to capture all amplification products within a detection range, and a plurality of detection lines (T-lines) each having a distance of 2mm each indicate a point mutation where a substance for capture is immobilized in association with an amplification reaction Primer, for example, an amplification Primer 5 'recognizing PLP sequence of Isoniazid (INH) drug-resistant KatG315 modifies biotin, avidin immobilized on the first detection line T 1 captures a product containing biotin, indicating whether or not KatG315 mutation occurs by color change, a Rifampin (RFP) drug-resistant rpo531 modified digoxin at the end of the associated Primer 5', an antibody immobilized on the second detection line T 2 by antigen-antibody reaction captures the associated amplification product to produce color change, thereby indicating whether or not rpo531 mutation occurs, so that what kind of the modified mutation is achieved by immobilization of the capture substance on the T-lines depending on the substance modified on the Primer, and the number of the modified mutation of the line C-resistant Primer is indicated by the color change of the control line and the color change of the amplification reaction line C-modified amplification probe when the line and the amplification reaction conditions of the amplification reaction occur.
Detecting a card: as shown in fig. 4. The relevant DNA sequence: 1-6, and the colloidal gold bonding pad is modified with the nucleic acid sequence shown in SEQ ID NO: 11. SEQ ID NO: 16. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15; streptavidin is fixed on all control lines; the detection lines are respectively fixed with SEQ ID NO: 23. SEQ ID NO: 28. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27.
And the related DNA sequence is modified by a colloidal gold bonding pad to form a universal sequence SEQ ID NO: 11, T 1 fixed avidin and T 2 fixed digoxin indicate whether KatG315 and inhA-15 single base mutation occur or not according to different primer modifications, and C line fixed SEQ ID NO: 23 (all base sequences are in a solution state when used).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Zhang Yang
<120> multiple detection method of drug-resistant gene of mycobacterium tuberculosis based on RCA amplification
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tggtgatcgc gtcctgtata cgcttcttcg gtgcccatgc gctgcgacct cagcatcgac 60
ctgtctagac ctcgatgccg g 81
<210> 2
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tctcgccgcg gccgggtata cgcttcttcg gtgcccatgc gcgaccacct tgcgatcggg 60
tagtctaccg acaacctatc a 81
<210> 3
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acagtcggcg cttggtatac gcttcttcgg tgcccatgcg ccagcggtag accacctatc 60
ggtctaccca gcgcca 76
<210> 4
<211> 82
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcggagtggt ggtgtacgta tacgcttctt cggtgcccat gcgcgaccgg tatgcgacct 60
ggtagtctag agttcggctt cc 82
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aggtcgatgc tgaggtcgca 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tacccgatcg caaggtggtc 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cgataggtgg tctaccgctg 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
taccaggtcg cataccggtc 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ggataggaca taataagcga 20
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cgcttcttcg gtgcccat 18
<210> 11
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cgcttcttcg gtgcccat 18
<210> 12
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tcctgcgcta gtggtggacg tagctccag 29
<210> 13
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ggccggcgcc gctctactat ccaacagcc 29
<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gttcgcggct gacaaccgcg accc 24
<210> 15
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
catgtggtgg tgaggctcct tcggcttgag 30
<210> 16
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cgtaagtctc cgaggttgcc atcgatgatt tgaacttcat c 41
<210> 17
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tggcaccgga accggtaagg acgcgatcac cagcggcatc gaggtcgtat ggacgaa 57
<210> 18
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tggcaccgga accggtaagg acgcgatcac caccggcatc gaggtcgtat ggacgaa 57
<210> 19
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
taccgatttc ggcccggccg cggcgagatg ataggttgtc ggggtgactg 50
<210> 20
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tcggggttga cccacaagcg ccgactgttg gcgctggggc ccggcggtct 50
<210> 21
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gcacccgcgt gtacaccacc actccgagga agccgaactc ggcgcttcgg 50
<210> 22
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tgtgtaacaa catgaagatt gtaggtcaga actcacctgt tagaaactgt gaagatcgct 60
tattatgtcc tatcctcagc 80
<210> 23
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gcgaagaagc cacgggta 18
<210> 24
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
aggacgcgat caccaccggc atcgaggtc 29
<210> 25
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ccggccgcgg cgagatgata ggttgtcgg 29
<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
caagcgccga ctgttggcgc tggg 24
<210> 27
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gtacaccacc actccgagga agccgaactc 30
<210> 28
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gcattcagag gctccaacgg tagctactaa acttgaagta c 41
Claims (9)
1. the paper microfluidic chip for nucleic acid detection is characterized in that the nucleic acid sequences involved in the paper microfluidic chip for nucleic acid detection are as follows: SEQ ID NO: 1 to SEQ ID NO: 28.
2. A paper-based microfluidic analytical device for nucleic acid detection comprising the method of claim 1, wherein the paper-based microfluidic analytical device, i.e. a single detection strip, comprises: the device comprises a sample pad, a colloidal gold combination pad, a detection line, a control line, an NC membrane, a water absorption pad and a bottom plate.
3. the paper-based microfluidic analytical device according to claim 2, wherein the sample pad, the gold colloidal conjugate pad, the NC membrane, and the bibulous pad overlap each other by 2mm, and have a width of 4 mm.
4. The paper-based microfluidic analytical device according to claim 2, wherein the sample pad, the colloidal gold conjugate pad, the NC membrane, and the water absorbent pad are a mylar or cellophane membrane, a nitrocellulose membrane, a water absorbent paper, and a bottom plate of PVC having viscosity, respectively.
5. A Mycobacterium tuberculosis drug-resistant gene multiple detection method based on RCA amplification reaction is characterized by comprising the following steps: detecting a card: the detection card consists of 6 detection strips, but only needs to be dripped once for detecting a sample, 6 different nano-gold probes are respectively fixed on 6 colloidal gold bonding pads, and substances fixed at other positions are the same; no. 1: performing multiplex detection; indicating whether a mutation has occurred within a detectable range, but which mutation or mutations have occurred is unknown; no. 2: negative control; the probes on the bonding pad can not be complementarily combined with all product sequences, but can be captured by complementary sequences on a control line; no. 3-6: and (3) single base mutation detection, which can verify the detection result of the position 1, determine whether the mutation really occurs, and indicate which mutation occurs.
6. The RCA amplification reaction-based Mycobacterium tuberculosis drug-resistant gene multiplex detection method of claim 5, wherein the related DNA sequence: 1-6, and the colloidal gold bonding pad is modified with the nucleic acid sequence shown in SEQ ID NO: 11. SEQ ID NO: 16. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15; streptavidin is fixed on all control lines; the detection lines are respectively fixed with SEQ ID NO: 23. SEQ ID NO: 28. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27.
7. A Mycobacterium tuberculosis drug-resistant gene multiple detection method based on RCA amplification reaction is characterized in that the Mycobacterium tuberculosis drug-resistant gene multiple detection method based on RCA amplification reaction further comprises the following steps: detection card 2: the multiplex detection is concentrated on a detection card, wherein a universal probe is modified on the colloidal gold bonding pad to capture all amplification products in the detection range; the detection lines are multiple, each line is 2mm away from the other line, each line indicates a point mutation, substances fixed at the point for capturing are related to amplification reaction primers, different capture substances are fixed on the T line according to different substances modified on the primers, detection of multiple drug-resistant mutation sites can be realized, and redundant universal probes modified by nano-gold flow to the control line fixed with complementary bases; when the amplification reaction solution is detected, under the condition that the color of the C line changes, the change of the number and the position of the T line can indicate the occurrence of the drug resistance mutation.
8. The method for multiplex detection of drug-resistant gene of Mycobacterium tuberculosis based on RCA amplification reaction of claim 7, wherein the related DNA sequences are that the universal sequence SEQ ID NO: 11 is modified on a colloidal gold conjugate pad, T 1 fixes avidin, T 2 fixes digoxin and indicates whether KatG315 and inhA-15 single base mutation respectively occur according to the different primer modifications, and the C line fixes SEQ ID NO: 23.
9. Use of the paper microfluidic chip for nucleic acid detection according to any one of claims 5 or 7 in the detection of drug-resistant genes of mycobacterium tuberculosis.
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