CN112094929A - Streptococcus agalactiae amplification primer group, application and streptococcus agalactiae detection method - Google Patents
Streptococcus agalactiae amplification primer group, application and streptococcus agalactiae detection method Download PDFInfo
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Abstract
The invention relates to a streptococcus agalactiae amplification primer group, application and a streptococcus agalactiae detection method, and belongs to the technical field of microbial detection. The detection method of the streptococcus agalactiae comprises the following steps: 1) designing an MCDA amplification primer group of a specific gene groEL: CP1, CP2, F1, F2, D1, D2, C1, C2, R1 and R2; 2) under a reaction system comprising the amplification primer group, performing MCDA (micro-isothermal amplification) isothermal amplification by taking the genome of the streptococcus agalactiae sample to be detected as a template to obtain an amplification product; 3) detecting the amplification product in the step 2) by using a gold nano biosensor to obtain a detection result. The method is based on multi-cross isothermal amplification, combines a nano biological detection technology, and can be used for quickly, sensitively and highly specifically detecting and identifying the streptococcus agalactiae directly from clinical samples.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of microbiology and molecular biology, in particular to a streptococcus agalactiae amplification primer group, application and a streptococcus agalactiae detection method.
[ background of the invention ]
Streptococcus agalactiae (s. agalactiae) is a β -hemolytic gram-positive bacterium. The pathogen can be colonized in vagina, intestinal tract and urethra of pregnant women, and cause urinary tract infection, bacteremia, chorioamnionitis and placental premature peeling of pregnant women, and may further induce premature and dead fetus, and also cause neonatal pneumonia, sepsis and meningitis. Thus, prenatal screening and early diagnosis of Streptococcus agalactiae in pregnant women helps to prevent the occurrence of serious diseases in infants.
The detection of Streptococcus agalactiae at present relies mainly on traditional isolation culture and biochemical identification. The method is long in time (2-7 days), comprises secondary enrichment, selective culture and subsequent biochemical identification, and has the defects of time and labor consumption although the biochemical identification can be judged by a full-automatic microorganism identification instrument. With the rapid development of molecular diagnostic techniques, some enrichment culture-based PCR diagnostic techniques (such as general PCR technique and fluorescence PCR technique) are used for screening Streptococcus agalactiae, however, these methods require expensive thermocycler equipment and expensive probe synthesis, subsequent electrophoresis operation, and skilled operators. However, some primary hospitals and field tests cannot meet the above conditions, and the application of the technologies is limited. Although the enrichment culture combined PCR technology and the fluorescence PCR technology increase the detection sensitivity, the detection period is still long, and the requirement of premature delivery or pregnant women about to be born cannot be met. Therefore, in order to provide accurate and rapid diagnosis for the parturient, it is necessary to develop a detection method which is time-saving, labor-saving and highly specific and can simultaneously detect and identify streptococcus agalactiae.
In order to overcome the disadvantages of PCR-based amplification techniques, a number of isothermal amplification techniques have been developed. Compared with the PCR technology, the isothermal amplification technology does not depend on thermal cycle amplification equipment, is simple, and has high reaction speed and good sensitivity. Therefore, the isothermal amplification technology is beneficial to realizing rapid amplification, convenient detection and on-site diagnosis. Currently, isothermal techniques that are widely used include Rolling Circle Amplification (RCA), tandem displacement amplification (SDA), helicase-dependent isothermal amplification (HDA), loop-mediated isothermal amplification (LAMP), cross amplification (CPA), and the like. However, these isothermal techniques have the disadvantages of simultaneous operation of multiple enzymes, expensive reagents, complex operation steps, etc., and the practicability, convenience and operability of these methods are to be improved, especially in the field of rapid diagnosis and resource-limited areas.
The Chinese patent publication CN106399517A respectively designs MCDA amplification primers aiming at a specific gene ipaH of shigella and a specific gene invA of salmonella, and combines the MCDA technology with the nano biological detection technology (LFB) to realize the detection of two strains; chinese patent publication CN 108220398A. An MCDA amplification primer is designed aiming at the Klebsiella pneumoniae specific gene rcsA, and the specific detection of the Klebsiella pneumoniae is realized by combining a nano biological detection technology.
At present, no research exists for designing MCDA amplification primers aiming at specific genes of streptococcus agalactiae and combining an LFB technology to carry out specific detection. Therefore, it is necessary to combine MCDA-LFB to study the feasibility of specific detection of Streptococcus agalactiae, and further design specific primers, thereby providing a more sensitive, objective, portable and simple detection method.
[ summary of the invention ]
Aiming at the defects of the prior art, the invention provides an MCDA amplification primer group designed based on the specific gene of the streptococcus agalactiae, and combines the LFB technology to realize the efficient detection of the specificity of the streptococcus agalactiae.
In order to solve the above problems, the present invention provides a streptococcus agalactiae amplification primer set, comprising:
as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2 as shown in SEQ ID NO: 3, and the cross inner primer CP1 is shown as SEQ ID NO: 4, and a cross inner primer CP2 as shown in SEQ ID NO: 5-10, wherein the amplification primers are C1, C2, D1, D2, R1 and R2; wherein, the 5' end of the amplification primer D1 is marked with fluorescein to obtain D1; biotin is marked at the 5' end of the amplification primer R1 to obtain R1.
Further, the amplification primer group is used for multi-crossover isothermal amplification of the characteristic gene groEL of the streptococcus agalactiae.
Another objective of the present invention is to provide an application of the amplification primer set for Streptococcus agalactiae in the detection of Streptococcus agalactiae.
It is still another object of the present invention to provide a method for detecting Streptococcus agalactiae, comprising the steps of:
1. designing and providing an amplification primer group of the streptococcus agalactiae: f1, F2, CP1, CP2, C1, C2, D1, D2, R1, R2;
2. under the reaction system comprising the amplification primer group, carrying out MCDA constant temperature amplification by taking the genome of the streptococcus agalactiae sample to be detected as a template to obtain an amplification product;
3. and (3) detecting the amplification product in the step (2) by using a gold nano biosensor to obtain a detection result.
Further preferably, the temperature of the MCDA amplification reaction in the step 2 is 60-66 ℃;
preferably, the MCDA amplification reaction in the step 2 is performed at a constant temperature of 61 ℃ for 30min, and the reaction is terminated at 85 ℃ for 5 min;
further preferably, in the amplification primer set, the concentration of the cross primers CP1 and CP2 is 60pmol, the concentration of the displacement primers F1 and F2 is 10pmol, the concentration of the amplification primers R1, R2, D1 and D2 is 30pmol, and the concentration of the amplification primers C1 and C2 is 20 pmol;
as a further preference, the MCDA amplification reaction system comprises, 10mM Betain, 6mM MgSO4, 1mM dNTP, 12.5. mu.L of 10 XBst DNA polymerase buffer, 10U of strand displacement DNA polymerase, 1. mu.L of template, supplemented with deionized water to 25. mu.L;
as a further preferred, the gold nano biosensor comprises a sample pad, a gold label pad, a fiber membrane, a water absorption pad and a back plate; the sample pad, the gold mark pad, the fiber membrane and the water absorption pad are sequentially assembled on the back plate; coating the gold nanoparticle-coupled streptomycin avidin on a gold label pad, coating an anti-fluorescein antibody on a detection line, and coating biotin-coupled bovine serum albumin on a control line;
as a further preference, the gold nano-biosensor comprises 30nm of gold nanoparticle-coupled streptavidin.
Compared with the prior art, the invention has the beneficial effects that:
the invention designs a set of MCDA amplification primer group aiming at specific gene groEL of streptococcus agalactiae: f1, F2, CP1, CP2, C1, C2, D1, D2, R1, R2; to construct a detectable product, fluorescein was labeled at the 5 'end of primer D1 and designated D1, and biotin at the 5' end of primer R1 and designated R1; the cross primers CP1 and CP2 are the main primers mediating MCDA amplification; the replacement primers F1 and F2 play a replacement role in MCDA reaction, and replace the cross primers CP1 and CP 2; the six amplification primers D1, C1, R1, D2, C2 and R2 can accelerate the MCDA reaction and increase the MCDA product; the method for detecting the streptococcus agalactiae aims at that the amplification product of the specific gene groEL of the streptococcus agalactiae can be visually detected by the gold nano biosensor, is convenient, fast, sensitive and specific, is suitable for being popularized to various specific nucleotide fragments and is applied to detection of pathogenic bacteria corresponding to the specific nucleotide fragments.
[ description of the drawings ]
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of the position and orientation of the MCDA-LFB primer design of the present invention;
FIG. 2 is a graph showing the results of MCDA primer verification and MCDA-LFB detection in the example of the present invention;
FIG. 3 is a graph showing the results of a standard MCDA-LFB optimum reaction temperature test according to an embodiment of the present invention;
FIG. 4 is a graph showing the results of sensitivity of MCDA-LFB detection of Streptococcus agalactiae according to example of the present invention;
FIG. 5 is a graph of the test results of the optimal detection time of MCDA-LFB according to the embodiment of the present invention;
FIG. 6 is a specific detection evaluation chart of MCDA-LFB technology in an embodiment of the invention.
[ detailed description ] embodiments
The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
The invention provides a streptococcus agalactiae amplification primer group, which is designed according to the following steps: the streptococcus agalactiae has a plurality of housekeeping genes, such as sodA, gyrB, groEL and the like, the characteristics of the housekeeping genes are different, the groEL gene is used as one housekeeping gene, the differences among streptococcus species are large, the conservation and the specificity are good, and the streptococcus agalactiae can be distinguished from other closely similar strains. And (3) designing an MCDA primer by using primer design software, and performing sequence comparison analysis on the obtained specific primer in an NCBI database to eliminate possible non-specific matching between the primer and other species sequences, thereby finally obtaining the optimized MCDA amplification primer. The position and orientation of the primer design is shown in FIG. 1.
Examples
1. Genome extraction
The extraction of genomic DNA from Streptococcus agalactiae and other bacteria was carried out using the DNA extraction kit from Tianzhu Biotech, Inc., according to the instructions. Determination of the concentration and purity of genomic DNA by UV spectrophotometer, DNA from Streptococcus agalactiae by ddH2O serial dilutions (from 3ng,300pg,30pg,3pg,300fg, to 3fg/μ l). The various genomic DNAs are packaged in small quantities and stored at-20 ℃ for further use.
2. Primer design
In order to verify, evaluate the MCDA-LFB technology and establish a rapid, sensitive and specific MCDA-LFB detection system aiming at the streptococcus agalactiae pathogen. In this example, a set of MCDA amplification primers (Primer design software: PrimeExplorer V4(Eiken Chemical) (http:// PrimeExplorer. jp/e /) and Primer Premier 5.0) were designed for the specific gene groEL of Streptococcus agalactiae, and the detection of Streptococcus agalactiae was performed by combining the MCDA-LFB technique, and the feasibility, sensitivity, specificity and reliability were verified. The schematic design of the primers is shown in FIG. 1; primer sequences and modifications are shown in table 1.
Table 1: primer information
Note:1nt, nucleotide;2mer, monomeric (monomeric units);3d1, the primer is used for an MCDA-LFB detection system, and fluorescein is marked at the 5' end;4r1, which was used in the MCDA-LFB detection system and labeled with biotin at the 5' end.
MCDA amplification
Standard MCDA reaction system: the concentrations of the cross primers CP1 and CP2 were 60pmol, the concentrations of the displacement primers F1 and F2 were 10pmol, the concentrations of the amplification primers R1, R2, D1 and D2 were 30pmol, the concentrations of the amplification primers C1 and C2 were 20pmol, 10mM Betain, 6mM MgSO4, 1mM dNTP, 12.5. mu.L of 10 XBst DNA polymerase buffer, 10U of strand displacement DNA polymerase, 1. mu.L of template, and supplemented with deionized water to 25. mu.L. The whole reaction was kept at a constant temperature of 61 ℃ for 1 hour and at 85 ℃ for 5min to terminate the reaction.
4. Design of a biodetector (LFB)
In this example, LFB was simply modified to optimize the LFB detection target sequence, mainly based on the concentration of each marker. The LFB comprises five parts, a sample pad, a gold label pad, a fibrous membrane, a bibulous pad, and a backing sheet. Firstly, a sample pad, a gold mark pad, a fiber membrane and a water absorption pad are sequentially assembled on a back plate. Then, SA-G (30nm, streptavidin coupled with gold nanoparticles), anti-FITC (anti-fluorescein antibody) and B-BSA (bovine serum albumin coupled with biotin) are coated on a gold label pad, a detection line and a Control Line (CL) respectively, and the gold label pad, the detection line and the control line are dried for later use.
Detection principle of LFB: agalactiae-MCDA product was added directly to the LFB pad area, 120ul of detection buffer was added to the pad area, and MCDA product was wicked from the pad to the absorbent pad. When the MCDA product reached the gold-labeled pad, one end of the double-labeled product (i.e., the biotin-labeled end) reacted with SA-G (streptavidin coupled to gold nanoparticles). When the product continues to move, the other end (i.e. the fluorescein labeled end) of the dual-standard product is combined with the antibody of the detection line area, and the dual-standard product is fixed in the detection line area. With the accumulation of the product in the detection line area, the color reaction is carried out through SA-G (gold nanoparticle coupled streptomycin avidin) at the other end, so that the MCDA product is visually detected. In addition, the surplus SA-G (gold nanoparticle-coupled strepavidin) can be directly subjected to color reaction with B-BSA (gold nanoparticle-coupled strepavidin) in a CL (quality control line) region to judge whether the function of LFB is normal or not.
Interpretation of LFB results (fig. 2 c): red bands appeared only in the CL region, indicating a negative control, no positive product (fig. 2c2, 2c3, 2c 4); red bands appeared in the CL and TL regions indicating a positive result for detection of the target (fig. 2c 1); when the LFB does not have the red line strip, indicating that the LFB is failed; only when TL appears with red stripes, CL has no red stripes, representing that the result is not feasible and needs to be re-detected.
5. Verification of feasibility of MCDA primers
The three detection methods are used for judging MCDA products, firstly, a visible dye Malachite Green is added in the preparation of a reaction system, after the amplification is finished, the color of a positive reaction tube system is changed from dark Green to bright Green, and the color of a negative reaction tube system is changed to colorless. Secondly, detecting the MCDA product by agarose electrophoresis, wherein the electrophoresis pattern of the positive amplification product is in a specific step shape and a step-shaped band does not appear in the negative reaction because the product contains amplification fragments with different sizes. The third method is the specific and rapid LFB method.
1) Visual color change method: as the amplification progresses, the reaction temperature gradually rises, Malachite Green is cracked into A groups and B groups, and the reaction system is colorless. When MCDA amplicons appear, the A group is embedded into the double chain, so that the reaction system presents bright green. Therefore, the result can be judged by the color change of the reaction system, the positive reaction system changes from dark green to light green, and the negative reaction system changes to colorless, as shown in FIG. 2 a. a1 shows positive amplification (3 pg of Streptococcus agalactiae template was added to the reaction system as a positive control), a2 shows negative amplification (3 pg of Streptococcus pyogenes template was added to the reaction system as a negative control), a3 shows negative amplification (3 pg of Streptococcus pneumoniae template was added to the reaction tube as a negative control), and a4 shows a blank control reaction (1. mu.l of double distilled water was used instead of 3pg of template as a blank control). Only the positive control showed positive amplification, indicating that MCDA primers designed for groEL can be used to detect Streptococcus agalactiae.
2) Electrophoresis detection method: the product after the MCDA amplification is subjected to electrophoresis detection, and the MCDA amplification product contains a DNA fragment mixture of a stem-loop structure and a multi-loop cauliflower-like structure, wherein the short fragments are different in size, and the DNA fragment mixture is composed of a series of inverted repeated target sequences, and a stepped map composed of zones with different sizes is displayed on a gel after electrophoresis, and the stepped map is shown in a figure 2 b. The MCDA amplification result is judged and read through an electrophoresis detection method, the expected result appears in the positive reaction, and any amplification band does not appear in the negative reaction and the blank control, so that the MCDA primer designed by the embodiment is further verified to be feasible and can be used for target sequence amplification detection.
3) LFB detection: the product of 2a was subjected to LFB detection. Positive detection of Streptococcus agalactiae was indicated when TL and CL appeared as red bands. While negative reactions and blanks showed only a CL red band. The LFB, MCDA-LFB technology and MCDA primer designed in the embodiment are feasible and can be used for detecting a target sequence (2 c).
5. Determination of the optimum reaction temperature for the MCDA technique
Under the condition of a standard reaction system, adding a DNA template aiming at the streptococcus agalactiae and a corresponding MCDA primer which is designed, wherein the template concentration is 3 pg/ul. The reaction was carried out at constant temperature (60-67 ℃) and the results were detected using agarose gel electrophoresis and a fluorescent indicator, as shown in FIG. 3. The detection results of the fluorescent indicators showed that the reaction system showed a distinct bright green color at 60-66 deg.C (a 1-a7 in FIG. 3). The results of agarose gel electrophoresis detection showed that the MCDA reaction showed brighter stepwise bands at 60-61 deg.C (b 1-b2 in FIG. 3), and thus 61 deg.C was recommended as the optimal reaction temperature for the MCDA primer; subsequent validation in the present invention selects 61 ℃ as the isothermal condition for MCDA amplification.
Sensitivity of MCDA-LFB to detection of Streptococcus agalactiae
After 1h of standard MCDA amplification using serial diluted streptococcus agalactiae genomic DNA, LFB detection showed: the detection range of MCDA-LFB is 3 ng-300 fg, and the LFB has red lines (4c) in TL and CL areas. When the amount of the genomic template in the reaction system was reduced to 300fg or less, LFB appeared as a red line only in the CL region, indicating a negative result (4c6-4c 8). FIG. 4c read MCDA amplification results using LFB visualization; 4c1 to 4c8 show template amounts of Streptococcus agalactiae of 3ng,300pg,30pg,3pg,300fg,30fg,3fg and a blank (1. mu.l double distilled water).
In order to further verify the sensitivity of MCDA-LFB in detecting Streptococcus agalactiae, another 2 detection methods were used for interpretation of the MCDA amplification results, and the detection sensitivity of MCDA-LFB was further confirmed. Firstly, the MCDA product can be subjected to agarose electrophoresis and then the amplicon is detected, and because the product contains amplified fragments with different sizes, the electrophoresis pattern of the positive amplified product is in a specific ladder shape, and no band appears in the negative reaction. And (3) detection and display: the detection range of agalactiae-MCDA is 3 ng-300 fg, and a step band (4b1-4b5) appears in positive reaction. When the amount of the genomic template in the reaction system was reduced to 300fg or less, no specific ladder band appeared, indicating a negative result (4b6-4b 8). FIG. 4b shows the reading of the result of the MCDA amplification by electrophoresis; 4a1 to 4a8 indicate template amounts of Streptococcus agalactiae of 3ng,300pg,30pg,3pg,300fg,30fg,3fg and a blank (1. mu.l double distilled water). Secondly, the visible dye MG is added into the reaction mixture in advance, the color of the positive reaction system is changed from dark green to bright green, and the color of the negative reaction system is changed to colorless. And (3) detection and display: the detection range of agalactiae-MCDA is 3 ng-300 fg, and the positive amplification system turns bright green (4a1-4a 5). When the amount of the genomic template in the reaction system was decreased to 300fg or less, the reaction became colorless, indicating a negative result (4a6-4a 8). FIG. 4a is a graph showing the visual reading of the result of MCDA amplification; 4a1 to 4a8 indicate template amounts of Streptococcus agalactiae of 3ng,300pg,30pg,3pg,300fg,30fg,3fg and a blank (1. mu.l double distilled water).
7. Determination of optimum detection time for MCDA technology
MCDA reaction is carried out for 10min,20min,30min and 40min at 61 ℃ by using the serially diluted streptococcus agalactiae genome DNA. 1 to 8 represent template amounts of Streptococcus agalactiae of 3ng,300pg,30pg,3pg,300fg,30fg,3fg and a blank (1. mu.l double distilled water). The MCDA product was detected by LFB and fluorescence indicator methods. When MCDA was reacted for 10min, as shown in FIG. 5a, when the amount of template DNA was 30pg or more, both CL and TL appeared on LFB, giving a positive result (5a1-5a 3); below 30pg, only CL appeared on LFB with negative results (5a4-5a 8). When the initial amount of DNA template is 3ng or less, the fluorescent indicator cannot judge whether MCDA amplification has occurred.
When MCDA was reacted for 20min, as shown in FIG. 5b, when the template amount of DNA was 3pg or more, both CL and TL appeared on LFB, with a positive result (5b1-5b 4); below 3pg, only CL appeared on LFB with negative results (5b5-5b 8). When the initial amount of the DNA template was 3pg or less, the fluorescent indicator was colorless and did not determine whether MCDA amplification occurred (5b5-5b 8).
When MCDA was reacted for 30min, as shown in FIGS. 5c and 5d, when template amount of DNA was 300fg and above, both CL and TL appeared on LFB with positive results (5c1-5c 5); below 300fg, only CL appeared on LFB with negative results (5c6-5c 8). When the initial amount of DNA template was 3pg or less, the fluorescent indicator showed no color, and it could not be judged whether MCDA amplification occurred (5c6-5c 8).
When MCDA was reacted for 40min, as shown in FIG. 5d, when template amount of DNA was 300fg and above, both CL and TL appeared on LFB with positive results (5d1-5d 5); below 300fg, only CL appeared on LFB with negative results (5d6-5d 8). When the initial DNA template amount was 300fg or less, the fluorescent indicator showed no color and it could not be judged whether MCDA amplification occurred (5d6-5d 8). Therefore, 30min was determined as the optimal detection time for the MCDA-LFB assay.
8. Determination of specificity of S.agalactiae-MCDA-LFB technique
The specificity of the S.agalactiae-MCDA-LFB technology is evaluated by taking other strains in streptococcus and DNA (streptococcus pyogenes, streptococcus pneumoniae, streptococcus mitis, streptococcus salivarius, streptococcus sanguis, streptococcus dysgalactiae, streptococcus grignard, streptococcus sinensis, streptococcus constellations and the like) which are pathogenic bacteria possibly causing vaginal infection as templates. The strain information is detailed in table 2. The agalactiae-MCDA-LFB technique can accurately identify the streptococcus agalactiae, and the specificity of the MCDA-LFB method is good, as shown in FIG. 6. In FIG. 6, LFB 1-8: a streptococcus agalactiae template; LFB 9-44, a non-Streptococcus agalactiae template. The results show that MCDA-LFB can correctly detect the target sequence.
Table 2: the strains used in this example
1Positive; negative.
2THH, Taihe Hospital; sa, Streptococcus agalactiae.
Application of S.agalactiae-MCDA-LFB technology in clinical specimens
To verify the clinical utility of the method, 260 vaginal and rectal swabs were collected. The detection is carried out by adopting an MCDA-LFB method, a chromogenic medium method and an enrichment culture coupled qPCR method. The results show that 20 positive samples were detected by MCDA-LFB method, which is consistent with the detection results of the enrichment qPCR method, while 8 positive samples were detected by chromogenic medium method, as shown in Table 3. Compared with the qPCR method coupled with enrichment culture, the sensitivity of the MCDA-LFB method is 100 percent, and the specificity is 100 percent. Compared with the chromogenic medium method, the sensitivity of the MCDA-LFB method is 100%, and the specificity is 95.2%. Moreover, the MCDA-LFB method requires only 50 minutes for each clinical sample, including sample collection (3min), DNA preparation (15min), MCDA reaction (30min), and LFB (2min), which is significantly shorter than the 25.6 hours required for enrichment culture coupled qPCR and the 24 hours required for chromogenic culture. The whole process only needs one thermostatic device, and expensive thermal cycling equipment is not needed. Therefore, the MCDA-LFB method is superior to the qPCR method and the chromogenic medium method coupled with enrichment culture, and is a valuable field and laboratory detection method for vagina and rectum swabs.
Table 3: clinical manifestations of MCDA-LFB
From the application results and data of the agalactiae-MCDA-LFB technology in clinical specimens, compared with the chromogenic medium method and the qPCR method coupled with enrichment culture in the prior art, the MCDA-LFB method has the advantages of remarkable technical effect and higher popularization and application values.
The invention selects the groEL gene in the streptococcus agalactiae to design the amplification primer, because the groEL gene is used as a housekeeping gene and has great difference among streptococcus species, the conservation and the specificity are good, the streptococcus agalactiae can be distinguished from other closely similar species, and the groEL gene has more discriminative property than other housekeeping genes. In addition, the condition that other genes (such as cfb and sip) are selected to cause missing detection because the sample strain does not contain the genes can be avoided. The invention is based on the design primer of groEL gene, and combines MCDA amplification technology, thus realizing the rapid amplification of specific gene; the amplification product of the specific gene groEL is combined with the visual detection of a gold nano biosensor to realize the convenient, quick, sensitive and specific detection of the streptococcus agalactiae. The method is suitable for being popularized to various specific nucleotide fragments and applied to the detection of pathogenic bacteria corresponding to the specific nucleotide fragments.
The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative embodiments, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.
Sequence listing
<110> Taihe hospital in Shiweir City (subsidiary hospital of Hubei pharmaceutical institute)
<120> Streptococcus agalactiae amplification primer group, application thereof and detection method of Streptococcus agalactiae
<141> 2020-08-18
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Claims (10)
1. A streptococcus agalactiae amplification primer set, comprising: as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2 as shown in SEQ ID NO: 3, and the cross inner primer CP1 is shown as SEQ ID NO: 4 and cross inner primers CP2 shown in (4),
as shown in SEQ ID NO: 5-10, wherein the amplification primers are C1, C2, D1, D2, R1 and R2;
wherein, biotin is marked at the 5' end of the amplification primer D1 to obtain D1; the 5' end of the amplification primer R1 is marked with fluorescein, and R1 is obtained.
2. The set of amplification primers for Streptococcus agalactiae according to claim 1, wherein: the amplification primer group is used for multi-cross isothermal amplification of the characteristic gene groEL of the streptococcus agalactiae.
3. Use of the amplification primer set for Streptococcus agalactiae according to any one of claims 1 to 2 for detecting Streptococcus agalactiae.
4. A method for detecting streptococcus agalactiae, comprising the steps of:
s1, designing and providing an amplification primer group of the streptococcus agalactiae: f1, F2, CP1, CP2, C1, C2, D1, D2, R1, R2;
s2, under the reaction system comprising the amplification primer group, carrying out MCDA constant temperature amplification by taking the genome of the streptococcus agalactiae sample to be detected as a template to obtain an amplification product;
and S3, detecting the amplification product obtained in the step S2 by using a gold nano biosensor to obtain a detection result.
5. The method for detecting Streptococcus agalactiae according to claim 4, wherein the temperature of the MCDA amplification reaction in step S2 is 60 to 66 ℃.
6. The method for detecting Streptococcus agalactiae according to claim 4, wherein the MCDA amplification reaction in step S2 is incubated at 61 ℃ for 30min and at 85 ℃ for 5min to terminate the reaction.
7. The method of claim 4, wherein the concentration of cross primers CP1 and CP2, the concentration of displacement primers F1 and F2, the concentration of amplification primers R1, R2, D1 and D2 is 30pmol, and the concentration of amplification primers C1 and C2 is 20 pmol.
8. The method of claim 4, wherein the MCDA amplification reaction comprises 10mM Betain, 6mM MgSO4, 1mM dNTP, 12.5. mu.L of 10 XBst DNA polymerase buffer, 10U of strand displacement DNA polymerase, 1. mu.L of template, and 25. mu.L of deionized water.
9. The method for detecting Streptococcus agalactiae according to claim 4, wherein the gold nanobiosensor comprises a sample pad, a gold label pad, a fibrous membrane, a water absorbent pad and a back plate; the sample pad, the gold mark pad, the fiber membrane and the water absorption pad are sequentially assembled on the back plate; coating the gold nanoparticle-coupled streptomycin avidin on a gold-labeled pad, coating an anti-fluorescein antibody on a detection line, and coating the biotin-coupled bovine serum albumin on a control line.
10. The method for detecting Streptococcus agalactiae according to claim 4, wherein the gold nanosensor comprises 30nm gold nanoparticle-coupled streptavidin.
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| CN106399517A (en) * | 2016-09-30 | 2017-02-15 | 中国疾病预防控制中心传染病预防控制所 | Multi-cross isothermal amplification and nanogold biosensing combined nucleic acid detection technology |
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Non-Patent Citations (3)
| Title |
|---|
| W.J. LEIGH等: "Development and evaluation of a quantitative polymerase chain reaction for aquatic Streptococcus agalactiae based on the groEL gene", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| XUEQIN CHENG 等: "Highly Sensitive and Rapid Identification of Streptococcus agalactiae Based on Multiple Cross Displacement Amplification Coupled With Lateral Flow Biosensor Assay", 《FRONTIERS IN MICROBIOLOGY》 * |
| YI WANG 等: "Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification", 《SCIENTIFIC REPORTS》 * |
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