CN117589739A - Visual quantitative detection platform based on CRISPR Cas-portable detector-smart phone and application thereof - Google Patents
Visual quantitative detection platform based on CRISPR Cas-portable detector-smart phone and application thereof Download PDFInfo
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Abstract
The invention discloses a visual quantitative detection platform based on CRISPR Cas-portable detector-smart phone and application thereof, wherein the specific detection steps of the detection platform are as follows: firstly extracting genomic DNA of a clinical sample, adding the genomic DNA into a CRISPR Cas system, carrying out timing constant-temperature reaction on the system, then placing the system into the portable detector, taking a picture by using a smart phone, calling a mobile phone applet capable of identifying the RGB value of a fluorescent picture, converting a color image into an RGB component image, substituting the corresponding color channel value into a linear fitting regression curve, and obtaining the detected pathogen gene concentration by the mobile phone applet. The method can complete the rapid quantitative detection of pathogen genes in samples, and the detection platform has wide application value in the rapid detection in the field.
Description
Technical Field
The invention relates to the technical field of pathogen detection, in particular to a visual quantitative detection platform based on CRISPR Cas-portable detector-smart phone and application thereof.
Background
Recently, detection systems based on CRISPR Cas detection methods are widely accepted, compared with PCR and traditional detection technologies, the special collateral splitting activity of the CRISPR Cas biological sensing system has a signal amplification function, the high sensitivity and ultra-fast biological sensing capability of the CRISPR Cas biological sensing system can be further enhanced, and meanwhile, the biological sensing system can be combined with an isothermal heating device and a signal output assembly, so that a rapid and multifunctional instant detection technology is developed.
The CRISPR Cas biosensing system mainly comprises four systems, CRISPR Cas9, CRISPR Cas12, CRISPR Cas13 and CRISPR Cas 14. Existing studies have focused mainly on the use of CRISPR Cas12a to detect viruses, and scientists have also used CRISPR Cas systems to detect pathogens and their resistance and virulence genes.
In recent years, more and more diseases caused by pathogenic microorganisms are rapidly popularized and rapidly transmitted, and rapid and accurate detection of pathogens plays a role in better controlling disease sources and avoiding large-scale transmission. The existing detection method has the defects that the results are required to be identified and presented by means of large-scale instruments such as a qPCR instrument, an enzyme-labeled instrument, a Raman spectrum detector and the like, and the results can be quantitatively analyzed, but the method has extremely high requirements on operators, so that the method is not beneficial to the clinical in-situ detection of pathogens, and is easy to generate nonspecific amplification and influence the experimental results. Therefore, there is an increasing need to develop a detection method that can be applied to clinical in situ detection of pathogens.
At present, smart phones play an increasing role in the field of analysis and detection. The method can be used for shooting and recording experimental results, can also be used for calling an applet or an app to directly analyze pictures, develop different application programs according to different requirements, can realize the digitization, visualization, micromation and convenience of detection, and is further applied to the detection fields of food and drug supervision, environmental monitoring, medical diagnosis and the like.
In summary, the CRISPR Cas detection method has great potential in rapid detection of new diseases, but at present, there is no portable detection device, and the CRISPR Cas detection method can be used and matched with a smart phone to perform real-time, quantitative and rapid intelligent detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a visual quantitative detection platform based on CRISPR Cas-portable detector-smart phone and application thereof.
The first object of the invention is to provide a visual quantitative detection platform based on colorimetric CRISPR Cas.
The second object of the invention is to provide an application of a visualized quantitative detection platform based on colorimetric CRISPR Cas in non-disease therapeutic diagnosis.
The third object of the invention is to provide a portable detector for visual quantitative detection based on a colorimetry.
In order to achieve the above object, the present invention is realized by the following technical scheme:
the utility model provides a visual quantitative detection platform based on colorimetry CRISPR Cas, includes portable detector, CRISPR Cas reaction system, image acquisition equipment and image data processing program based on the visual quantitative detection of colorimetry, but portable detector based on the visual quantitative detection of colorimetry includes airtight box (2), power (4) and is used for supporting shooting with image acquisition equipment's support (5), the upper plate and the lower plate of box (2) are parallel, and the upper plate of box (2) is provided with the observation window, is provided with light filter (1) on the observation window, is provided with sample placement area (8) on the lower plate, image acquisition equipment shoots the sample that awaits measuring of placing in sample placement area (8) through the observation window, obtains the sample fluorescent image that awaits measuring; sample feeding ports (3) which can be opened and closed are also arranged on the two side walls of the box body (2); two groups of parallel strip-shaped light sources (7) irradiating the sample placing area (8) are oppositely arranged on two side surfaces inside the box body (2); the strip-shaped light sources (7) are connected with the box body through opaque connecting plates (6), the connecting plates (6) are respectively positioned on two side walls of the box body, and an included angle theta between the mounting surfaces of the two groups of strip-shaped light sources (7) and the side walls is 15-60 degrees; the image processing program can convert the fluorescent part in the fluorescent image of the sample to be detected into a G value in RGB values, and substitutes the G value result into a linear fitting regression curve to output the values, wherein the linear fitting regression curve is drawn according to the relation between the concentration of the DNA standard of the gene to be detected and the G value.
Preferably, the CRISPR Cas reaction system comprises a crRNA whose nucleotide sequence is as shown in any one of the following groups: SEQ ID NO: 1-2, SEQ ID NO: 7-8, SEQ ID NO: 13-14, SEQ ID NO: 19-20, SEQ ID NO: 25-26, SEQ ID NO: 31-32 or SEQ ID NO:37 to 38.
More preferably, the CRISPR Cas reaction system further comprises a CRISPR Cas nuclease, a 10 x Cas nuclease buffer, fluorescently labeled ssDNA, an RNase Inhibitor, and enzyme-free water.
More preferably, the CRISPR Cas nuclease is Lbcpf1.
Even more preferably, the 10×cas nuclease buffer is 10×lbcpf1 buffer.
Most preferably, the CRISPR Cas reaction system is a CRISPR Cas12a reaction system.
Even most preferably, the CRISPR Cas12a reaction system comprises crRNA, 10 x Lbcpf1buffer, lbcpf1, fluorescently labeled ssDNA, RNase Inhibitor, and enzyme-free water.
Preferably, the fluorescent labeled ssDNA is FAM labeled at the 5 'end and BHQ labeled at the 3' end.
More preferably, the nucleotide sequence of the fluorescently labeled ssDNA is set forth in SEQ ID NO: 43.
Preferably, the image acquisition device is a smart phone.
More preferably, the image acquisition device is a smart phone of any brand of Hua Cheng, apple or millet.
Preferably, the linear fit regression curve is y=0.1512x+161.3.
The application of the visual quantitative detection platform in detection of non-disease diagnosis or treatment purpose is also within the protection scope of the invention, and the detection steps of the visual quantitative detection platform are as follows:
s1, extracting genome DNA of a sample to be detected, mixing the genome DNA with the CRISPR Cas reaction system, and then placing the mixture at a constant temperature of 28-37 ℃ for reaction for 10-30 min to obtain a reacted sample;
s2, placing the reacted sample in the portable detector based on the visual quantitative detection of the colorimetric method, and shooting the reacted sample in the portable detector by using image acquisition equipment to obtain a colorimetric result diagram;
s3, converting the fluorescent part of the reacted sample in the colorimetric result diagram into a G value in RGB values, taking the G value result as an x value to be taken into a linear fitting regression curve, and carrying out quantitative analysis, wherein the linear fitting regression curve is drawn according to the relation between the concentration of the DNA standard of the gene to be detected and the G value.
Preferably, in step S1, the sample to be tested is heated at 100℃for 10min, and genomic DNA of the sample to be tested is extracted.
Preferably, in step S1, the CRISPR Cas reaction system is a CRISPR Cas12a reaction system.
More preferably, in step S1, the genomic DNA of the sample to be tested is extracted, mixed with the CRISPR Cas reaction system, and then reacted at a constant temperature of 37 ℃ for 30min to obtain a reacted sample.
Most preferably, the sample to be tested is a sample containing klebsiella pneumoniae.
Preferably, in step S2, the image capturing device is a smart phone.
Preferably, in step S3, the linear fit regression curve is y=0.1512x+161.3.
Preferably, the visual quantitative detection platform is used for detecting pathogen genes in the detection of non-disease diagnosis or treatment purposes.
More preferably, the pathogen genes are klebsiella pneumoniae (k.p.) virulence gene rmpA, iucA, iroB, peg gene family and drug resistance gene blaNDM, blaKPC, blaOXA gene family.
The portable detector for visual quantitative detection based on the colorimetry is also in the protection scope of the invention.
Preferably, the strip light source (7) is a 470nm blue light source or a 620-750 nm red light source.
Preferably, the distance between the strip-shaped light source (7) and the lower plate is 3-6.5 cm.
Preferably, the strip-shaped light sources (7) are 1-6 constant-voltage LED light strips which are arranged in parallel.
More preferably, the strip-shaped light sources (7) are 6 constant-voltage LED lamp strips which are arranged in parallel, and the distances between the center points of the lamp strips on the left side and the right side and the bottom surface are 3cm, 3.6cm, 4.4cm, 5.2cm, 5.8cm and 6.5cm respectively.
Preferably, the symmetry axis of the strip-shaped light source (7) is perpendicular to the upper and lower plates of the box (2).
Preferably, the two groups of parallel strip-shaped light sources (7) are connected in series and then connected in series with the power supply (4), and the power supply (4) is a constant voltage power supply with the voltage of 5-12V.
More preferably, the lamp beads of the two groups of parallel strip-shaped light sources (7) are connected in series in the constant-voltage LED lamp strip.
Even more preferably, the power supply (4) is a 5V constant voltage power supply.
Most preferably, the power supply (4) is a 5V constant voltage battery.
Preferably, the connecting plates (6) are respectively positioned on two side walls of the box body, and an included angle theta between the mounting surfaces of the two groups of strip-shaped light sources (7) and the side walls is 30 degrees.
Preferably, the box body (2) is a cuboid of 9.9-10.4 cm multiplied by 9.9-10.1 cm.
Most preferably, the box (2) is a cube of 10cm by 10 cm.
Preferably, the sample placement area (8) is located at the center line of the lower plate.
Preferably, the sample placement area (8) can be used for placing a PCR tube or an ELISA plate or the like containing a sample.
The use of the portable detector in a gene quantification and/or analysis system is also within the scope of the present invention.
Compared with the prior art, the invention has the following beneficial effects:
the portable detector disclosed by the invention is simple in preparation process, easy to obtain and convenient to carry, the portable detector disclosed by the invention is used for detecting by matching with an image data processing program in a smart phone, the concentration of pathogen genomic DNA (deoxyribonucleic acid) and a fluorescence value have a linear relation in a certain range, picture results of different fluorescence colors are digitized by the image data processing program through algorithms, and the picture results are substituted into a standard curve to finish rapid quantitative detection of pathogen genes in clinical samples, so that the rapid quantitative detection of the clinical samples can be separated from traditional large-scale equipment such as qPCR (quantitative polymerase chain reaction), a microplate reader and a Raman spectrum detector, and can be used for quantitatively detecting experimental samples after real-time constant-temperature reaction. In addition, the mobile phones with different operating systems are suitable for the portable detector claimed by the invention, no extra equipment or complex operation is needed, the remote data transmission can be realized by utilizing the network connection of the mobile phones, and the real-time monitoring and the remote collaboration are convenient. Meanwhile, the quantitative detection result can be intuitively presented, and more intuitive data display and analysis can be provided.
The visual quantitative detection platform disclosed by the invention comprises a CRISPR Cas12a detection system, a detection object is pyrolyzed to obtain pathogen genome DNA, when target DNA exists in a sample to be detected, specific crRNA can guide Cas12a enzyme to cut the target DNA, and simultaneously, the trans-cutting activity of the Cas12a enzyme is excited, so that single-stranded DNA in the system is cut. Therefore, when the detection platform disclosed by the invention is used for detecting pathogen genes, after DNA is extracted in one step by a thermal cracking method, detection signals can be amplified at a constant temperature, and the CRISPR Cas12a detection system and the portable detector can be used for detection, wherein the detection duration is only 10 minutes at least, and compared with the traditional pathogen detection method, the detection speed is high; the experimental flow is simplified, and non-specific amplification is not easy to generate in a detection system.
The visual quantitative detection platform based on the CRISPR Cas12 a-smart phone can detect in scenes outside a laboratory, realizes clinical rapid in-situ detection, improves the flexibility and practicality of pathogen detection, can rapidly detect pathogens in a field environment, and is beneficial to timely taking targeted medical prevention and control measures; the visual quantitative detection platform provided by the invention enables the pathogen gene detection to be simpler, faster and more flexible, and is expected to play a great role in the fields of pathogen detection and the like.
Drawings
Fig. 1 is a schematic structural diagram of a portable detector for visual quantitative detection by colorimetry.
Fig. 2 is an exploded view of fig. 1.
FIG. 3 is a bar graph of percent fluorescence brightness for different lamp strip numbers and positions.
Fig. 4 is a schematic diagram of an independently developed image processing program interface display and sample quantitative detection.
Fig. 5 is a standard curve of the mobile phone.
Fig. 6 is a standard curve of the apple phone.
Fig. 7 is a millet handset standard curve.
Fig. 8 is a linear fit regression curve of iucA gene detection for different handset models.
Fig. 9 is a result of sensitivity detection of the iucA gene in klebsiella pneumoniae clinical samples by a visual quantitative detection platform based on CRISPR Cas12 a-smart phone.
Fig. 10 is a detection flow of a visual quantitative detection platform based on CRISPR Cas12 a-smart phone.
Detailed Description
The invention will be further elaborated in connection with the drawings and the specific embodiments described below, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 design and Synthesis of virulence Gene crRNA
1. Experimental method
The complete sequences of the virulence genes rmpA, iucA, iroB and peg344 gene families were obtained from the NCBI database and high frequency sequences were obtained by sequence alignment. The high frequency sequence of rmpA gene family was analyzed to be NZ_CP026012.1, the high frequency sequence of iucA gene family was analyzed to be NZ_CP026012.1, the high frequency sequence of iroB gene family was analyzed to be NZ_CP026012.1, and the high frequency sequence of peg344 gene family was analyzed to be MZ245622.1.
And performing multi-species reverse verification through online blast comparison, and determining that the target DNA template is not overlapped with a host and other common pathogens, thereby obtaining the target DNA template. And determining the used Cas12a enzyme (Lbcpf 1) and the specific scaffold sequence and PAM sequence thereof according to the target DNA template sequence, respectively designing 3 groups of crRNAs aiming at rmpA, iucA, iroB and peg344 gene families by utilizing the conserved target region sequence obtained by analysis, wherein the designed crRNAs are shown in a table 1.
TABLE 1 sequence information of crRNA primers for different virulence genes
The following reaction system was configured:
| component (A) | Final concentration | Dosage of |
| 10×Lbcpf1 buffer | - | 3μL |
| Lba Cas12a | 30nM | 1μL |
| Different virulence genes crRNA | 35nM | Each 3. Mu.L |
| DNA of different virulence genes | 3nM | 3μL |
| 5`FAM-TZ-1-GAT(SEQ ID NO：43) | 300nM | 0.9μL |
| RNase Inhibitor(0.4U/μL) | - | 3μL |
| Enzyme-free water | - | To 30 mu L |
And (3) carrying out constant-temperature reaction for 30min at 37 ℃ in the metal bath, further screening the synthesized crRNA according to the slope standard curve result in the qPCR instrument, and selecting the crRNA with the highest fluorescence value increasing speed in unit time for subsequent quantitative detection.
2. Experimental results
Slope standard curves obtained by constructing different virulence genes are shown in table 2.
TABLE 2 slope standard curve results for different virulence genes
The increase rate of the fluorescence value can be calculated by the tangential slope of each primer per unit time, and the larger the tangential slope is, the faster the increase rate of the fluorescence value per unit time is. As can be seen from Table 2, the tangential slopes of the primers A of the virulence genes rmpA, iucA, iroB and the peg344 are the largest, i.e. the fluorescent values of the primers A of the virulence genes rmpA, iucA, iroB and the peg344 increase at the highest rate in unit time, so the primers A are selected for subsequent quantitative detection.
EXAMPLE 2 design and Synthesis of drug resistant Gene crRNA
1. Experimental method
The complete sequences of the drug-resistant gene blaNDM, blaKPC and blaOxa48 gene families were obtained from the NCBI database and high frequency sequences were obtained by sequence alignment. The high frequency sequence of the blaNDM gene family was analyzed to be FN396876.1, the high frequency sequence of the blaKPC gene family was analyzed to be KJ151293.1, and the high frequency sequence of the blaOxA48 gene family was analyzed to be AY236073.2.
And performing multi-species reverse verification through online blast comparison, and determining that the target DNA template is not overlapped with a host and other common pathogens, thereby obtaining the target DNA template. And determining the used Cas12a enzyme (Lbcpfl) and the specific scaffold sequence and PAM sequence thereof according to the target DNA template sequence, respectively designing 3 groups of crRNAs aiming at blaNDM, blaKPC and blaOXA48 gene families by utilizing the sequences of the conserved target regions obtained by analysis, wherein the designed crRNAs are shown in Table 3.
TABLE 3 sequence information of crRNA primers for different drug resistance genes
The following reaction system was configured:
| component (A) | Final concentration | Dosage of |
| 10×Lbcpf1 buffer | - | 3μL |
| Lba Cas12a | 30nM | 1μL |
| Different drug resistance genes crRNA | 35nM | Each 3. Mu.L |
| Different drug resistance genes DNA | 3nM | 3μL |
| 5`FAM-TZ-1-GAT(SEQ ID NO：43) | 300nM | 0.9μL |
| RNase Inhibitor(0.4U/μL) | - | 3μL |
| Enzyme-free water | - | To 30 mu L |
And (3) carrying out constant-temperature reaction for 30min at 37 ℃ in the metal bath, further screening the synthesized crRNA according to the slope standard curve result in the qPCR instrument, and selecting the crRNA with the highest fluorescence value increasing speed in unit time for subsequent quantitative detection.
2. Experimental results
Slope standard curves obtained by constructing different drug resistance genes are shown in table 4.
TABLE 4 slope standard curve results for different drug resistance genes
The increase rate of the fluorescence value can be calculated by the tangential slope of each primer per unit time, and the larger the tangential slope is, the faster the increase rate of the fluorescence value per unit time is. As shown in Table 4, the tangential slopes of the A-group primers of the drug-resistant gene blaNDM, blaKPC and blaOxA48 were the largest, i.e., the fluorescence value of the A-group primers of the drug-resistant gene blaNDM, blaKPC and blaOxA48 increased at the highest rate per unit time, so that the A-group primers were selected for subsequent quantitative detection.
Example 3 Portable detector for visual quantitative detection of colorimetry, autonomously developed image processing program and test of different models of mobile phones
1. Portable detector for visual quantitative detection by colorimetric method
The portable detector is shown in fig. 1 and 2, and comprises a sealable box body (2), a power supply (4) and a bracket (5) for supporting image acquisition equipment for shooting, wherein an upper plate and a lower plate of the box body (2) are parallel, an observation window is arranged on the upper plate of the box body (2), an optical filter (1) is arranged on the observation window, a sample placement area (8) is arranged on the lower plate, and the image acquisition equipment can shoot a sample to be detected placed in the sample placement area (8) through the observation window; the box body (2) is also provided with a sample feeding port (3) which can be opened and closed; two groups of parallel strip-shaped light sources (7) are arranged on two sides of the inside of the box body (2).
The two groups of strip-shaped light sources (7) are constant-voltage LED lamp strips with the left side and the right side both arranged on the inclined plane of the triangular prism in parallel, the constant-voltage LED lamp strips are connected with the box body through opaque connecting plates (6), the connecting plates (6) are respectively positioned on the two sides of the box body, an included angle theta between the mounting surfaces and the side walls of the two groups of strip-shaped light sources (7) is 30 degrees, 6 constant-voltage LED lamp strips are arranged on each side, and the distances between the center points of the lamp strips on the left side and the right side and the bottom surface are 3cm, 3.6cm, 4.4cm, 5.2cm, 5.8cm and 6.5cm respectively; the lamp beads are connected in series in the constant-voltage LED lamp strip. The two groups of strip light sources (7) are connected in series and then connected in series with a power supply (4), the strip light sources (7) are 470nm blue light sources or 620-750 nm red light sources, and the power supply (4) is a 5V constant voltage battery.
A transparent PCR tube or an ELISA plate containing a sample can be placed on the sample placement area (8).
2. Influence of lamp strip number and position of portable detector for visual quantitative detection of colorimetry on fluorescent picture result
(1) Experimental method
The overall structure of the portable detector is the same as the "portable detector for visual quantitative detection by colorimetric method" in this embodiment, except that in the internal structure, the distances between the center points of the light strip and the bottom surface are shown in table 5.
As shown in table 5, the G value obtained by the test of the device built in the lamp strip with the highest brightness is taken as a reference value (recorded as 100%), and the G value obtained by the test of the device built in the lamp strip with the different numbers of the remaining lamp strips (one, two, three, four or six) and the lamp strips with the different lamp strip positions (upper, middle or lower) is normalized, and the devices built in the different lamp strip positions with the same lamp strip number are taken as a group, and the total number is 5; after the intra-group comparison, a bar graph is drawn by taking the highest percentage of each of the 5 groups for the inter-group comparison. The specific parameters are shown in Table 5.
TABLE 5 summary of distances from center to bottom for lamp strip at different numbers and positions (units: cm)
(2) Experimental results
The transparent PCR tube is characterized by light transmission and reflection, is easily interfered by external environment light, and can generate light reflection in the light path direction of a light source on the wall of the transparent PCR tube, so that the reflection noise of a target area in a shot picture is increased, and the recognition effect is interfered. The position with the highest brightness of different lamp bands is selected as a representative example, a fluorescence brightness percentage histogram is drawn, as shown in fig. 3, the portable detector is provided with 6 lamp bands, and when the distances between the central point of each lamp band and the bottom surface are respectively 3, 3.6, 4.4, 5.2, 5.8 and 6.5cm, the brightness percentage is the highest, and the fluorescence identification effect is the best.
3. Influence of different condensing devices on detection effect of portable detector for visual quantitative detection of colorimetric method
(1) Experimental method
The overall structure of the portable detector is the same as the portable detector for visual quantitative detection of the first and second embodiments, and the difference is that in the internal structure, two groups of strip-shaped light sources (7) are constant-voltage LED light strips with both left and right sides being arranged on the side wall of the box body (2) in parallel, the two groups of strip-shaped light sources (7) are fixed on the side wall of the box body (2) through a lamp holder, and as a light-gathering structure, a plastic diffuser is arranged below the lamp holder, and a concave lens is arranged below the plastic diffuser.
(2) Experimental results
The light of the two groups of strip-shaped light sources (7) passing through the concave lens can be dispersed to different degrees, the reflection noise point and the shadow area of a target area in a shot picture are increased, and interference is generated on the identification effect, so that the opaque connecting plates (6) are selected as light condensing devices, the connecting plates (6) are respectively positioned at two sides of the box body, and the included angle theta between the mounting surfaces and the side walls of the two groups of strip-shaped light sources (7) is ensured to be 30 degrees.
4. Self-developed image processing program
The image processing program used by the invention is a WeChat applet which is independently developed based on JavaScript and Python languages, and the WeChat applet comprises four plates of 'selecting a photo', 'starting to detect', 'taking a color from a region', 'detecting and recording'.
The image processing program interface developed independently is shown in fig. 4, wherein a in fig. 4 is a main interface, B in fig. 4 is a model selection interface, C in fig. 4 is a program front page, D in fig. 4 is a recognition result interface, E in fig. 4 is an image analysis interface, and F in fig. 4 is a quantitative detection interface.
The image processing program independently researched and developed can identify the picture containing a plurality of test tubes at one time, accurately identify the area with irregular shape and automatically cut the picture by automatically identifying the edge of a sample to be identified through an automatic identification frame, automatically thresholding the picture and morphologically processing the picture according to mean filtering and bilateral filtering, extracting the roi value of the brightest area in the middle of the picture, taking the measuring point on the basis of the roi, and achieving the technical effect of intelligently removing the reflection point, so that the identification is more accurate.
5. Testing of different mobile phone models
(1) Experimental method
0.01mg of fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) powder was weighed, dissolved in 5mL of PBS buffer, and mixed well to prepare a 2. Mu.g/mL FITC solution. Taking 30 mu L of the FITC solution as a positive control group, diluting the solution with an exponential multiple of 10 to prepare FITC concentrations of 2 mu g/mL, 0.2 mu g/mL, 0.02 mu g/mL and 2X 10 respectively -3 μg/mL、2×10 -4 Mu g/mL and 2X 10 -5 The sample to be measured in mug/mL is placed in the portable detector for visual quantitative detection of the first and second colorimetry methods, and different brands of mobile phones (apples, hua being or millet) are used for shooting the colorimetric result diagrams of the same group of sample to be measured. And converting the colorimetric result diagram into an RGB component image by using an independently developed image processing program in the second embodiment, identifying to obtain a required G value, and drawing three standard curves according to the G value of the colorimetric result diagram of the sample to be detected.
(2) Experimental results
As shown in a in fig. 5 and B in fig. 5, bloom is a cell phone standard curve: y=37.66x+279.3, r 2 =0.98; as shown in a in fig. 6 and B in fig. 6, the standard curve of the apple phone: y=36.05x+288.1, r 2 =0.97; as shown by a in fig. 7 and B in fig. 7, smallStandard curve of rice mobile phone: y=25.36x+252.8, r 2 =0.98. The logarithmic concentration (x) and the G value (y) of the fluorescein of different brands of mobile phones are in good linear relation.
Example 4 application of CRISPR Cas-portable detector-smart phone-based visualized quantitative detection platform in clinical sample detection
1. Drawing of a Linear fitting regression Curve
Taking klebsiella pneumoniae pathogen gene iucA as an example:
s1, preparing klebsiella pneumoniae pathogen gene iucA DNA standard substances with the concentration of 500pM, 400pM, 300pM, 200pM, 100pM and 0pM, and obtaining gene standard substances to be detected with different concentrations;
s2, placing the gene standard to be detected obtained in the step S1 in a portable detector for visual quantitative detection of the first embodiment and the colorimetric method, placing the gene standard to be detected in a bottom sample placing bin of the portable detector, opening a battery box switch, adjusting a smart phone into a shooting mode, shooting the smart phone from top to bottom through a hole right above, selecting 2.5 times of amplification factor, shooting by using a shooting function of the smart phone to obtain a colorimetric result diagram of the gene standard to be detected, and taking possible differences of shooting effects of different mobile phones into consideration, respectively shooting colorimetric result diagrams of the gene standard to be detected by using Huacheng, apple and millet mobile phones, wherein each mobile phone repeatedly shoots three times;
s3, recognizing the G value of the fluorescent part in the colorimetric result diagram of the standard sample of the gene to be detected, which is obtained by shooting in the step S2, through an independently developed image processing program in the step S3, taking the average of three G values obtained by the same sample on the same device, obtaining the G average value of the standard samples shot by different mobile phones as original data, and drawing a linear fitting regression curve y=0.1512x+161.3 suitable for quantitative detection of the iucA gene by different devices through optimization processing of the original data, wherein the obtained linear fitting regression curve is shown in fig. 8.
2. Clinical sample detection method based on CRISPR Cas-portable detector-smart phone visual quantitative detection platform
S1.100 ℃ heating a sample to be detected for 10min to obtain genome DNA of the sample to be detected;
s2, taking the genome DNA obtained in the step S1 as a template, mixing the CRISPR-Cas12a reaction system according to the formula shown in the table 6, placing the prepared sample containing the CRISPR Cas12a reaction system into a metal bath preheated in advance, reacting at the constant temperature of 37 ℃ for 30min, taking out a PCR tube, uniformly mixing and centrifuging to obtain a reacted sample;
s3, placing the reacted sample obtained in the step S2 in a portable detector for visual quantitative detection of the first embodiment and the colorimetric method, placing the reacted sample in a bottom sample placing bin of the portable detector, opening a battery box switch, adjusting the smart phone into a photographing mode, photographing the smart phone from top to bottom through a hole right above, selecting 2.5 times of magnification, and photographing a colorimetric result chart by using a photographing function of the smart phone;
s4, converting the colorimetric result diagram obtained in the step S3 into an RGB component image by using an independently developed image processing program in the second embodiment, obtaining a required G value, taking the numerical result of the G value of the colorimetric result diagram of the sample to be detected as an x value by using the image processing program, taking the numerical result of the G value of the colorimetric result diagram of the sample to be detected as the x value, carrying out quantitative analysis on a fluorescence result in a linear fitting regression curve drawn according to the relation between the concentration of the DNA standard sample of the gene to be detected and the G value, and outputting the gene concentration of the sample to be detected.
TABLE 6CRISPR-Cas12a reaction System
3. Sensitivity of visual quantitative detection platform in clinical detection based on CRISPR Cas-portable detector-smart phone
(1) Experimental method
Taking 5 mu L of a glycerin strain of Klebsiella pneumoniae (K.P.) which is clinically obtained and stored in a refrigerator at-20 ℃, adding 5 mu L of liquid LB medium containing ampicillin, culturing for 8-12 h at 150rpm at 37 ℃ in a shaking table, and measuring OD 600 Value, guarantee OD 600 The value is in the range of 0.2 to 0.8. Taking 1.4mL of bacterial liquid, centrifuging to remove supernatant, adding 100 mu L of deionized water for resuspension, sealing an EP tube with a sealing film, and boiling at 100deg.CBoiling for 10min, centrifuging the thermal cracking solution at 12000rpm for 2min, and obtaining the supernatant as clinical klebsiella pneumoniae DNA.
The supernatant was subjected to gradient dilution to obtain a thermal lysate supernatant after the gradient dilution at DNA concentrations of 310 ng/. Mu.L, 155 ng/. Mu.L, 77.5 ng/. Mu.L, 40 ng/. Mu.L, 20 ng/. Mu.L and 10 ng/. Mu.L. According to the second embodiment, a clinical sample detection method based on a CRISPR Cas-portable detector-smart phone visual quantitative detection platform is used for detecting the supernatant of the thermal cracking liquid after gradient dilution. Three tubes were repeatedly arranged for each concentration of sample, and the mixture was centrifuged.
(2) Experimental results
Taking the virulence gene iucA as an example, fig. 9 shows that the visual quantitative detection platform of the CRISPR Cas-portable detector-smart phone detects the sensitivity of the iucA gene in the klebsiella pneumoniae clinical sample, and as can be seen from fig. 9, the clinical sample detection method of the second embodiment, which is based on the visual quantitative detection platform of the CRISPR Cas-portable detector-smart phone, has high sensitivity in clinical detection.
4. CRISPR Cas-portable detector-smart phone-based visual quantitative detection platform detection result accuracy
(1) Experimental method
Taking three known clinical samples containing the genes as an example of the virulence gene iucA, according to the first embodiment of the clinical sample detection method based on the CRISPR Cas-portable detector-smart phone visual quantitative detection platform, detecting thermal cracking to extract DNA, configuring a CRISPR Cas12a reaction system, and performing constant-temperature reaction at 37 ℃ for 30min. The clinical sample. And substituting the signal intensity identified by the portable detector into a standard curve, calculating to obtain a concentration value and a coincidence degree, and judging the accuracy of the quantitative detection method.
(2) Experimental results
Taking the virulence gene iucA as an example, three clinical samples containing the genes are known, the concentration of the DNA obtained after thermal cleavage extraction is 500pM,200pM and 45pM, and the linear fitting regression curve y=0.1512x+161.3 is obtained by drawing the linear fitting regression curve according to the first embodiment. G values detected by a CRISPR Cas-portable detector-smart phone based visual quantitative detection platform clinical sample detection method are 233.50, 192.00 and 168.00 respectively, and are substituted into linear fitting regression curves to obtain concentration values of 477.5pM, 203.00pM and 44.3pM, wherein the coincidence degrees are 95.5%, 101.5% and 98.4% respectively, and the coincidence degrees are within an error allowable range. The detection result obtained by the clinical sample detection method based on the CRISPR Cas-portable detector-smart phone visual quantitative detection platform is accurate.
The invention constructs a visual quantitative detection platform based on a CRISPR Cas12 a-smart phone by using an independently developed image processing program and matching with a CRISPR Cas12 a-pathogen gene detection system and a portable detector for visual quantitative detection of a colorimetric method, and the visual quantitative detection platform is used for detecting K.P. samples containing virulence genes rmpA, iucA, iroB and peg344, drug-resistant genes blaNDM, blaKPC and blaOXA48, and the detection flow is shown in figure 10. The color recognition software of the intelligent mobile phone can recognize and display the values in an R (red) red channel, a G (green) green channel and a B (blue) blue channel in a color picture RGB color space, and substitutes the values of the G (green) green channel into an established linear fitting regression curve to obtain the concentration values of virulence genes and drug-resistant genes.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The visual quantitative detection platform based on the colorimetric CRISPR Cas is characterized by comprising a portable detector based on the visual quantitative detection of the colorimetric method, a CRISPR Cas reaction system, an image acquisition device and an image data processing program, wherein the portable detector based on the visual quantitative detection of the colorimetric method comprises a sealable box body (2), a power supply (4) and a bracket (5) for supporting the image acquisition device for shooting, an upper plate and a lower plate of the box body (2) are parallel, an observation window is arranged on the upper plate of the box body (2), an optical filter (1) is arranged on the observation window, a sample placement area (8) is arranged on the lower plate, and the image acquisition device shoots a sample to be detected in the sample placement area (8) through the observation window to obtain a fluorescent image of the sample to be detected; sample feeding ports (3) which can be opened and closed are also arranged on the two side walls of the box body (2); two groups of parallel strip-shaped light sources (7) irradiating the sample placing area (8) are oppositely arranged on two side surfaces inside the box body (2); the strip-shaped light sources (7) are connected with the box body through opaque connecting plates (6), the connecting plates (6) are respectively positioned on two side walls of the box body, and an included angle theta between the mounting surfaces of the two groups of strip-shaped light sources (7) and the side walls is 15-60 degrees; the image processing program can convert the fluorescent part in the fluorescent image of the sample to be detected into a G value in RGB values, and substitutes the G value result into a linear fitting regression curve to output the values, wherein the linear fitting regression curve is drawn according to the relation between the concentration of the DNA standard of the gene to be detected and the G value.
2. The visual quantitative detection platform of claim 1, wherein crRNA is included in the CRISPR Cas reaction system, the nucleotide sequence of the crRNA being as set forth in any one of the following: SEQ ID NO: 1-2, SEQ ID NO: 7-8, SEQ ID NO: 13-14, SEQ ID NO: 19-20, SEQ ID NO: 25-26, SEQ ID NO: 31-32 or SEQ ID NO:37 to 38.
3. The visual quantitative detection platform of claim 1, wherein the image acquisition device is a smart phone.
4. The use of the visual quantitative detection platform according to claim 1 for the detection of non-disease diagnosis or treatment purposes, wherein the detection steps of the visual quantitative detection platform are as follows:
s1, extracting genome DNA of a sample to be detected, mixing the genome DNA with the CRISPR Cas reaction system, and then placing the mixture at a constant temperature of 28-37 ℃ for reaction for 10-30 min to obtain a reacted sample;
s2, placing the reacted sample in the portable detector based on the visual quantitative detection of the colorimetric method, and shooting the reacted sample in the portable detector by using image acquisition equipment to obtain a colorimetric result diagram;
s3, converting the fluorescent part of the reacted sample in the colorimetric result diagram into a G value in RGB values, taking the G value result as an x value to be taken into a linear fitting regression curve, and carrying out quantitative analysis, wherein the linear fitting regression curve is drawn according to the relation between the concentration of the DNA standard of the gene to be detected and the G value.
5. A portable detector for colorimetric-based visual quantitative detection as claimed in claim 1.
6. The portable detector according to claim 5, characterized in that the strip light source (7) is a 470nm blue light source or a 620-750 nm red light source.
7. The portable detector according to claim 5, characterized in that the distance of the strip light source (7) from the lower plate is 3-6.5 cm.
8. The portable detector according to claim 5, wherein the strip-shaped light sources (7) are 1-6 constant voltage LED strips arranged in parallel.
9. The portable detector according to any one of claims 7 or 8, wherein the strip-shaped light sources (7) are 6 constant-voltage LED light strips arranged in parallel, and the distances between the center points of the light strips on the left side and the right side and the bottom surface are 3cm, 3.6cm, 4.4cm, 5.2cm, 5.8cm and 6.5cm respectively.
10. The portable detector according to any one of claims 5 to 9, wherein the two parallel groups of strip-shaped light sources (7) are connected in series and then connected in series to the power supply (4), and the power supply (4) is a constant voltage power supply of 5 to 12V.
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