A crRNA combination for detecting Epstein-Barr virus based on microdroplet digital analysis and its application

Abstract

The present invention discloses a crRNA for detecting Epstein-Barr virus based on microdroplet digital analysis, wherein the crRNA is selected from one or more of the crRNAs shown in nucleotide sequences such as SEQ ID NO: 1 to SEQ ID NO: 11. The quantitative detection kit for Epstein-Barr virus nucleic acid constructed using the crRNA of the present invention does not require a DNA amplification step in the process of detecting Epstein-Barr virus, thus avoiding the result bias and possible contamination risk generated during the amplification process; and the result collection is completed using a fluorescent microscope, which is convenient and fast, and is suitable for rapid detection at the screening site. In addition, the quantitative detection kit for Epstein-Barr virus nucleic acid can achieve absolute quantification of the single-copy Epstein-Barr virus DNA level, without the need for internal reference correction and the establishment of a standard curve, thus avoiding the error caused by the use of different standards, and compared with the traditional qPCR detection method, the screening results have significant advantages.

Description

Technical Field

[0001] The present invention relates to the technical field of viral nucleic acid detection, and in particular to a crRNA combination for detecting Epstein-Barr virus based on microdroplet digital analysis and an application thereof. Background Art

[0002] Nasopharyngeal carcinoma is a head and neck malignant tumor originating from the nasopharyngeal epithelium. The early symptoms are atypical, and patients are often in the late stage when they seek medical treatment. Epstein-Barr virus infection is closely related to the development of nasopharyngeal carcinoma. The main methods for early screening and detection of EBV in nasopharyngeal carcinoma include: plasma EBV-related antibody detection (such as VCA-IgA, EBNA-IgA, etc.), EBV DNA copy number detection (including plasma and nasopharyngeal swabs) and EBV methylation detection.

[0003] Existing studies have shown that nasopharyngeal carcinoma screening based on efficient plasma free EBV DNA copy number can significantly prolong patient survival. Real-time quantitative polymerase chain reaction (real-time qPCR) is currently the most commonly used method for EBV DNA copy number detection. The BamHI-W region is a repetitive fragment region of the EBV genome, which is easy to detect low copy number samples and is the most commonly used target for PCR detection. However, there is currently a lack of internationally standardized absolute quantitative detection conditions and protocols, a lack of universal quantitative standards and detection sequences, resulting in large differences in test results from different institutions and difficulty in mutual verification; and EB virus detection methods based on nucleic acid amplification all involve large-scale replication of nucleic acids (tens of thousands of times), which will bring a series of problems, especially the amplification deviation caused by errors in the sequence replication process and the false positive results that may be caused by contamination of the amplification product.

[0004] Nucleic acid detection technology based on microdroplet digital analysis can divide the reaction sample into hundreds of thousands of tiny reaction units of droplets, so that each droplet reaction unit contains at most one target molecule to be tested, and then detects the target and generates a signal through CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas protein and its probe at a constant temperature. The original copy number or concentration of the target can be calculated by Poisson distribution algorithm based on the proportion of its fluorescence or other signals in the total reaction unit. Compared with traditional methods, nucleic acid detection technology based on microdroplet digital analysis can achieve absolute nucleic acid quantification at the single molecule level without relying on positive standard references. Because its constant temperature reaction is easy to implement and the reaction system is not affected by amplification efficiency, it can ensure good convenience, stability and quantitative accuracy, which helps to solve the problems of rapid nucleic acid detection in primary medical structures, retesting of low-concentration clinical samples and repeatability of detection structures in different institutions. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide a crRNA combination for detecting Epstein-Barr virus based on microdroplet digital analysis and its application.

[0006] The first object of the present invention is to provide a crRNA combination for detecting Epstein-Barr virus based on microdroplet digital analysis.

[0007] The second object of the present invention is to provide the use of the above-mentioned crRNA combination in the preparation of an EB virus nucleic acid quantitative detection kit.

[0008] The third object of the present invention is to provide a quantitative detection kit for EB virus nucleic acid.

[0009] In order to achieve the above object, the present invention is implemented by the following scheme:

[0010] A crRNA combination for detecting Epstein-Barr virus based on microdroplet digital analysis, wherein the crRNA is one or more of the crRNAs with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 11.

[0011] Preferably, the crRNA is one or more of the crRNAs having nucleotide sequences such as SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and / or SEQ ID NO: 8.

[0012] More preferably, the crRNA is a crRNA having a nucleotide sequence as shown in SEQ ID NO: 3 and a crRNA having a nucleotide sequence as shown in SEQ ID NO: 7.

[0013] The present invention also seeks to protect the use of the above-mentioned crRNA combination in the preparation of an EB virus nucleic acid quantitative detection kit.

[0014] The present invention also seeks to protect a quantitative detection kit for Epstein-Barr virus nucleic acid, which is based on microdroplet digital analysis and achieves absolute quantification of the Epstein-Barr virus DNA copy number in clinical samples at the single-molecule level under non-amplification conditions, without the need for quantitative standards, thereby reducing the risk of false positives and reducing the detection differences between different testers under different quantitative PCR detection conditions (standards and reaction systems).

[0015] A kit for quantitative detection of Epstein-Barr virus nucleic acid, comprising the above-mentioned crRNA combination.

[0016] Preferably, it also contains Cas12a protein, single-stranded DNA reporter probe and / or reaction buffer;

[0017] The sequence of the single-stranded DNA reporter probe is: 5'-CCCCCC-3'.

[0018] More preferably, the reaction buffer comprises 10-200 mM Tris-HCl, 50-250 mM MgCl2, 50-150 mM KCl and / or PEG200 with a mass / volume ratio of 25-50%.

[0019] Further preferably, the reaction buffer comprises 100 mM Tris-HCl, 150 mM MgCl2, 100 mM KCl and / or 50% PEG200.

[0020] More preferably, the pH of the reaction buffer is 8.5-9.5.

[0021] More preferably, the pH of the reaction buffer is 9.0.

[0022] Preferably, the method for using the EB virus nucleic acid quantitative detection kit is specifically as follows:

[0023] S1. Collect the DNA sample to be tested and mix it with the components of the EB virus nucleic acid quantitative detection kit to obtain a reaction mixture;

[0024] S2. The reaction mixture obtained in step S1 is dispersed into microdroplets with a diameter of 20 to 30 μm and a number of not less than 30,000, and incubated at a constant temperature of 37 to 45 ° C for 60 to 100 min;

[0025] S3. Use a fluorescence microscope to take bright field and fluorescent images of the droplets, observe the number of fluorescent droplets in the droplets after the incubation, determine the ratio of fluorescent droplets, and calculate the number of EB virus DNA copies according to the Poisson distribution algorithm;

[0026] Fluorescent microdroplet ratio = number of positive droplets / total number of droplets;

[0027] The Poisson distribution algorithm is shown in Formula I;

[0028] Formula I:

[0029] Wherein, c is the EB virus DNA copy number, in copies per microliter (copies / μL); PR represents the ratio of fluorescent microdroplets; r represents the average radius of a droplet; V represents the average volume of a droplet, in μL.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention provides a crRNA for detecting Epstein-Barr virus based on microdroplet digital analysis, wherein the crRNA is selected from one or more of the crRNAs with nucleotide sequences such as SEQ ID NO: 1 to SEQ ID NO: 11. The quantitative detection kit for Epstein-Barr virus nucleic acid constructed using the crRNA of the present invention does not require a DNA amplification step in the process of detecting Epstein-Barr virus, thus avoiding the result bias and possible contamination risk generated during the amplification process; and a fluorescence microscope can be used to complete the result collection, so the detection is convenient and fast, and is suitable for rapid detection at the screening site.

[0032] In addition, the EB virus nucleic acid quantitative detection kit can achieve absolute quantification of single-copy EB virus DNA levels without the need for internal reference correction and establishment of a standard curve, thus avoiding errors caused by the use of different standards, and compared with traditional qPCR detection methods, the screening results are significantly superior. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Fluorescence-time curve diagram of EB virus detected by each crRNA;

[0034] Figure 2 This is a fluorescence-time curve diagram of crRNA combination detection of EB virus;

[0035] Figure 3 is the number graph of fluorescent microdroplets;

[0036] Figure 4 is the concentration curve diagram;

[0037] Figure 5 The figure shows the quantitative test results of saliva samples from 11 patients with nasopharyngeal carcinoma;

[0038] Figure 6 The quantitative test results of plasma samples from 100 nasopharyngeal carcinoma patients and 108 healthy subjects are shown in the figure;

[0039] Figure 7 A is the ROC curve of EBV DNA copy number based on microdroplet digital analysis and EBV DNA copy number corresponding to qPCR technology; A is the ROC curve of EBV DNA copy number based on microdroplet digital analysis; B is the ROC curve of EBV DNA copy number corresponding to qPCR technology;

[0040] Figure 8 It is a correlation curve diagram between the EBV DNA copy number based on microdroplet digital analysis and the EBV DNA copy number corresponding to the qPCR technology;

[0041] Fig. 9A is a box plot and ROC curve diagram of EBV DNA copy number corresponding to samples of different stages; A is a box plot of EBV DNA copy number corresponding to different clinical stages based on microdroplet analysis; B is a box plot of EBV DNA copy number corresponding to different clinical stages based on qPCR technology; C is a ROC curve diagram of EBV DNA copy number corresponding to different clinical stages based on microdroplet analysis; D is a ROC curve diagram of EBV DNA copy number corresponding to different clinical stages based on qPCR technology. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0043] The BamHI-W region of the EBV genome described in the present invention is the BamHI-W region reported by Dennis Lo's team (PMID: 15014025; 23459720; 28792880).

[0044] Example 1 Screening of crRNA for EB virus based on microdroplet digital analysis

[0045] 1. Experimental methods

[0046] Based on the BamHI-W region of the EBV (Epstein-Barr virus) genome, Benchling was used to design and screen crRNA sequences with off-target scores greater than 95, and a total of 8 23bp crRNAs (BamHI-W-crRNA T1 to BamHI-W-crRNAT8) and 3 20bp crRNAs (BamHI-W-crRNA T1', BamHI-W-crRNAT4' and BamHI-W-crRNA T8') were obtained.

[0047] Specifically, crRNA and its nucleotide sequence are shown in Table 1.

[0048] Table 1 crRNA and its nucleotide sequence information

[0049]

[0050]

[0051] Taking BamHI-W-crRNA T1 as an example, it is used as crRNA to perform EB detection on BamHI-W quantitative reference. The detection method is as follows:

[0052] (1) The DNA sample in this example is a BamHI-W quantitative reference substance, and the preparation method is as follows:

[0053] Using BamHI-W Linear-F having a nucleotide sequence such as SEQ ID NO: 15 and BamHI-W Linear-R having a nucleotide sequence such as SEQ ID NO: 16, PCR amplification was performed on the Epstein-Barr virus PCR amplification standard (EB002) according to the reaction system shown in Table 2 and the reaction procedure shown in Table 3. After the amplification, the amplified products were collected and electrophoresed on a 1.0% agarose gel, and then recovered using a GeneJET gel recovery kit (ThermoFIsher) to obtain a BamHI-W quantitative reference (nucleotide sequence as shown in SEQ ID NO: 17).

[0054] BamHI-W Linear-F (SEQ ID NO: 15): 5'-CCCACGGTGGACAGGC-3';

[0055] BamHI-W Linear-R (SEQ ID NO: 16): 5'-GGAGCGGAGACCAGGAG-3'.

[0056] Table 2 PCR amplification reaction system

[0057]

[0058]

[0059] Table 3 PCR amplification reaction program

[0060]

[0061] (2) Prepare 5 μL of reaction premix according to the system shown in Table 4.

[0062] Table 4 Reaction system

[0063] Components Volume / final concentration Cas12a protein 200nM crRNA (BamHI-W-crRNA T1, SEQ ID NO: 1) 200nM Single-stranded DNA reporter probe 500nM 1× reaction buffer, pH 9.0 0.5μL RNase inhibitors 1U / μL DEPC water To 5μL

[0064] The sequence of the single-stranded DNA reporter probe is: 5'-CCCCCC-3'; 1× reaction buffer is obtained by diluting 10× reaction buffer, and 10× reaction buffer contains Tris-HCl with a final concentration of 100mM, MgCl2 of 150mM, KCl of 100mM and PEG200 of 50% by volume.

[0065] Thoroughly mix 5 μL of the reaction premix and 5 μL of the DNA sample from step S1 to obtain a reaction mixture;

[0066] (3) adding the reaction mixture obtained in step (2) to a 96-well plate and placing it in a fluorescence quantitative PCR instrument (ROCHE 480, 96-well) for detection at a constant temperature of 45° C., collecting fluorescence signals every 2 minutes, and collecting them continuously for 45 times to obtain real-time fluorescence signal results;

[0067] (4) dispersing the reaction mixture obtained in step (2) into microdroplets with a diameter of 20 to 30 μm and a number of microdroplets of not less than 30,000, heating the mixture at a constant temperature of 45° C. for reaction, and taking photos and observing the number of fluorescent droplets using an inverted fluorescence microscope (Nikon, ECLIPSETi2-U) after 60 minutes;

[0068] (5) plotting a fluorescence-time curve using the fluorescence signal of the real-time fluorescence signal collected in step (3) as the vertical axis and time as the horizontal axis; calculating the positive droplet ratio (fluorescent droplet number ratio) of step (4), and calculating the DNA concentration of the DNA sample using a Poisson distribution algorithm based on the positive droplet ratio p and the droplet radius r. The Poisson distribution algorithm is shown in Formula I;

[0069] Formula I:

[0070] Wherein, c is the EB virus DNA copy number, in copies per microliter (copies / μL); PR represents the ratio of fluorescent microdroplets; r represents the average radius of a droplet; V represents the average volume of a droplet, in μL.

[0071] The negative control was set up without adding DNA sample and the detection was performed according to the same steps.

[0072] BamHI-W-crRNA T2 to BamHI-W-crRNA T8, BamHI-W-crRNA T1', BamHI-W-crRNA T4' and BamHI-W-crRNA T8' were used to replace BamHI-W-crRNA T1, and the same treatment was performed, and the corresponding fluorescence-time curves were drawn.

[0073] 2. Experimental results

[0074] The fluorescence-time curves obtained using BamHI-W-crRNA T1 to BamHI-W-crRNA T8, BamHI-W-crRNA T1', BamHI-W-crRNA T4' and BamHI-W-crRNA T8' were summarized. The results are as follows: Figure 1As shown; The results show that the fluorescence effects of BamHI-W-crRNA T3, BamHI-W-crRNA T5, BamHI-W-crRNA T7 and BamHI-W-crRNA T8 are significantly better than other crRNAs. Therefore, BamHI-W-crRNA T3, BamHI-W-crRNA T5, BamHI-W-crRNA T7 and BamHI-W-crRNA T8 are the optimal crRNA sequences. Example 2 Screening of crRNA combinations for detecting Epstein-Barr virus based on microdroplet digital analysis

[0075] The optimal sequences obtained in Example 1: BamHI-W-crRNA T3 (SEQ ID NO: 3), BamHI-W-crRNAT5 (SEQ ID NO: 5), BamHI-W-crRNA T7 (SEQ ID NO: 7) and BamHI-W-crRNA T8 (SEQ ID NO: 8) were used as experimental objects, and the EB virus detection effect was obtained using (BamHI-W-crRNA T3 + BamHI-W-crRNA T5), (BamHI-W-crRNA T3 + BamHI-W-crRNA T7) and (BamHI-W-crRNA T3 + BamHI-W-crRNA T8) as crRNA.

[0076] 1. Experimental methods

[0077] Taking (BamHI-W-crRNA T3 + BamHI-W-crRNA T5) as crRNA as an example, detection was performed. The difference between the detection method and the detection method described in Example 1 is:

[0078] The crRNA in the reaction system shown in Table 4 was replaced with BamHI-W-crRNAT3 (SEQ ID NO: 3) with a final concentration of 200 nM and BamHI-W-crRNA T5 (SEQ ID NO: 5) with a final concentration of 200 nM. The rest of the treatment methods were the same, and the corresponding fluorescence-time curves were drawn, and the fluorescent microdroplet number images were recorded.

[0079] BamHI-W-crRNA T5, BamHI-W-crRNA T7, BamHI-W-crRNA T8, (BamHI-W-crRNA T3+BamHI-W-crRNA T7) and (BamHI-W-crRNA T3+BamHI-W-crRNA T8) were used to replace (BamHI-W-crRNA T3+BamHI-W-crRNA T5), respectively, and the same detection was performed and the corresponding fluorescence-time curves were plotted.

[0080] The negative control was set up without adding crRNA and the same treatment was performed.

[0081] 2. Experimental results

[0082] The fluorescence-time curves drawn separately are summarized, and the results are as follows Figure 2 As shown; the results show that when (BamHI-W-crRNAT3+BamHI-W-crRNA T7) are used together as crRNA, the fluorescence effect of detecting EB virus is significantly better than other combinations. Therefore, the combination of (BamHI-W-crRNA T3+BamHI-W-crRNA T7) as crRNA is the optimal combination for detecting EB virus;

[0083] (BamHI-W-crRNA T3+BamHI-W-crRNA T5), (BamHI-W-crRNA T3+

[0084] The fluorescence microdroplet number images of (BamHI-W-crRNA T7) and (BamHI-W-crRNA T3+BamHI-W-crRNA T8) are shown in Figure 3 As shown, the results show that (BamHI-W-crRNA T3+BamHI-W-crRNA T7) is the largest number of fluorescent microdroplets observed in the crRNA fluorescent microdroplet image.

[0085] Example 3 A kit for quantitative detection of EB virus nucleic acid

[0086] 1. Composition

[0087] Including Cas12a protein with a final concentration of 200nM, 200nM BamHI-W-crRNA T3 (SEQ ID NO: 3), 200nM BamHI-W-crRNA T7 (SEQ ID NO: 7), 500nM single-stranded DNA reporter probe, 1× reaction buffer and 1U / μL RNase inhibitor.

[0088] The sequence of the single-stranded DNA reporter probe is: 5'-CCCCCC-3', 1× reaction buffer is obtained by diluting 10× reaction buffer, and 10× reaction buffer contains Tris-HCl with a final concentration of 100mM, MgCl2 with 150mM, KCl with 100mM and PEG200 with a volume ratio of 50%.

[0089] 2. Usage

[0090] The DNA sample to be tested is collected, added to the EB virus nucleic acid quantitative detection kit and mixed thoroughly to obtain a reaction mixture; the reaction mixture is dispersed into microdroplets with a diameter of 20 to 30 μm and a number of not less than 30,000, and incubated at a constant temperature of 45°C for 60 minutes. After the incubation, the number of fluorescent microdroplets is observed, and the ratio of fluorescent microdroplets is calculated, and the EB virus DNA copy number is calculated according to the Poisson distribution algorithm.

[0091] The Poisson distribution algorithm is shown in Formula I;

[0092] Formula I:

[0093] Wherein, c is the EB virus DNA copy number, in copies per microliter (copies / μL); PR represents the ratio of fluorescent microdroplets; r represents the average radius of a droplet; V represents the average volume of a droplet, in μL.

[0094] Example 4 Absolute quantitative detection of EBV DNA BamHI-W quantitative reference

[0095] 1. Experimental methods

[0096] (1) The DNA sample used in this example is the BamHI-W quantitative reference (purchased from Guangzhou Exon Biotechnology Co., Ltd.), which contains the sequence fragment of 12001-15072 bp in the BamHI-W region. The DNA sample is diluted with enzyme-free water in equal proportions to a concentration of 5×10 5 copies / μL, 1×10 5 copies / μL, 5×10 4 copies / μL, 1×10 4 copies / μL, 5×10 3 copies / μL, 5×10 2 copies / μL, 5×10 1 copies / μL and 5×10 0 copies / μL of DNA sample 1 to DNA sample 8.

[0097] (2) Prepare 5 μL of reaction premix according to the system shown in Table 5.

[0098] Table 5 Reaction system

[0099] Components Volume / final concentration Cas12a protein 200nM crRNA (BamHI-W-crRNA T3, SEQ ID NO: 5) 200nM crRNA (BamHI-W-crRNA T7, SEQ ID NO: 7) 200nM Single-stranded DNA reporter probe 500nM 1× reaction buffer, pH 9.0 0.5μL RNase inhibitors 1U / μL DEPC water To 5μL

[0100] The sequence of the single-stranded DNA reporter probe is: 5'-CCCCCC-3', 1× reaction buffer is obtained by diluting 10× reaction buffer, and 10× reaction buffer contains Tris-HCl with a final concentration of 100mM, MgCl2 with 150mM, KCl with 100mM and PEG200 with a volume ratio of 50%.

[0101] Thoroughly mix 5 μL of reaction premix and 5 μL of DNA sample 1 to obtain reaction mixture 1;

[0102] (3) dispersing the reaction mixture 1 obtained in step (2) into microdroplets with a diameter of 20 to 30 μm and a number of microdroplets of not less than 30,000, heating the mixture at a constant temperature of 45° C. for reaction, and taking photos and observing the number of fluorescent droplets using an inverted fluorescence microscope (Nikon, ECLIPSETi2-U) after 60 minutes;

[0103] (4) calculating the positive droplet ratio (the ratio of the number of fluorescent droplets) in step (3), and calculating the DNA concentration of the DNA sample using a Poisson distribution algorithm based on the positive droplet ratio p and the droplet radius r. The Poisson distribution algorithm is shown in Formula I;

[0104] Formula I:

[0105] Wherein, c is the EB virus DNA copy number, in copies per microliter (copies / μL); PR represents the ratio of fluorescent microdroplets; r represents the average radius of a droplet; V represents the average volume of a droplet, in μL.

[0106] The DNA samples 2 to 8 obtained by dilution in step (1) were used to replace the DNA sample 1 in step (2) respectively, and the corresponding DNA concentrations were calculated.

[0107] (5) Draw a concentration curve graph with the actual concentration of each DNA sample as the vertical axis (expected concentration) and the DNA concentration obtained in step (4) as the horizontal axis (measured concentration).

[0108] 2. Experimental results

[0109] The concentration curve is shown in Figure 4 As shown in the results, the DNA concentration calculated in this example is not much different from the actual concentration of each DNA sample, and the linear range is 5 to 100,000 copies / μL (R 2 =0.9955), indicating that the scheme described in this example can accurately detect the DNA concentration of the DNA sample.

[0110] Example 5 Quantitative detection of EBV DNA in clinical saliva samples

[0111] 1. Experimental methods

[0112] Nucleic acid samples extracted from saliva of 11 nasopharyngeal carcinoma patients collected from the Cancer Center of Sun Yat-sen University were used. The total DNA in each saliva sample was extracted using a DNA extraction kit, and the samples were designated as samples 1 to 11. Sample 1 was used as an example for EBV DNA quantitative detection.

[0113] (1) Prepare 5 μL of reaction premix according to the reaction system shown in Table 5 in step (2) of Example 4, and thoroughly mix 5 μL of reaction premix with 5 μL of sample 1 to obtain reaction mixture 2.

[0114] (2) dispersing the reaction mixture 2 obtained in step (1) into microdroplets with a diameter of 20 to 30 μm and a number of microdroplets of not less than 30,000, heating the mixture at a constant temperature of 45° C. for reaction, and taking photos after 60 minutes to observe the number of fluorescent droplets;

[0115] (3) calculating the positive droplet ratio (the ratio of the number of fluorescent droplets) in step (2), and calculating the DNA concentration of the DNA sample using a Poisson distribution algorithm based on the positive droplet ratio p and the droplet diameter d. The Poisson distribution algorithm is shown in Formula I;

[0116] Formula I: The volume of each droplet

[0117] Sample 1 in step (1) is replaced with samples 2 to 11 respectively, and the DNA concentrations in the corresponding samples are calculated.

[0118] The control group was set up as follows: using qPCR technology to perform quantitative detection on samples 1 to 11 respectively.

[0119] 2. Experimental results

[0120] The quantitative test results of saliva samples from 11 patients with nasopharyngeal carcinoma are as follows Figure 5 As shown, the results show that the method of the present invention has good consistency in detection results compared with qPCR technology, and has higher detection sensitivity in samples with low copy concentrations.

[0121] Example 6 Quantitative detection of EBV DNA in clinical plasma samples

[0122] 1. Experimental methods

[0123] The plasma of 108 healthy subjects (HC) and 100 nasopharyngeal carcinoma patients collected from the Physical Examination Center of the Cancer Prevention and Treatment Center of Sun Yat-sen University were used, and the free DNA in the plasma was extracted using a magnetic bead DNA extraction kit as the DNA samples of this example.

[0124] The specific method for quantitative detection of EBV DNA based on microdroplet digital analysis is as follows: taking free DNA (free DNA sample) extracted from the plasma of one of 100 nasopharyngeal carcinoma patients as an example, EBV quantitative detection is performed.

[0125] (1) Prepare 5 μL of reaction premix according to the reaction system shown in Table 5 in step (2) of Example 4, and thoroughly mix 5 μL of reaction premix with 5 μL of free DNA sample to obtain reaction mixture 3.

[0126] (2) dispersing the reaction mixture 3 obtained in step (1) into microdroplets with a diameter of 20 to 30 μm and a number of microdroplets of not less than 30,000, heating the mixture at a constant temperature of 45° C. for reaction, and taking photos after 60 minutes to observe the number of fluorescent droplets;

[0127] (3) calculating the positive droplet ratio (the ratio of the number of fluorescent droplets) in step (2), and calculating the DNA concentration of the DNA sample using a Poisson distribution algorithm based on the positive droplet ratio p and the droplet diameter d. The Poisson distribution algorithm is shown in Formula I;

[0128] Formula I: The volume of each droplet

[0129] The free DNA extracted from the remaining plasma of NPC patients and healthy subjects is used to replace the free DNA sample in step (1), and the corresponding DNA concentration is detected and calculated, which is the EBV DNA copy number based on microdroplet digital analysis.

[0130] The control group was set up for quantitative detection using the traditional qPCR detection method. Specifically, the free DNA extracted from the plasma of NPC patients and healthy subjects was quantitatively detected using qPCR technology to obtain the EBVDNA copy number corresponding to the qPCR technology.

[0131] The qPCR primers corresponding to the qPCR technique are shown in SEQ ID NO: 12 and SEQ ID NO: 13; the corresponding qPCR probe is shown in SEQ ID NO: 14;

[0132] BamHI-WF (SEQ ID NO: 12): 5'-CCCAACACTCCACCACACC-3';

[0133] BamHI-WR (SEQ ID NO: 13): 5'-TCTTAGGAGCTGTCCGAGGG-3';

[0134] BamHI-W probe (SEQ ID NO: 14):

[0135] FAM-CACACACTACACACACCCACCCGTCTC-TAMRA.

[0136] ROC curves were drawn and AUC values ​​were calculated for the EBV DNA copy number based on the microdroplet digital analysis and the EBV DNA copy number corresponding to the qPCR technology, respectively; and the correlation curve of the EBV DNA copy number of the two detection methods was drawn.

[0137] The 100 NPC patients used in this example were divided into different clinical AJCC stages for NPC, including 15 cases in stage I+II, 40 cases in stage III, and 45 cases in stage IV. Box plots and ROC curves of the EBVDNA copy numbers corresponding to healthy controls (HC) and samples of different stages were drawn, and the area under the curve (AUC) was calculated.

[0138] 2. Experimental results

[0139] The experimental results are as follows Figure 6 As shown, the results showed that compared with the traditional qPCR detection method, the EBV DNA quantitative detection method based on droplet digital analysis has higher detection accuracy in healthy people, can better distinguish healthy people from nasopharyngeal carcinoma patients, and has better specificity; in nasopharyngeal carcinoma patients, the detection sensitivity is higher in samples with low copy concentrations.

[0140] The ROC curve of the EBV DNA copy number of the EBV DNA quantitative detection method based on microdroplet digital analysis and the EBV DNA copy number corresponding to the traditional qPCR detection method is shown in the figure. Figure 7 As shown, Figure 7 A is the ROC curve corresponding to the EBV DNA copy number of the EBV DNA quantitative detection method based on microdroplet digital analysis, Figure 7 B is the ROC curve of EBV DNA copy number corresponding to the traditional qPCR detection method; the results showed that the AUC value of the ROC curve of EBV DNA copy number corresponding to the EBV DNA quantitative detection method based on microdroplet digital analysis reached 0.9883 (P < 0.0001), while the AUC of the ROC curve of EBV DNA copy number corresponding to the traditional qPCR detection method was only 0.909 (P < 0.0001);

[0141] And through Figure 7 The ROC curves for the critical values ​​(COV) of the EBV DNA quantitative detection method based on microdroplet digital analysis (Digital assay) and the traditional qPCR detection method (BamHI-W qPCR) are shown in Table 6.

[0142] Table 6 Sensitivity and specificity at different critical values

[0143]

[0144] COV1: for highest accuracy; COV2: for highest specificity; CI: confidenceinterval.

[0145] The results showed that when the highest accuracy (optimal COV) was guaranteed, the sensitivity of the EBV DNA quantitative detection method based on microdroplet digital analysis (COV1 = 224.6 copies / mL) was (99.0%, 95% CI: 94.6% to 99.9%), and the specificity was (92.6%, 95% CI: 85.9% to 96.8%); the sensitivity of the traditional qPCR detection method (COV1 = 107.5 copies / mL) was (86.0%, 95% CI: 77.6% to 92.1%), and the specificity was (88.9%, 95% CI: 81.4% to 94.1%).

[0146] While maintaining high specificity (99.1%, 95% CI: 95.0%-99.9%), the sensitivity of the EBV DNA quantitative detection method based on droplet digital analysis (COV2=1491.0 copies / mL) was (86.0%, 95% CI: 77.6%-92.1%); the sensitivity of the traditional qPCR detection method (COV2=1026.0 copies / mL) was (70.0%, 95% CI: 60.0%-78.8%).

[0147] The results show that the EBV DNA quantitative detection method based on droplet digital analysis has higher diagnostic sensitivity and specificity than the traditional qPCR detection method.

[0148] The correlation curve between the EBV DNA copy number of the EBV DNA quantitative detection method based on microdroplet digital analysis (droplet digital analysis) and the EBV DNA copy number corresponding to the traditional qPCR detection method (qPCR) is shown in Figure 8 The results show that: after the rank correlation test, the quantitative results of the two methods are highly correlated (r s =0.8364, p<0.0001), indicating that there is no difference between the quantitative detection technology of EBV DNA based on microdroplet digital analysis and the traditional qPCR detection method in the quantitative detection of plasma EBV DNA.

[0149] The box plots and ROC curves of EBV DNA copy numbers corresponding to samples of different stages are shown in the figure below. Fig. 9As shown, COV1 represents the COV value at the highest accuracy, and COV2 represents the COV value at the highest specificity; Fig. 9 A is a box plot of EBV DNA copy numbers at different clinical stages corresponding to the EBV DNA quantitative detection method based on microdroplet digital analysis. Fig. 9 C is the corresponding ROC curve graph; Fig. 9 B is a box plot of EBV DNA copy numbers at different clinical stages corresponding to the traditional qPCR detection method. Fig. 9 D is the corresponding ROC curve.

[0150] The results showed that in patients with NPC, the plasma EBV DNA level gradually increased with the aggravation of clinical stage, indicating that the plasma EBV DNA level was directly related to tumor complexity; in patients with early NPC (stage I-II), the AUC of the EBV DNA quantitative detection method based on microdroplet digital analysis was 0.988 (95% CI: 0.974-1.000, p < 0.0001), while the AUC of qPCR technology was 0.768 (95% CI: 0.611-0.925, p = 0.0008); for patients with stage III NPC, the EBV DNA quantitative detection method based on microdroplet digital analysis was 0.988 (95% CI: 0.974-1.000, p < 0.0001), while the AUC of qPCR technology was 0.768 (95% CI: 0.611-0.925, p = 0.0008). The AUC of the DNA quantitative detection method was 0.994 (95% CI: 0.9858-1.000, p < 0.0001), while the AUC of the traditional qPCR detection method was 0.909 (95% CI: 0.841-0.977, p < 0.0001); for patients with stage IV nasopharyngeal carcinoma, the AUC of the EBV DNA quantitative detection method based on droplet digital analysis was 0.984 (95% CI: 0.9573-1.000, p < 0.0001), while the AUC of the traditional qPCR detection method was 0.956 (95% CI: 0.908-1.000, p < 0.0001).

[0151] The results show that for patients with nasopharyngeal carcinoma stages I to III, the EBV DNA quantitative detection method based on droplet digital analysis has better predictive efficacy for nasopharyngeal carcinoma than the traditional qPCR detection method. For patients with nasopharyngeal carcinoma stage IV, there is no significant difference between the two detection methods, and the predictive efficacy is equivalent.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A crRNA combination for detecting Epstein-Barr virus based on microdroplet digital analysis, characterized in that: The crRNA is one or more of the crRNAs with nucleotide sequences such as SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and / or SEQ ID NO:

8.

2. The crRNA combination according to claim 1, characterized in that The crRNA is a combination of a crRNA having a nucleotide sequence as shown in SEQ ID NO: 3 and a crRNA having a nucleotide sequence as shown in SEQ ID NO:

7.

3. Use of the crRNA combination described in any one of claims 1 to 2 in the preparation of an Epstein-Barr virus nucleic acid quantitative detection kit.

4. A quantitative detection kit for EB virus nucleic acid, characterized in that: The kit comprises the crRNA combination described in any one of claims 1 to 2.

5. The EB virus nucleic acid quantitative detection kit according to claim 4, characterized in that: Also contains Cas12a protein, single-stranded DNA reporter probe and / or reaction buffer; The sequence of the single-stranded DNA reporter probe is: 5'-CCCCCC-3'.

6. The EB virus nucleic acid quantitative detection kit according to claim 5, characterized in that: The reaction buffer comprises Tris-HCl with a concentration of 10 to 200 mM, MgCl2 with a concentration of 50 to 250 mM, KCl with a concentration of 50 to 150 mM and / or PEG200 with a volume ratio of 25 to 50%.

7. The EB virus nucleic acid quantitative detection kit according to claim 6, characterized in that: The reaction buffer comprises 100 mM Tris-HCl, 150 mM MgCl2, 100 mM KCl and / or 50% PEG200 by volume.

8. The EB virus nucleic acid quantitative detection kit according to claim 7, characterized in that: The pH of the reaction buffer is 8.5-9.

5.

9. The EB virus nucleic acid quantitative detection kit according to claim 8, characterized in that: The pH of the reaction buffer was 9.0.

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