CN115029485A - bKV virus detection kit based on micro-drop digital PCR - Google Patents
bKV virus detection kit based on micro-drop digital PCR Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly discloses a BKV virus detection kit based on droplet digital PCR. The primers for detecting the BKV virus consist of a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2. The nucleotide sequence of the probe matched with the primer is shown as SEQ ID NO. 3. The invention designs targets aiming at the specific conserved region of the BKV virus, eliminates the interference of common pathogens and genome nucleic acid in a sample, avoids false positive, and simultaneously, the selection of the targets of the conserved region can effectively avoid false negative caused by the influence of factors such as BKV virus variation and different serotypes, and the like, thereby improving the accuracy and the sensitivity of detection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a BKV virus detection kit based on droplet digital PCR.
Background
BK Virus (BKV) belongs to a subtype of polyoma cytomegalovirus family, and the main transmission path of the BK Virus is considered to be respiratory tract and digestive tract at present, and the other transmission paths comprise placenta, organ transplantation, dirty food, blood products and the like. BKV is a double-stranded closed circular DNA virus, non-enveloped, containing about 5300 base pairs. Can be replicated in the cell nucleus of a host under the condition of immunosuppression, and the virus has certain immune resistance. BK virus was first isolated from urine in the ureter of a 39 year old male patient receiving a kidney transplant at the virus research laboratory in London, UK in 1971 and is named under the acronym for that patient. According to records, the virus positive rate of BK virus in serum of ten-year-old children can reach 75% on average, and the BK virus is detected in urine more generally than blood. Primary infection with BK virus occurs mostly in childhood, via respiratory infection, usually without symptoms or some minor symptoms of the upper respiratory tract. When infected with BK virus, it can hide in kidney, urinary tract, lymph tissue and brain, and when the host immunity is deteriorated, it can be activated again to cause disease, so that the virus can be detected in urine of normal people.
The BKV primary infection is mostly concentrated in infants, and usually has no obvious clinical symptoms or symptoms such as fever and slight respiratory tract infection. The virus then remains dormant throughout the body for many organs or tissues, with the common sites being renal tubules and urothelial cells. The onset of BK virus can lead to hemorrhagic cystitis, ureteral stenosis and BK-related renal disease, and in renal transplant patients can lead to abnormal transplant renal function and even loss of transplant renal function. BKV is not pathogenic to healthy people, but when the immunity of the body is reduced or in an immunosuppressed state, the BKV latent in the body can be reactivated. Especially after organ transplantation, BKV latent in the urothelium and the tubular epithelium is activated to start high-level replication, and a large amount of replicated virus particles are excreted from the urinary tract, resulting in BKV uropathy. In a kidney transplant recipient, as the disease progresses, the BKV can enter the nucleus of renal tubular epithelial cells and replicate a large amount of progeny viruses to cause cell necrosis and release, so that tissues are infiltrated with immunity and inflammation; when tubular epithelial cells are shed and local basement membranes are exposed, the virus begins to disrupt tubular capillaries into the blood, forming BKV blood disease (BKV viremia). BKV continues to be expressed in high loads in the blood, further destroying transplanted kidney tissue leading to tubular atrophy and interstitial fibrosis, ultimately forming BKVN.
As the BKVN shows BKV urine disease and BKV blood disease at the early stage, the quantitative polymerase chain reaction method for detecting the BKV DNA load in the urine and peripheral blood of a kidney transplant recipient becomes an important method for detecting the disease change at the early stage in clinic. The BKVN has close relation with the BKV DNA load in urine and blood, when the BKV DNA load in urine is more than 1.0 multiplied by 10 7 copies/mL and blood BKV DNA loading > 1.0 × 10 4 At copies/mL, the risk of lesion development into BKVN is extremely high. Domestic data also proves that the BKV DNA carrying capacity of the blood is more than or equal to 1.0 multiplied by 10 5 The positive predictive value of copies/mL serving as a positive index for predicting the generation of BKVN is as high as 83.3%. Thus, the higher the BKV DNA load in blood, the greater the risk of developing BKVN; patients who are negative in blood tests but have high urine BKV DNA loads also need to be reviewed and alerted to the worsening of the disease on a regular basis.
The real-time fluorescent quantitative PCR technology is the most widely applied technology for the current BKV DNA detection. As rapid and sensitive detection can be realized, a large number of documents prove that the fluorescent quantitative PCR detection of the BKV DNA has important significance for monitoring the BKV loading change caused by the reduction of immunocompetence caused by organ transplantation, immune diseases and the like. However, due to the lack of a unified detection execution standard among different laboratory PCR platforms, no matter what the sample type, quantitative traceability, DNA extraction method, DNA fragment to be detected, quality control, detection equipment, detection limit value, etc. lack of a unified standard, the detection results of the same sample in different laboratories, especially the BKV DNA threshold value setting, have large differences, resulting in the situation that the experimental results cannot be repeated, even the results are contradictory. And dependence on Ct value remains the largest technical bottleneck in current quantitative PCR, in this sense the so-called "quantification" is also only relative. In addition, the sensitivity and accuracy of detection are limited under the conditions of low copy target molecules and small concentration difference of the template. The accuracy of the detection report results directly influences the diagnosis of clinical diseases and the judgment of treatment effects, so that the development of more sensitive and accurate quantitative methods and reagents for the BKV DNA in urine and blood and the formulation of standardized detection and result judgment methods are urgently needed to improve the reliability of the clinical application of the BKV DNA detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a BKV virus detection kit based on droplet digital PCR.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a primer for detecting BKV virus, which consists of a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2.
In a second object, the invention provides a probe matched with the primer, wherein the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
According to the invention, bioinformatics analysis is carried out on the genome sequences of 565 cases of the BKV viruses, target point design is carried out on specific conserved regions of the BK viruses, common pathogens and genome nucleic acid interference in a sample are eliminated, false positive is avoided, and meanwhile, the target point selection of the conserved regions can effectively avoid false negative caused by the influence of factors such as BKV virus variation and different serotypes, so that the primers as shown in SEQ ID NO. 1-2 and the probes as shown in SEQ ID NO. 3 are screened out.
According to the invention, experimental screening verifies that the combination of the forward primer, the reverse primer and the probe can achieve an expected result, the quantitative result, microdroplet grouping condition, fluorescent signal, quantitative repeatability, quantitative linear range and the like of the BKV template with different concentrations are superior to those of other primer and probe combinations, and meanwhile, nonspecific detection of homologous interference pathogen nucleic acid is not seen, so that the forward primer, the reverse primer and the probe are used as BKV target primer and probe for system optimization.
In a preferred embodiment of the probe of the present invention, the probe is labeled with a fluorescent group at the 5 'end and a quencher group at the 3' end.
As a preferred embodiment of the probe of the present invention, the fluorescent group comprises at least one of FAM, VIC/HEX, ROX, CY3, CY5, CY5.5, CY7, JOE, TAMRA, TET, Texas Red, Quasar 570, Quasar 670, Atto 425, Atto 590, Alexa flow fluorescent group; the quencher comprises at least one of Dabcyl, Eclipse, MGB, BHQ1, BHQ2, BHQ3, BBQ 650, TAMRA and TQ1-TQ7 quencher.
Preferably, the fluorophore is FAM; the quencher group is BHQ 1.
The invention also provides a method for detecting the BKV virus based on the micro-droplet digital PCR, wherein the method comprises the steps of detecting the BKV virus by using the primer and the probe.
Digital PCR (digital PCR) is a new technology in recent years, and can determine the absolute number of target molecules to be detected as low as a single copy by performing a micro-unit process on a sample, and the digital PCR can directly count the number of target molecules without depending on any calibrator or external standard, which is an absolute quantitative PCR technology. Digital PCR is therefore particularly suitable for applications which are not well resolved by virtue of Ct values: copy number variation, mutation detection, gene relative expression research, second-generation sequencing result verification, miRNA expression analysis, single-cell gene expression analysis and the like.
Compared with real-time fluorescent quantitative PCR, digital PCR (digital PCR) has the advantages of precision, ultrahigh sensitivity, insusceptibility to PCR inhibitors and the like. However, no related research for absolute quantitative detection of the BKV virus by using a digital PCR technology and no finished product kit suitable for industrialization are available at present. The invention develops a kit and a detection method for absolute quantitative detection of BK virus based on a digital PCR (polymerase chain reaction) technology by taking a droplet digital PCR (ddPCR) technology as a basis, and proves the application value of the kit in aspects of organ transplantation, immunosuppression and the like related to the BKV virus. The kit can realize the rapid and accurate quantification of the BKV virus in a short time, provides powerful technical support for monitoring the virus load of BKV virus infection related diseases, and has wide application prospect and industrialization prospect.
As a preferred embodiment of the BK virus detection kit, the kit further comprises an internal standard primer and a probe, wherein the nucleotide sequence of the internal standard primer is shown as SEQ ID NO. 4-5, and the nucleotide sequence of the internal standard probe is shown as SEQ ID NO. 6.
Preferably, the probe of the internal standard is labeled with a fluorescent group at the 5 'end and a quenching group at the 3' end. The fluorescent group comprises at least one of FAM, VIC/HEX, ROX, CY3, CY5, CY5.5, CY7, JOE, TAMRA, TET, Texas Red, Quasar 570, Quasar 670, Atto 425, Atto 590 and Alexa Flour fluorescent group; the quencher comprises at least one of Dabcyl, Eclipse, MGB, BHQ1, BHQ2, BHQ3, BBQ 650, TAMRA and TQ1-TQ7 quencher.
More preferably, the fluorophore of the probe of the internal standard is VIC; the quencher group is MGB.
The working concentration of the primer of the internal standard is 900nM, and the working concentration of the probe of the internal standard is 375 nM.
In a preferred embodiment of the kit for detecting a BK virus according to the present invention, the kit further comprises a micro-drop enzyme mixture for digital PCR, a positive control mixture and a negative control mixture, wherein the positive control mixture is diluted with positive urine containing a BK virus, the negative control mixture comprises mixed negative urine of a normal person, and the micro-drop enzyme mixture for digital PCR comprises a taq enzyme, a UDG enzyme and a restriction endonuclease.
In a preferred embodiment of the kit for detecting a BK virus according to the present invention, the kit further comprises a positive control mixture diluted with positive urine containing a BK virus, and a negative control mixture including mixed negative urine of a normal person.
Preferably, the kit of the invention uses two groups of positive controls, and the copy number of the two groups of positive controls has a difference of several orders of magnitude, and can be respectively used as a strong positive control and a weak positive control, so as to avoid the situation that the sample DNA copy number is too high or too low to be quantitative.
As a preferred embodiment of the BK virus detection kit according to the present invention, the use of the kit comprises the steps of:
1) obtaining total DNA of a sample;
2) preparing the total volume of the reaction mixed solution of the micro-drop digital PCR according to the preset reaction number;
3) preparing each reaction system according to a preset reaction number;
4) and (3) carrying out micro-droplet digital PCR amplification on each reaction system subjected to micro-droplet treatment, and then calculating the nucleic acid concentration of each sample according to the amplification result.
Preferably, the reaction mixture of the micro-drop digital PCR comprises an enzyme mixture for micro-drop digital PCR and a BKV virus detection mixture (including primers and probes for BKV virus detection, primers and probes for internal standard).
The reaction number N is equal to the number of samples to be detected (N) + the quality control number (3) + 1.
In the above system, the volume of the BKV virus detection mixture was 9 XN. mu.L, and the volume of the enzyme mixture for the digital PCR in the form of a droplet was 1 XN. mu.L.
The procedure for the droplet digital PCR amplification was: reacting at 50 ℃ for 2 min; reacting at 95 ℃ for 5 min; reaction at 95 ℃ for 30sec, reaction at 60 ℃ for 30sec, 45 cycles; reacting at 98 ℃ for 10 min; the temperature rise and fall speed of 1 ℃/sec was set for each step.
In a fourth aspect, the present invention provides a non-diagnostic use of the above kit for detecting BKV virus in serum, plasma or urine derived from human samples.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a BKV virus detection kit based on droplet digital PCR, which has the advantages of sensitivity, accuracy, wide applicability and the like;
(1) the result is accurate: the method realizes absolute quantification, does not depend on Ct value, does not need a standard curve, and more accurately detects the virus content, thereby providing accurate diagnosis basis for BK virus related disease diagnosis and virus load monitoring.
(2) The sensitivity is high: a single copy template can be detected.
(3) The applicability is wide: is suitable for detecting complex samples and is not easily influenced by PCR inhibitors.
(4) The specificity is high: through bioinformatics analysis of the genome sequences of the 565 recorded BKV viruses, target point design is carried out aiming at the specific conserved region of the BKV viruses, common pathogens and genome nucleic acid interference in a sample are eliminated, false positive is avoided, and meanwhile, false negative caused by the influence of factors such as BKV virus variation and different serotypes can be effectively avoided through target point selection of the conserved region.
Drawings
FIG. 1 is a diagram showing the results of primer and probe screening;
FIG. 2 is a one-dimensional diagram of the present invention for detecting a gradient diluted BKV international standard substance;
FIG. 3 is a graph of a linear correlation analysis of the detection of a gradient diluted BKV international standard substance according to the present invention;
FIG. 4 is a two-dimensional graph of the results of a clinical sample for detecting urine BKV DNA positivity by using a droplet-type digital PCR according to the present invention;
FIG. 5 is a two-dimensional graph of the results of a clinical sample for detecting urine BKV DNA negativity by using a droplet-type digital PCR according to the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.
Example 1 design and screening of primers and probes for BKV Virus detection
1) Designing a primer and a probe: the genetic sequence of the BKV virus is obtained by an on-line tool of the National Center for Biotechnology Information (NCBI), the recorded 565 genome sequences of the BKV virus are subjected to bioinformatics analysis, target point design is carried out aiming at a specific conserved region of the BKV virus, common pathogens and genome nucleic acid interference in a sample are eliminated, false positive is avoided, and meanwhile, false negative caused by BK virus variation, different serotypes and other factors can be effectively avoided by target point selection of the conserved region.
The sequences of the designed primers and probes are shown in Table 1.
TABLE 1
2) And (3) primer and probe screening: experimental screening verifies that the combination of the forward primer BKV-F2(SEQ ID NO:1), the reverse primer BKV-R1(SEQ ID NO:2) and the probe BKV-FP3(SEQ ID NO:3) can achieve an expected result, the quantitative result, micro-colony grouping condition, fluorescent signals, quantitative repeatability, quantitative linear range and the like of the BKV template with different concentrations are superior to those of other primer-probe combinations, and non-specific detection of homologous interference pathogen nucleic acid is not found, so that the forward primer BKV-F2 (shown in the specification) is used as a primer and a probe for detecting BKV virus for system optimization, and the method is concretely analogized with reference to FIG. 1 (wherein F2R2P2 represents that BKV-F2, BKV-R2 and BKV-FP2, and other F2R3P2, F2R4P2 and the like are adopted).
Example 2A BKV virus detection kit based on droplet digital PCR
The kit comprises:
(1) enzyme mixture for micro-droplet digital PCR: including taq enzyme (taq DNA polymerase), UDG enzyme (uracil-DNA glycosylase), restriction enzymes (EcoR I, EcoR-HF, EcoR K, BamH I, Hind, Alu I, BsuR I, Bal I, Hal II, Hal III, HPa I, Sma I; preferably EcoR I); the enzyme mixed solution for the micro-drop digital PCR is adapted to equipment for carrying out the digital PCR, and can be replaced by other enzyme mixed solutions for the digital PCR.
(2) B KV detection reaction solution: comprises detecting buffer (reaction buffer), dNTPs, BKV DNA primer and probe combination, and internal label primer and probe combination;
(3) internal standard: artificially synthesized exogenous recombinant plasmid for monitoring extraction and detection process;
(4) weak positive control mixture: is prepared by diluting positive urine containing the BKV virus;
(5) strong positive control mixture: is prepared by diluting positive urine containing the BKV virus;
(6) negative control mixture: mixing the negative urine of normal person;
the sequence of the BKV DNA primer and probe combination is as follows:
BKV-F2：5'-ACTGCTCCTCAATGGATGT-3'(SEQ ID NO:1)；
BKV-R1：5'-GAGCTGCCCCTGGACAC-3'(SEQ ID NO:2)；
BKV-FP3：5'-FAM-TAGGCCTGTACGGGACTGTAACACC-BHQ1-3'(SEQ ID NO:3)；
in this example, the final concentration of BKV-F2 was 2.22. mu. M, BKV-R1 was 2.22. mu. M, BKV-FP3 was 0.56. mu.M.
The internal standard primer and probe combination has the following sequences:
Control-F：5'-CTACCAGCAGAACACCCCCA-3'(SEQ ID NO:4)；
Control-R：5'-GCGGCGGTCACGAACTC-3'(SEQ ID NO:5)；
Control-Probe：5'-VIC-CCCAACGAGAAGCG-MGB-NFQ3'(SEQ ID NO:6)。
in this example, the final concentration of Control-F was 2. mu. M, Control-R and the final concentration of 2. mu. M, Control-Probe was 1.11. mu.M.
The difference between the weak positive control and the strong positive control is that the copy number has a difference of several orders of magnitude, and the weak positive control and the strong positive control can be respectively used as the strong positive control and the weak positive control, so that the condition that the sample DNA copy number is too high or too low to be quantified can be avoided, and preferably, the copy number of the strong positive control is at least 100 times of that of the weak positive control.
Example 3 detection method Using the above-described BKV Virus detection kit
The detection method comprises the following steps:
(1) collecting samples: suitable sample types are serum, plasma or urine.
a. The serum is extracted from the bent part of the arm or the back of the hand of the examinee by a sterile syringe to obtain 2ml of venous blood, and after the sample automatically separates out the serum, the sample is directly centrifuged at 1600rpm at room temperature for 5 minutes to separate out the serum, and then the serum is transferred to a 1.5ml sterilized centrifugal tube for standby.
b. 2ml of venous blood is extracted from the bent part of the arm or the back of the hand of a detected person by using a sterile syringe for plasma, the venous blood is injected into a sterile collecting tube containing EDTA, the sterile collecting tube is immediately and slightly inverted and mixed evenly, after the plasma is automatically separated out from the sample, the sample is centrifuged at 1600rpm for 5 minutes at room temperature to separate out the plasma, and then the plasma is transferred into a 1.5ml sterile centrifuge tube for standby.
c. And (4) reserving 10-20 mL of middle-stage morning urine in a urine cup to a sterile storage tube, and slightly reversing and uniformly mixing for later use.
Samples were stored immediately after collection in ice cubes or placed at 4 ℃. The sample can be stored at 4 ℃ for less than 72 hours at-20 ℃ for several months, and the sample is placed at-70 ℃ for long-term storage.
(2) Sample processing and nucleic acid extraction (sample processing zone):
it is recommended to use nucleic acid extraction or purification reagent (Yue Chan Ji 20220055) produced by Guangdong Yongno medical science and technology Limited company to extract clinical samples, positive and negative controls according to the instruction of nucleic acid extraction kit, and when extracting, an internal standard template is added into the samples, and the elution volume is 80 μ L.
(3) Reagent preparation (reagent preparation area):
a. the detection reaction is respectively provided with positive quality control and negative quality control.
b. And (5) preparing.
1) Thawing all the components to room temperature, fully dissolving all the components, then oscillating and mixing uniformly, and centrifuging for a short time;
2) and determining the reaction number N, wherein N is the number of samples to be detected (N) + the quality control number (3) + 1. The amount of each reagent added to the reaction mixture was calculated as follows:
3) preparing a reaction system by taking 1.5ml of sterile centrifuge tubes, adding all reagents, shaking and mixing uniformly, and centrifuging for a plurality of seconds.
4) Then, 10. mu.l/tube of the mixture was dispensed into 0.2ml of PCR reaction tubes.
c. Sample application (sample preparation zone).
Respectively adding 10 mul of BKV negative control, BKV positive control and clinical sample DNA into a 0.2ml PCR reaction tube, covering a tube cover tightly, shaking and mixing uniformly, throwing all liquid on the tube wall to be low (avoiding generating bubbles) by short-time centrifugation, and then preparing micro-droplets.
d. Micro-droplets (sample preparation area) were prepared.
And taking 20 mu L of the mixed solution in the reaction tube to generate micro-droplets, wherein the specific operation steps are carried out according to the instruction of the biochip analyzer.
Pcr amplification (amplify detection zone).
Taking a prepared detection plate containing micro-droplets of a sample to be detected, putting the detection plate into a PCR amplification instrument, and carrying out PCR amplification, wherein the amplification procedure is as follows: reacting at 50 ℃ for 2 min; reacting at 95 ℃ for 5 min; reaction at 95 ℃ for 30sec, reaction at 60 ℃ for 30sec, 45 cycles; reacting at 98 ℃ for 10 min; the temperature rise and fall speed of 1 ℃/sec is set in each step.
(4) Micro-droplet detection and result analysis (amplification zone and analysis zone).
after PCR amplification, placing the detection plate in a biochip analyzer, starting QuantDrop software, and carrying out detailed experimental steps according to the instruction of the analyzer.
b. And (4) setting. Clicking 'setting', assigning a name and a sample type to each sample including a negative quality control and a positive quality control in a 'sample definition' module, and selecting 'unknown sample' from 'type'. The channel 1 is a FAM channel for detecting BK virus; channel 2 is an internal detection standard for the VIC channel. Click "ok" after setup is complete.
c. And (4) operating. And clicking 'operation', automatically operating the instrument, displaying the progress, and forming a data file after the operation is finished.
d. And (6) analyzing. Clicking 'analysis', opening and analyzing data, displaying experimental data of a selected hole (sample), and obtaining a concentration result of the BKV DNA of each sample.
Example 4 Performance test of quantitative determination of BKV DNA International Standard substance by BKV Virus detection kit
The accuracy and sensitivity of the BKV virus detection kit obtained in example 2 for quantitatively detecting BKV DNA international standard substance (NIBSC code #14/212) were compared with the performance of the fluorescent PCR platform already on the market.
The nominal concentration of the international standard substance is 1.58E +07IU/ml, and the gradient dilution is 1.58E +06, 1.58E +05, 1.58E +04, 1.58E +03, 1.58E +02, 7.90E +01 and 4.74E +01 IU/ml.
1) Sample extraction: the BKV DNA international standard substance is extracted using a nucleic acid extraction or purification reagent (yue chan jie 20220055) produced by guangdong yono medical science and technology ltd. And (3) extracting 300 mu l of sample according to the instruction of the nucleic acid extraction kit, adding an internal standard template into the sample during extraction, and extracting nucleic acid according to the instruction of the kit operation, wherein the elution volume is 80 mu l.
2) Detecting the extracted BKV DNA international standard substance by using the BKV virus detection kit in the embodiment 2;
3) the BKV international standard substance is synchronously detected by using a commercially available BK virus nucleic acid assay kit (PCR-fluorescent probe method) (from Beijing Xinnuomeidei gene detection technology Co., Ltd.).
The results are shown in Table 2 below and FIGS. 2-3:
TABLE 2
The result shows that the BBKV virus detection kit has high accuracy in detecting the BKV DNA international standard substance, the linear correlation coefficient R2 is 0.998 within the concentration range of 4.74E +01 IU/ml-1.58E +06IU/ml, the absolute value of the deviation of the detection result and the theoretical value is less than or equal to 0.2, and the sensitivity can reach 4.74E +01 IU/ml. The detection result of the fluorescent PCR kit which is already on the market is greatly different from the theoretical value, and the sensitivity is only 1.58E +03 IU/ml.
Example 5 Performance test of quantitative determination of human urine BKV DNA by BKV Virus detection kit
The BKV virus detection kit obtained in example 2 was used to quantitatively detect BKV DNA in human urine, and compared with the fluorescent PCR kit already on the market for performance.
In this embodiment, the BKV virus detection kit of embodiment 2 is used to detect 191 cases of clinical urine samples suspected of being infected by BKV viruses, and the specific detection method is as follows:
1) sample extraction: 191 clinical human urine samples, positive and negative controls were extracted using a nucleic acid extraction or purification reagent (Yuetchan Ji 20220055) produced by Guangdong Yongno medical science and technology Co., Ltd. 300ul of sample is taken according to the instruction of the nucleic acid extraction kit for extraction, an internal standard template is added into the sample during extraction, the extraction of nucleic acid is carried out according to the instruction of the kit, and the elution volume is 80 mul.
2) 191 clinical urine samples suspected of being infected by the BKV viruses are detected by adopting the BKV virus detection kit in the embodiment 2.
3) The same 191 clinical specimens were simultaneously tested using the commercially available BKV viral nucleic acid assay kit (PCR-fluorescent probe method) from xinnuomeidi gene detection technology ltd, beijing.
The results are shown in Table 3 below and FIGS. 4-5:
TABLE 3
45 samples of urine with positive BKV DNA are detected by the BKV virus detection kit, while 29 samples of urine with positive BKV DNA are detected by fluorescence PCR, and if the detection is carried out by only using the fluorescence PCR method, the missed diagnosis of 35.6 percent (16/45) of urine positive samples can be caused.
Example 6 Performance test for quantitative detection of human serum BKV DNA with BKV Virus detection kit
The BKV virus detection kit obtained in example 2 was used to quantitatively detect human serum BKV DNA, and compared with the fluorescent PCR kit already on the market for performance.
In this embodiment, the BKV virus detection kit of embodiment 2 is used to detect 162 cases of clinical serum samples suspected of being infected by BKV viruses, and the specific detection method is as follows:
1) sample extraction: 162 clinical human serum samples, positive and negative controls were extracted using nucleic acid extraction or purification reagents (Yuetchan Ji 20220055) from Guangdong Yongno medical science and technology Limited. 300ul of sample is taken according to the instruction of the nucleic acid extraction kit for extraction, an internal standard template is added into the sample during extraction, the extraction of nucleic acid is carried out according to the instruction of the kit, and the elution volume is 80 mul.
2) Detecting 162 cases of clinical serum samples suspected of being infected by the BKV virus by using the BKV virus detection kit in the embodiment 2;
3) the same 162 clinical specimens were simultaneously tested using a commercially available BK virus nucleic acid assay kit (PCR-fluorescent probe method) from Beijing Xinnaomeidei Gene detection technology, Inc.
The results are given in Table 4 below:
TABLE 4
39 BKV DNA positive serum samples are detected by adopting the BKV virus detection kit, only 7 BKV DNA positive serum samples are detected by fluorescence PCR, and 82% (32/39) of serum positive samples can be missed to be diagnosed if the detection is carried out by only using the fluorescence PCR method.
Example 7 specificity of quantitative detection of common pathogens in urine Using the BKV Virus detection kit of example 2
In this embodiment, the BKV virus detection kit described in embodiment 2 is used to specifically detect 14 common interfering pathogens in urine and blood, and the specific detection method is as follows:
1) preparation of interfering pathogen: adopting a BKV virus negative urine sample and a positive urine sample to respectively carry out multiple dilution on Chlamydia Trachomatis (CT), Ureaplasma Urealyticum (UU), ureaplasma micans (UP), gonococcus (NGH), Mycoplasma Hominis (MH), cytomegalovirus Human (HCMV), Epstein-Barr virus (EBV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), immunodeficiency virus I (HIV-1), staphylococcus aureus, candida albicans, Escherichia coli and JCV virus, and finally obtaining the content of the interference pathogen not lower than 1 × 10 3 BKV virus positive sample (concentration 5X 10) with copies/mL concentration 4 copies/mL) and a content of interfering pathogens of not less than 1X 10 3 BKV virus negative samples at copies/mL concentration.
2) Sample extraction: the urine sample containing the interference sample, positive and negative controls were extracted using a nucleic acid extraction or purification reagent (Yuetchan mechanical arm 20220055) produced by Guangdong Yongno medical science and technology Limited. Taking 300ul of sample according to the instruction of the nucleic acid extraction kit for extraction, adding an internal standard template into the sample during extraction, and extracting nucleic acid according to the instruction of the kit operation, wherein the elution volume is 80 mul.
3) The 14 common pathogens were detected using the BKV virus detection kit based on the droplet digital PCR described in example 2:
the results show the following table 5:
TABLE 5
The result shows that the BKV virus detection kit based on the droplet digital PCR of the invention can not non-specifically identify 14 common interfering pathogens of urine and blood, and the 14 pathogens have no influence on the quantitative detection result of the BKV virus nucleic acid.
BKV virus is a major cause of complications after organ transplantation surgery, and when BK viral nephropathy (BKVN) develops, renal function disappears. At present, an effective treatment means is lacked for treating the BKV virus infection, so that BK virus detection on a patient subjected to a transplantation operation becomes a key work, and a good prevention effect can be achieved by matching with nursing intervention.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Guangdong Yongnuo medical science and technology Limited
<120> BKV virus detection kit based on droplet type digital PCR
<130> 2022-06-02
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> BKV-F2
<400> 1
ctcctcaatg gatgttgcc 19
<210> 2
<211> 15
<212> DNA
<213> BKV-R1
<400> 2
gggagctgcc cctgg 15
<210> 3
<211> 22
<212> DNA
<213> BKV-FP3
<400> 3
tctaggcctg tacgggactg ta 22
<210> 4
<211> 20
<212> DNA
<213> Control-F
<400> 4
ctaccagcag aacaccccca 20
<210> 5
<211> 17
<212> DNA
<213> Control-R
<400> 5
gcggcggtca cgaactc 17
<210> 6
<211> 14
<212> DNA
<213> Control-Probe
<400> 6
cccaacgaga agcg 14
Claims (10)
1. A primer for detecting the BKV virus consists of a forward primer and a reverse primer, and is characterized in that the nucleotide sequence of the forward primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2.
2. A probe used in combination with the primer of claim 1, wherein the nucleotide sequence of the probe is as shown in SEQ ID NO. 3.
3. The probe of claim 2, wherein the probe is labeled with a fluorescent group at the 5 'end and a quencher group at the 3' end.
4. The probe of claim 3, wherein the fluorophore comprises at least one of FAM, VIC/HEX, ROX, CY3, CY5, CY5.5, CY7, JOE, TAMRA, TET, Texas Red, Quasar 570, Quasar 670, Atto 425, Atto 590, Alexa flow fluorophore; the quencher comprises at least one of Dabcyl, Eclipse, MGB, BHQ1, BHQ2, BHQ3, BBQ 650, TAMRA and TQ1-TQ7 quencher.
5. A BKV virus detection kit based on droplet digital PCR, comprising the primer of claim 1 and the probe of any one of claims 2 to 4.
6. The BKV virus detection kit of claim 5, further comprising an internal standard primer and a probe, wherein the nucleotide sequence of the internal standard primer is shown in SEQ ID nos. 4-5, and the nucleotide sequence of the internal standard probe is shown in SEQ ID No. 6.
7. The BKV virus detection kit of claim 6, wherein the working concentration of the primer of the internal standard is 900nM, and the working concentration of the probe of the internal standard is 375 nM.
8. The BKV virus detection kit of claim 5, further comprising a micro-droplet enzyme mixture for digital PCR, the positive control mixture being diluted with positive urine containing BK virus, the negative control mixture comprising mixed negative urine of normal humans, the positive control mixture comprising taq enzyme, UDG enzyme and restriction endonuclease.
9. The BKV virus detection kit of any of claims 5-8, wherein use of the kit comprises the steps of:
1) obtaining total DNA of a sample;
2) preparing the total volume of the reaction mixed solution of the micro-drop digital PCR according to the preset reaction number;
3) preparing each reaction system according to a preset reaction number;
4) and (3) carrying out micro-droplet digital PCR amplification on each reaction system subjected to micro-droplet treatment, and then calculating the nucleic acid concentration of each sample according to the amplification result.
10. Use of a kit according to any of claims 5 to 8 for non-diagnostic purposes for the detection of BKV virus in serum, plasma or urine derived from human samples.
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