CN112226537A - Kit for simultaneously detecting BK virus and human cytomegalovirus and detection method thereof - Google Patents
Kit for simultaneously detecting BK virus and human cytomegalovirus and detection method thereof Download PDFInfo
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Abstract
The invention discloses a BK virus and human cytomegalovirus combined detection kit and a detection method thereof. The invention applies real-time fluorescence quantitative PCR technology, adopts primers and probes with high specificity of BK virus and human cytomegalovirus, can judge whether the two viruses exist in a sample to be detected through one PCR reaction, and is more convenient and quicker than a single fluorescence quantitative PCR method, and saves cost. The detection sensitivity of the kit to two viruses reaches 10copies/mL, the specificity is good, and the kit does not have cross reaction with other viruses; meanwhile, the kit has good amplification repeatability, and the precision is less than or equal to 2.8 percent.
Description
Technical Field
The invention belongs to the technical field of virus detection kits, relates to a real-time fluorescence RT-PCR detection kit, and particularly relates to a kit for simultaneously detecting BK virus and human cytomegalovirus and a detection method thereof.
Background
Human polyomavirus type BK (BK virus) is a circular double-stranded DNA virus with about 5300bp of membrane without vesicle, belongs to polyomavirus family with JC virus (JCV) and SV40 virus, and is a subgroup of polyomavirus, with Human natural host, the infection rate of BK virus population is up to 90%, but clinical symptoms are often limited to individuals with immune function deficiency. BK virus is associated with the following diseases: hemorrhagic cystitis, interstitial glomerulonephritis, ureteral stenosis, vasculopathy, pneumonia, encephalitis, retinitis and even multiple organ failure. Polyoma virus-associated nephropathy (PVAN) occurs in about 5% of the renal transplant recipient population, with about 50% to 70% of these populations experiencing graft loss, primarily due to the activated infection of BK virus in the transplanted kidney.
Human Cytomegalovirus (HCMV) is a β -herpes virus that widely infects humans. Although adults are generally asymptomatic or less symptomatic after infection with HCMV, infection in pregnant women often results in severe consequences such as abortion, stillbirth, fetal malformation, and developmental delay, while HCMV often causes fatal infections in individuals with suppressed immune function (e.g., organ transplant recipients). Therefore, the method has important significance for diagnosing the HCMV active infection quickly and accurately at an early stage.
Opportunistic infections are pathogens which are weak in pathogenicity and cannot cause diseases when the immune function of a human body is normal, but when the immune function of the human body is reduced, the pathogens enter the human body in a false manner and invade the human body, so that various diseases are caused. Both of the above viruses belong to DNA viruses, and are often associated with opportunistic infections and coexistence in the same host. In order to better guide the research on the two viruses, a product capable of detecting BK virus and human cytomegalovirus needs to be developed. The advent of PCR has greatly increased the sensitivity of the detection means, however, conventional detection methods or products can only detect one virus type at a time. The Multiplex PCR (M-PCR) means that a plurality of pairs of specific primers are added into the same reaction system, if a template which is specifically complementary with each primer pair exists, more than one target DNA fragment can be amplified in one reaction tube at the same time, and the aim of detecting a plurality of pathogens at one time is fulfilled. However, since the multiplex PCR is a PCR reaction in which a plurality of pairs of primers are added to the same PCR reaction system to amplify a plurality of DNA fragments, the plurality of pairs of primers in the same reaction system are prone to interact, such as forming hairpin structures, dimer structures, etc., and the more the number of primer pairs and primers is, the more the interaction between the primers is, thereby affecting the PCR amplification efficiency, and further affecting the wide application of the multiplex PCR. The present invention has been made in an effort to develop a reliable diagnostic reagent for detecting BK virus and human cytomegalovirus with high sensitivity in the same reaction.
Disclosure of Invention
In order to solve the problem that BK virus and human cytomegalovirus cannot be detected simultaneously in the prior art, the invention provides a primer and a probe for detecting nucleic acid of the BK virus and the human cytomegalovirus based on a fluorescence quantitative PCR technology, so as to realize quick, effective and accurate detection of the BK virus and/or the human cytomegalovirus.
The invention also aims to provide a double fluorescence quantitative kit for detecting BK virus and human cytomegalovirus, which is a one-step double real-time fluorescence quantitative RT-PCR detection kit.
The invention further aims to provide application of the kit in preparing a reagent for detecting BK virus and human cytomegalovirus.
The fourth purpose of the invention is to provide a using method of the kit.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, a detection primer and a fluorescent probe for nucleic acids of BK virus and human cytomegalovirus are provided, the detection primer and the fluorescent probe comprise specific amplification primer sequences of 2 virus genes and specific fluorescent probe sequences corresponding to the primers, a fluorescent reporter group is labeled at the 5 'end of each fluorescent probe sequence, a fluorescent quencher group is labeled at the 3' end, and the specific sequences are as follows:
BK virus:
BK upstream primer: the nucleotide sequence is shown as SEQ ID NO.1,
the nucleotide sequence of the BK downstream primer is shown as SEQ ID NO.2,
the BK specific fluorescent probe has a nucleotide sequence shown as SEQ ID NO.3,
human cytomegalovirus:
CMV upstream primer: the nucleotide sequence is shown as SEQ ID NO.4,
the CMV downstream primer has a nucleotide sequence shown as SEQ ID NO.5,
the nucleotide sequence of the CMV-specific fluorescent probe is shown as SEQ ID NO. 6.
TABLE 1 sequences of detection primers and fluorescent probes for BK virus and human cytomegalovirus nucleic acids
Preferably, the BK virus-specific fluorescent probe and the human cytomegalovirus-specific fluorescent probe have a fluorescent reporter group selected from the group consisting of 6-carboxyfluorescein (6-carboxyfluorescein, 6-FAM), Hexachloro-6-methylfluorescein (HEXACHLORO-6-methylfluorescein, HEX), VIC fluorescent dye, tetrachloro-6-carboxyfluorescein (tetrachloro-6-carboxyfluorescein, TE T), Carboxy-X-rhodamine, 6-carboxytetramethylrhodamine (6-carboxytetramethylrhodamine MRDA, TAA), sulforhodamine (S. mu. Lforhodamine 101, Texas Red), 6-Carboxy-4 ', 5 ' -dichloro-2 ', 7 ' -dimethoxyfluorescein succinimidyl ester (6-carboxyxy-4 ', 5 ' -dichoro-2 ', 7' -dimethoxfluorescein, JOE), cyanine 3(cyanine3, Cy3), cyanine3.5 (cyanine3.5, Cy3.5), cyanine 5(cyanine5, Cy5), and cyanine5.5 (cyanine5.5, Cy 5.5); the BK virus specific probe and the human cytomegalovirus specific probe are provided with fluorescence quenching groups selected from at least one of 6-carboxytetramethylrhodamine, 4- (4-dimethylamino phenylazo) benzoic acid, Black Hole Quencher 1(Black Hole Quencher 1, BHQ1), Black Hole Quencher 2(Black Hole Quencher 2, BHQ2) or Black Hole Quencher 3(Black Hole Quencher 3, BHQ 3).
Preferably, the BK virus-specific fluorescent probe and the human cytomegalovirus-specific fluorescent probe have a fluorescent reporter group selected from 6-carboxyfluorescein (6-carboxyfluorescein, 6-FAM) and Hexachloro-6-methylfluorescein (HEX); the fluorescence quenching group is selected from Black Hole Quencher 1(Black Hole Quencher 1, BHQ 1).
Most preferably, the fluorescence reporter group of the BK virus-specific fluorescent probe is Hexachloro-6-methyl fluorescein (HEX), and the fluorescence quencher group is BHQ 1; the fluorescent reporter group of the human cytomegalovirus specific fluorescent probe is 6-carboxyfluorescein (6-carboxyfluorescein, 6-FAM), and the fluorescent quencher group is BHQ 1.
In a second aspect, the invention provides a kit for detecting BK virus and human cytomegalovirus nucleic acid in the same reaction, which comprises detection primers and fluorescent probes of any one of the two viruses, wherein the BK virus-specific fluorescent probe and the human cytomegalovirus-specific fluorescent probe are respectively provided with different fluorescent reporter groups.
Preferably, the kit for detecting BK virus and human cytomegalovirus nucleic acid in the same reaction is used by preparing a mixture of primers and fluorescent probes according to the following ratio: the molar ratio of the BK virus amplification primer pair to the BK virus specific fluorescent probe is 3:1, the molar ratio of the human cytomegalovirus amplification primer pair to the human cytomegalovirus specific fluorescent probe is 2:1, and the molar ratio of the BK virus specific fluorescent probe to the human cytomegalovirus specific fluorescent probe is 2: 1.
Preferably, the kit for simultaneously detecting BK virus and human cytomegalovirus nucleic acid further comprises a positive control and a negative control, wherein the negative control does not contain a physiological saline solution of BK virus and human cytomegalovirus, and the negative control can also be a physiological saline solution without BK virus genes and human cytomegalovirus genes; the positive control includes a plasmid containing a BK virus target sequence obtained by amplifying the sequences shown as SEQ ID No.1 and SEQ ID No.2 (named as BK virus positive control), and a plasmid containing a human cytomegalovirus target sequence obtained by amplifying the sequences shown as SEQ ID No.4 and SEQ ID No.5 (named as human cytomegalovirus positive control).
Specifically, the preparation process of the positive control is as follows: extracting the DNAs of the BK virus and the human cytomegalovirus respectively as templates, and amplifying corresponding target sequences by using corresponding primers respectively; and connecting the amplified product into a vector to respectively obtain a plasmid containing a BK virus target sequence and a plasmid containing a human cytomegalovirus target sequence, wherein the vector is a pUC57-Kan vector.
In one specific example, the positive control is prepared as follows: respectively taking the DNA fragment of the BK virus and the DNA fragment of the human cytomegalovirus as templates, and respectively amplifying the corresponding DNA fragments by using corresponding primers; and connecting the amplified product into a vector to respectively obtain a plasmid containing the BK virus amplified fragment and a plasmid containing the human cytomegalovirus amplified fragment, namely a BK virus positive control and a human cytomegalovirus positive control.
In one embodiment, the negative control is a physiological saline solution that does not contain BK virus and human cytomegalovirus. The negative control may be a physiological saline solution containing no BK virus gene or human cytomegalovirus gene.
Preferably, the kit for simultaneously detecting BK virus and human cytomegalovirus further comprises a virus genome DNA extraction reagent, dNTPs, PCR reaction liquid and enzyme, wherein the PCR reaction liquid comprises 220 mM-280 mM Tris-base, 0.2% -0.3% TritonX-100 in percentage by mass and 20 mmol/L-30 mmol/L MgCl2(ii) a The enzyme is Taq enzyme.
More preferably, in the kit for simultaneously detecting BK virus and human cytomegalovirus, the PCR reaction solution comprises 250mM Tris-base, 0.25 percent by mass of TritonX-100 and 25mmol/L MgCl2。
In a third aspect, the application of the kit for detecting BK virus and human cytomegalovirus in the same reaction in preparing a reagent for detecting BK virus and human cytomegalovirus is provided.
In a fourth aspect, there is provided a method for detecting BK virus and human cytomegalovirus in the same reaction, the method being for non-diagnostic and non-therapeutic purposes, the method using any of the above kits for detecting BK virus and human cytomegalovirus in the same reaction, comprising the steps of:
adding the components in the kit for detecting BK virus and human cytomegalovirus in the same reaction by using a sample DNA to be detected as a template to perform multiple fluorescent quantitative PCR amplification reaction, performing detection analysis according to a reaction result, and judging that the two viruses are negative if S-type amplification curves do not appear in all detection channels; if the detection channel has an S-type amplification curve and the Ct value is less than or equal to 38, the corresponding virus is judged to be positive.
Preferably, the reaction system (25. mu.l total system, the same applies hereinafter) of the multiplex quantitative PCR amplification reaction in the method for detecting BK virus and human cytomegalovirus in the same reaction comprises: 5mM dNTPs, 5 mu L PCR reaction liquid, 0.3 mu mol BK virus amplification primer pair, 0.3 mu mol human cytomegalovirus amplification primer pair, 0.1 mu mol BK virus specific probe, 0.1 mu mol human cytomegalovirus specific probe, 1 mu L enzyme and 0.5 to 5 mu L DNA of a sample to be tested.
Preferably, in the method for detecting BK virus and human cytomegalovirus in the same reaction, the reaction conditions of the multiplex quantitative PCR amplification reaction are as follows: pre-denaturation at 93-95 ℃ for 2-10 min; denaturation at 93-95 ℃ for 10-30 s, annealing at 55-60 ℃, extension and signal acquisition for 30-60 s, and circulating for 40-45 times.
The multiple fluorescent quantitative PCR amplification reaction is carried out in a multiple fluorescent quantitative PCR instrument. Further, the multiple fluorescence quantitative PCR instrument is an ABI series multiple fluorescence quantitative PCR instrument, a Bio-Rad series (ICycler/MJ Opticon 2) multiple fluorescence quantitative PCR instrument, an S Gene MX series multiple fluorescence quantitative PCR instrument, an R oche Lightcycler multiple fluorescence quantitative PCR instrument, a Ccpheid smartcycler multiple fluorescence quantitative PCR instrument, a Corbett Rortor-Gene multiple fluorescence quantitative PCR instrument and a Hangzhou Japanese series multiple fluorescence quantitative PCR instrument. It should be noted that the multiplex quantitative PCR instrument is not limited to the PCR instrument mentioned above, and other multiplex quantitative PCR instruments can be used in the present invention.
Compared with the prior art, the invention has the following beneficial effects:
1. the detection primer pairs designed aiming at the BK virus and the human cytomegalovirus are matched with each other, so that the mutual interference among the primers can be avoided, the BK virus and the human cytomegalovirus can be simultaneously detected in one tube of specimen through multiple fluorescence quantitative PCR, the detection time is saved, only one operation is needed, and the pollution generation chance is reduced.
2. The detection method using the kit has high sensitivity, and the detection sensitivity of two viruses reaches 10 copies/mL. Meanwhile, the detection method of the invention has good specificity and does not have cross reaction with other viruses, such as adenovirus and coxsackie virus CA 16.
3. The kit has very good amplification repeatability, and the precision obtained by calculation is less than or equal to 2.8 percent.
Drawings
FIG. 1 is a graph showing the amplification curves of positive samples of different concentrations in example 2;
FIG. 2 is a graph showing the amplification curves of the FAM channels of BK viruses of positive samples at different concentrations in example 2;
FIG. 3 is a graph showing the amplification of the VIC channel of cytomegalovirus of different concentrations of positive test substances in example 2;
FIG. 4 is a graph showing the amplification curve of positive test samples of the experimental group in example 3;
FIG. 5 is a graph showing the amplification curve of the positive test sample of the control group in example 3;
FIG. 6 is a graph showing the amplification curve of the reproducibility test of the precision test substance in example 4;
FIG. 7 is the amplification curve of the FAM channel for BK virus reproducibility detection of the precision test substance in example 4;
FIG. 8 is a graph showing the amplification curve of the VIC channel in the cytomegalovirus reproducibility test of the precision test article in example 4;
FIG. 9 is a graph showing the amplification curve of the positive test substance in example 5;
FIG. 10 is a graph showing the amplification curve of negative test substances in example 5;
FIG. 11 is a graph showing the amplification curves of the samples to be tested in example 5;
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the following embodiments and accompanying drawings. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Example 1 provides a kit for detecting BK virus and human cytomegalovirus in the same reaction.
The kit comprises PCR reaction liquid, a nucleic acid composition for simultaneously detecting BK virus and human cytomegalovirus, hot start Taq enzyme, a positive control and a negative control. Wherein, the PCR reaction solution comprises 250mM Tris-base, 0.25 percent of TritonX-100 by mass percent and 25mmol/L MgCl2。
Wherein the nucleic acid composition comprises:
BK virus amplification primer pairs with sequences shown as SEQ ID No.1 and SEQ ID No. 2;
human cytomegalovirus amplification primer pairs with sequences shown as SEQ ID No.4 and SEQ ID No. 5;
the sequence of the BK virus specific fluorescent probe is shown as SEQ ID No.3, and both ends of the BK virus specific fluorescent probe are respectively connected with FAM and BHQ 1; the sequence of the fluorescent probe is shown as SEQ ID No.6, and both ends of the fluorescent probe are respectively connected with VIC and BHQ 1.
Positive controls included plasmids containing BK viral target sequence and plasmids containing human cytomegalovirus target sequence.
The construction method of BK virus target sequence plasmid comprises the following steps: amplifying a nucleic acid sample of the BK virus by a PCR method, wherein the sequence of a PCR primer is shown as SEQ ID NO: 1-2. And after purifying the amplified fragment, connecting the amplified product into a pUC57-Kan vector, carrying out sequencing identification, transforming a recombinant vector with a correct sequencing result into DH5 alpha, amplifying, and extracting a plasmid to obtain a BK virus positive control.
The construction method of the human cytomegalovirus target sequence plasmid comprises the following steps: amplifying a nucleic acid sample of the human cytomegalovirus by adopting a PCR method, wherein the sequence of a PCR primer is shown as SEQ ID NO: 4-5. And after purifying the amplified fragment, connecting the amplified product into a pUC57-Kan vector, carrying out sequencing identification, transforming a recombinant vector with a correct sequencing result into DH5 alpha, amplifying, and extracting a plasmid to obtain a human cytomegalovirus positive control.
The negative control was physiological saline solution containing no BK virus and human cytomegalovirus.
[ example 2 ] measurement of sensitivity of the kit of example 1 for detecting BK virus and human cytomegalovirus in the same reaction
(1) The BK virus positive control and the human cytomegalovirus positive control in the kit of example 1 were mixed and mixed to prepare a mixture, and positive test substances were obtained (the concentration of each virus positive control was 10)6copies/mL)。
(2) Carrying out 10-fold gradient dilution on the positive sample (namely 10 copies/mL-10)5copies/mL), the kit of example 1 was used to perform double fluorescence quantitative PCR detection on the positive samples under each gradient, and the system of the double fluorescence quantitative PCR reaction is shown in table 2. The reaction conditions of the double fluorescent quantitative PCR reaction are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃, extension, signal acquisition for 40s, and circulation for 40 times.
(3) And (4) analyzing results: and judging whether the detection result is positive or negative through a fluorescence amplification curve graph and the Ct value, and determining whether the sample contains BK virus and human cytomegalovirus. Specifically, when the Ct value of the amplification curve graph is less than or equal to 38 and the amplification curve shows obvious exponential increase (i.e. S type), the result is positive; when Ct value of amplification curve graph>38 or no Ct value, the result was negative. The detection results are shown in FIGS. 1 to 3. FIG. 1 is a graph showing the amplification curve of positive samples of different concentrations in example 2. FIG. 2 is the amplification curves of FAM channels of BK viruses at different concentrations of positive test substances in example 2In the figure, the curves indicated by the arrows (2-1), (2-2), (2-3), (2-4) and (2-5) in FIG. 2 correspond to concentrations of 105copies/m L、104copies/mL、103copies/mL、102Amplification curves at copies/mL, 10 copies/mL. FIG. 3 is a graph showing the amplification curve of the VIC channel of human cytomegalovirus of the positive test samples of different concentrations in example 2, wherein the curves indicated by the arrows (3-1), (3-2), (3-3), (3-4) and (3-5) in FIG. 3 correspond to concentrations of 105copies/mL、104copies/mL、103copies/mL、102Amplification curves at copies/mL, 10 copies/mL.
TABLE 2 System for Dual fluorescent quantitative PCR reaction
Wherein, the Taq enzyme is hot-started in the reaction system. ddH2And O is double distilled water. All reagents in the reaction system are placed in the same reaction tube for double fluorescent quantitative PCR detection. As can be seen from FIG. 1, the kit of example 1 can simultaneously detect BK virus and human cytomegalovirus in a sample to be detected at one time, and save detection time.
As can be seen from FIGS. 2 to 3, the BK virus and human cytomegalovirus are present at 10 copies/mL-105Amplification curves in the range of copies/mL are in good linear relation, the detection sensitivity of the two viruses reaches 10copies/mL, and the sensitivity is high.
Example 3 comparison of specificity of the kit of example 1 with known commercial kits
(1) The BK virus positive control and the human cytomegalovirus positive control (each virus positive control has a concentration of 10) in the kit of example 16copies/mL) and preparing a mixed solution to obtain a positive to-be-detected product; not containing the above BK virusAnd the normal saline of the human cytomegalovirus positive reference substance is a negative test substance.
(2) The experiment is divided into an experimental group and a control group, and the kit of the experimental group is the kit of the embodiment 1. The kit of the control group is substantially the same as the kit of example 1 (the kit components of the control group are all the components of the known commercial kit, namely dNTPs, PCR reaction solution, amplification primers and probes of BK virus and human cytomegalovirus, enzyme solution, positive control and negative standard, wherein the DNA regions of the BK virus positive control and the human cytomegalovirus positive control comprise the fragments of the kit amplification primer pairs of the control group).
(3) The kit of the experimental group and the kit of the control group are adopted to carry out double fluorescence quantitative PCR detection on the positive to-be-detected product and the negative to-be-detected product, and the reaction system, the reaction conditions and the result analysis are the same as those in the example 2. The detection results are shown in FIGS. 4 to 5. FIG. 4 is a graph showing the amplification curve of positive test samples, BK virus amplification curve and human cytomegalovirus amplification curve in the experimental group of example 3.
As can be seen from fig. 4, the experimental group obtained 2S-type amplification curves, and the negative test sample had no amplification curve, which indicates that the kit of the experimental group can simultaneously detect BK virus and human cytomegalovirus in the positive test sample, and no non-specific amplification curve occurs.
As can be seen from FIG. 5, the control group obtained an S-type amplification curve, which failed to amplify the positive control of human cytomegalovirus, indicating that there was mutual interference between the two primers and the two probes in the control group, resulting in failure to detect both BK virus and human cytomegalovirus.
Example 4 measurement of precision of kit of example 1
And (3) mixing the BK virus positive control and the human cytomegalovirus positive control in the kit of the embodiment 1 to prepare a mixed solution, so as to obtain a positive test substance. Carrying out 10-fold gradient dilution on the positive sample (the concentration gradient is 10)5copies/mL、104copies/mL、103copies/mL、102copies/mL, 10copies/mL) to obtain precision samples, as described in example 1The kit is used for carrying out double fluorescence quantitative PCR amplification, the amplification precision of a to-be-detected product is 10 multiple holes, and the detection result is shown in FIGS. 6-8. FIG. 6 is a graph showing the amplification curve of the reproducibility test of the precision assay sample in example 4. FIG. 7 is a graph showing the amplification of the FAM channel in the BK virus reproducibility test of the precision test substance in example 4. FIG. 8 is a graph showing the VIC channel amplification in the repeated detection of human cytomegalovirus in the samples with precision in example 4.
As can be seen from FIGS. 6 to 8, the precision of each channel of the kit of the above embodiment is very good, CT value data is derived, the precision obtained by calculation is less than or equal to 2.8%, and the amplification repeatability of the kit is very good.
Example 5 detection of clinical samples with the kit of example 1
(1) The BK virus positive control in the kit of example 1 and the human cytomegalovirus positive control were mixed and mixed to prepare a mixture, and a positive control was obtained with a concentration of 105copies/mL. The negative control was physiological saline without BK viral gene and human cytomegalovirus gene.
(2) The samples to be extracted are 12 samples including 5 BK virus samples, 4 human cytomegalovirus samples, 2 adenovirus samples and 1 measles virus sample, and all the samples are from a disease control center. And respectively extracting DNA in each sample to be extracted by adopting a virus DNA extraction kit (purchased from Tiangen Biochemical technology (Beijing) Co., Ltd.) to obtain corresponding samples to be detected.
(3) The kit of example 1 is used to perform double fluorescence quantitative PCR detection on the positive control, the negative control and each sample to be detected in step (2), and the reaction system, reaction conditions and result analysis are the same as those in example 2. The measurement results are shown in FIG. 9. FIG. 9 is a graph showing the amplification curve of the positive control in example 5. FIG. 10 is a graph showing the amplification curve of the negative control in example 5. FIG. 11 is a graph showing the amplification curves of the samples to be tested in example 5.
As can be seen from FIGS. 9 to 11, the kit of example 1 can detect two viruses in the positive control, and the negative control does not detect any virus, indicating that the kit of example 1 has high specificity.
As can be seen from fig. 11, when the kit of example 1 is used to detect a sample to be detected, 5 BK virus samples and 4 human cytomegalovirus samples can be detected, and adenovirus samples and CA16 coxsackie virus samples are not detected, and the accuracy of detecting the samples reaches 100%. In conclusion, the kit for simultaneously detecting BK virus and human cytomegalovirus in the embodiment can simultaneously detect two items of BK virus and human cytomegalovirus for one tube of specimen, has short detection time, high detection sensitivity, high accuracy, good specificity and good repeatability, and has higher guiding significance for clinical application.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (10)
1. A primer and a probe for double fluorescence PCR detection aiming at BK virus and human cytomegalovirus are characterized in that the 5 'end of a probe sequence is marked with a fluorescence reporter group, the 3' end is marked with a fluorescence quenching group, and the specific sequence is as follows:
BK virus:
BK upstream primer: the nucleotide sequence is shown as SEQ ID NO.1,
the nucleotide sequence of the BK downstream primer is shown as SEQ ID NO.2,
the BK specific fluorescent probe has a nucleotide sequence shown as SEQ ID NO.3,
human cytomegalovirus:
CMV upstream primer: the nucleotide sequence is shown as SEQ ID NO.4,
the CMV downstream primer has a nucleotide sequence shown as SEQ ID NO.5,
the nucleotide sequence of the CMV-specific fluorescent probe is shown as SEQ ID NO. 6.
2. The primer and probe for detecting the dual fluorescence PCR of BK virus and human cytomegalovirus according to claim 1, wherein the fluorescence reporter groups carried on the BK virus probe and the human cytomegalovirus probe are selected from at least one of 6-carboxyfluorescein, hexachloro-6-methylfluorescein, VIC fluorescent dye, tetrachloro-6-carboxyfluorescein, carboxy-X-rhodamine, 6-carboxytetramethylrhodamine, sulforhodamine, 6-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxyfluorescein succinimidyl ester, cyanine3, cyanine3.5, cyanine5, and cyanine 5.5; the BK virus specific probe and the human cytomegalovirus specific probe are provided with fluorescence quenching groups selected from at least one of 6-carboxytetramethylrhodamine, 4- (4-dimethylaminobenzeneazo) benzoic acid, a black hole quencher 1, a black hole quencher 2 or a black hole quencher 3.
3. The primer and probe for the dual fluorescence PCR detection of BK virus and human cytomegalovirus according to claim 2, wherein the fluorescent reporter group is selected from 6-carboxyfluorescein or hexachloro-6-methylfluorescein; the fluorescence quenching group is selected from a black hole quenching agent 1; preferably, the fluorescence reporter group of the BK virus probe is hexachloro-6-methyl fluorescein, and the fluorescence quenching group is BHQ 1; the fluorescent reporter group of the human cytomegalovirus probe is 6-carboxyfluorescein, and the fluorescent quencher group is BHQ 1.
4. A kit for combined detection of BK virus and human cytomegalovirus, which is characterized by comprising the primers and probes for detection of two viruses according to any one of claims 1 to 3, wherein the BK virus probe and the human cytomegalovirus probe are respectively provided with different fluorescent reporter groups.
5. The BK virus and human cytomegalovirus combined detection kit according to claim 4, wherein the kit is used for preparing a primer and probe mixed solution according to the following proportion: the molar ratio of the BK virus amplification primer pair to the BK virus probe is 3:1, the molar ratio of the human cytomegalovirus amplification primer pair to the human cytomegalovirus probe is 2:1, and the molar ratio of the BK virus probe to the human cytomegalovirus probe is 2: 1.
6. The combined detection kit for BK virus and human cytomegalovirus according to claim 4, which further comprises a positive control substance and a negative control substance, wherein the negative control substance is a physiological saline solution; the positive control comprises a plasmid containing a BK virus target sequence and a plasmid containing a human cytomegalovirus target sequence; preferably, the positive control comprises a plasmid containing a BK viral target sequence obtained by amplification of the sequences shown as SEQ ID No.1 and SEQ ID No.2, and a plasmid containing a human cytomegalovirus target sequence obtained by amplification of the sequences shown as SEQ ID No.4 and SEQ ID No. 5.
7. The BK virus and human cytomegalovirus combined detection kit according to claim 4, wherein the kit further comprises at least one of DNA extraction reagent, dNTPs, PCR reaction solution and enzyme, the PCR reaction solution comprises 220 mM-280 mM Tris-base, 0.2% -0.3% TritonX-100 in percentage by mass and 20 mmol/L-30 mmol/L MgCl2(ii) a The enzyme is Taq enzyme.
8. The BK virus and human cytomegalovirus combined detection kit according to claim 7, wherein the PCR reaction solution of the kit comprises 250mM Tris-base, 0.25% TritonX-100 in percentage by mass and 25mmol/L MgCl2。
9. Use of the combined detection kit for BK virus and human cytomegalovirus of any one of claims 4 to 8 in preparation of reagents for detecting BK virus and human cytomegalovirus.
10. A combined detection method of BK virus and human cytomegalovirus, wherein said detection method is for non-diagnostic, non-therapeutic purposes, comprising the steps of:
(1) extracting DNA of a sample to be detected;
(2) taking a sample DNA to be detected as a template, adding each component in the kit for detecting BK virus and human cytomegalovirus in the same reaction according to any one of claims 4 to 8 to perform multiple fluorescence quantitative PCR amplification reaction, performing detection analysis according to the reaction result, and judging that both viruses are negative if an S-type amplification curve does not appear in each detection channel; if the detection channel has an S-type amplification curve and the Ct value is less than or equal to 38, the corresponding virus is judged to be positive;
the reaction conditions of the multiplex fluorescent quantitative PCR amplification reaction in the detection method for detecting the BK virus and the human cytomegalovirus in the same reaction are as follows: pre-denaturation at 93-95 ℃ for 2-10 min; denaturation at 93-95 ℃ for 10-30 s, annealing at 55-60 ℃, extension and signal acquisition for 30-60 s, and circulating for 40-45 times.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344970A (en) * | 2010-07-27 | 2012-02-08 | 温州医学院附属第一医院 | Primer sequences and quantitative determination kit used for simultaneously detecting human CMV and BK virus DNA |
| CN102363818A (en) * | 2011-06-28 | 2012-02-29 | 同昕生物技术（北京）有限公司 | Treble real-time fluorescence quantitative polymerase chain reaction (PCR) method for simultaneously detecting epstein-barr virus (EBV), polyma virus (BKV) and cytomegalovirus (CMV) of people and kit |
| WO2020023776A2 (en) * | 2018-07-25 | 2020-01-30 | Icahn School Of Medicine At Mount Sinai | Compositions and methods for inhibiting cancers and viruses |
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2020
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344970A (en) * | 2010-07-27 | 2012-02-08 | 温州医学院附属第一医院 | Primer sequences and quantitative determination kit used for simultaneously detecting human CMV and BK virus DNA |
| CN102363818A (en) * | 2011-06-28 | 2012-02-29 | 同昕生物技术（北京）有限公司 | Treble real-time fluorescence quantitative polymerase chain reaction (PCR) method for simultaneously detecting epstein-barr virus (EBV), polyma virus (BKV) and cytomegalovirus (CMV) of people and kit |
| WO2020023776A2 (en) * | 2018-07-25 | 2020-01-30 | Icahn School Of Medicine At Mount Sinai | Compositions and methods for inhibiting cancers and viruses |
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