CN113373267A - Multiplex fluorescence quantitative RT-PCR kit for detecting blood-borne infectious viruses - Google Patents
Multiplex fluorescence quantitative RT-PCR kit for detecting blood-borne infectious viruses Download PDFInfo
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Abstract
The invention provides a multiplex fluorescence quantitative RT-PCR kit for detecting blood-borne infectious viruses, which comprises quantitative RT-PCR reaction liquid, enzyme Mix liquid, primer probe Mix liquid, standard substances (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P standard substances), positive and negative reference substances. The primer probes comprise four groups of primers and corresponding probes of different fluorescent labels. The invention designs high-specificity primer probes according to the highly conserved proteins of cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P respectively, and simultaneously detects whether the viruses exist in a serum sample through a PCR reaction. The method is simple, convenient and quick, saves cost, has good repeatability, and can effectively and quickly screen the blood-borne infectious virus. Can be applied to the rapid screening of the blood-borne infectious virus and the research of the epidemiology of the blood-borne infectious virus.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a fluorescent quantitative RT-PCR detection kit, and particularly relates to a multiple fluorescent quantitative RT-PCR detection kit for detecting blood-borne infectious viruses, which is a nucleic acid detection method for detecting Cytomegalovirus (CMV), Epstein-Barr virus (EBV) and Herpes Simplex Virus (HSV) in a serum sample in the same reaction tube by one-step multiple real-time fluorescent quantitative RT-PCR, and can be applied to the rapid screening of blood-borne infectious viruses and the research on the epidemiology of blood-borne infectious viruses.
Background
In the past decades, people are increasingly aware of the serious threat of infectious pathogens to blood safety, blood-borne infectious pathogens through blood transfusion become a main concern affecting clinical safety blood, and blood-borne infectious pathogens through blood transfusion are mainly transmitted through blood donation from blood donors carrying pathogens, so that the screening strategy of blood-borne infectious pathogens is an important measure for ensuring clinical safety blood. The american blood bank association (AABB) guidelines show: more than 70 infectious pathogens, of which more than 60 are the viruses (more than 50) and bacteria (more than 10), are transmitted by blood transfusion; common pathogens include Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), human immunodeficiency virus (HIV-1/2), human T-lymphotropic virus (HTLV-1/2), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Herpes Simplex Virus (HSV), human herpes virus type 8 (HHV-8), dengue virus (DENV-1/2/3/4), Hepatitis E Virus (HEV), avian influenza H7N9 virus, Ebola virus (EBOV), Treponema Pallidum (TP), malaria, and the like. The Chinese implemented blood product pathogen screening comprises the following steps: TP, HIV-1/2, HBV and HCV. Although the screening of four pathogens can effectively guarantee the in-bed blood safety of China and most countries and regions all over the world, with the obvious increase of the prevalence trend of new (sudden) pathogens and undetected pathogens and the increasing frequency of international cooperation and communication, the occurrence of the input blood-borne infectious pathogens is increased in recent years, the propagation risk of the pathogens is increased, the clinical safe blood consumption is seriously threatened, and the establishment of a screening project meeting the development requirements of the new era is urgent.
Currently, the Chinese pathogen screening for blood products mainly adopts ELISA and single or triple pathogen nucleic acid detection for TP, HIV-1/2, HBV and HCV. The nucleic acid detection technology is direct, has high specificity and sensitivity for detecting trace infectious pathogens, but has serious influence on the wide application of the technology due to factors such as expensive detection cost, long operation time and the like. The real-time fluorescence quantitative PCR technology has the advantages of full-closed detection, single-tube amplification real-time quantification, simplicity, rapidness, cost saving, good repeatability and the like, has high specificity and sensitivity, is more optimized compared with common PCR detection, and can be used as a rapid and effective detection means for screening blood-borne infectious pathogens.
Disclosure of Invention
The invention aims to provide a multiplex fluorescence quantitative RT-PCR kit for detecting blood-borne infectious viruses, which is a one-step multiplex fluorescence quantitative RT-PCR detection kit for detecting blood-borne infectious viruses (cytomegalovirus, Epstein-Barr virus and herpes simplex virus). The kit comprises quantitative RT-PCR reaction liquid, enzyme Mix liquid, primer probe Mix liquid, standard substances (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P), positive control substances (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P) and negative control substances. The box body is provided with container holes for respectively placing a quantitative RT-PCR reaction liquid tube, an enzyme Mix liquid tube, a primer probe Mix liquid tube, a standard sample tube (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P), a positive control sample tube (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P) and a negative control sample tube.
The quantitative RT-PCR reaction liquid tube comprises a PCR reaction buffer liquid tube (containing a mixture of magnesium chloride and deoxyribonucleotide triphosphate and the like), an enzyme Mix liquid tube (containing heat-resistant TaqDNA polymerase, RNase inhibitor, MMLV reverse transcriptase and the like), a primer probe Mix liquid tube comprises four groups of primers and four corresponding probes with different fluorescent labels, and the primer probe Mix liquid tube is a brown tube. The negative control is DEPC (diethyl pyrocarbonate) treated water sterilized at high temperature and high pressure, and the positive control is a positive plasmid sample of cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P.
The upstream primer and the downstream primer for the multiplex fluorescent quantitative RT-PCR detection and corresponding specific probe sequences are as follows: upstream primer CMV-F: 5'-CTGGGCATAAGCCATAATCTCATC-3'
Downstream primer CMV-R: 5'-CAGCCTTCCCTAAGACCACC-3'
Specific probe CMV-P5 '-FAM-TGCCGCCATGGCCTGACTGCAGCCA-BHQ 1-3'
Upstream primer EBV-F: 5'-CCAAGAAGGTGGCCCAGAT-3'
Downstream primer EBV-R of 5-TCCAGTGCTTGGGCCTTC-3'
Specific probe EBV-P5 '-VIC-TGAGCCTGACGTGCCCCCGGGAGCG-BHQ 1-3'
Upstream primer HSV-F: 5'-AGAGGGACATCCAGGACTTTG-3'
The downstream primer HSV-R: 5'-CTTGTAATACACCGTCAGGTGG-3'
Specific probe HSV-P5 '-CY 5-ACCGCCGAACTGAGCAGACACCCGC-BHQ 3-3'
The upstream primer RNase P-F: 5'-GATTTGGACCTGCGAGCG-3'
The downstream primer RNase P-R: 5'-GAGCGGCTGTCTCCACAAGT-3'
The specific probe RNase P-P is 5 '-ROX-TTCTGACCTGAAGGCTCTGCGCG-BHQ 2-3'.
The specific probes of cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P are respectively marked by four different fluorescences of FAM, VIC, CY5 and ROX.
The standard substance comprises cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNaseP standard substance, and the gene sequence of the conserved region is as follows:
the cytomegalovirus standard sequence is:
AATCTTAAAGATTTTCTGGGCATAAGACATAATCTCATCAGGAGAGCACTGAGGCAAGTTCTGCAATGCCGCCATGGCCTGACTGCAGCCATTGGTGGTCTTAGGGAAGGCTGAGTTCTTGGTAAAGA。
the Epstein-Barr virus standard sequence is:
TTATTTTGAATACCACCAAGAAGGTGGCCCAGATGGTGAGCCTGACGTGCCCCCGGGAGCGATAGAGCAGGGCCCCGCAGATGACCCAGGAGAAGGCCCAAGCACTGGACCCCGGGGTCAGGGT。
the herpes simplex virus standard product sequence is as follows:
GCATCACCGACCCGGAGAGGGACATCCAGGACTTTGTCCTCACCGCCGAACTGAGCAGACACCCGCGCGCGTACACCAACAAGCGCCTGGCCCACCTGACGGTGTATTACAAGCTCATGGCCCGCCGC。
the sequence of the RNase P standard product is as follows:
ATGGCGGTGTTTGCAGATTTGGACCTGCGAGCGGGTTCTGACCTGAAGGCTCTGCGCGGACTTGTGGAGACAGCCGCTCACCTTGGCTATTCAG。
the kit provided by the invention needs to be stored at-20 ℃, and the times of repeated freeze thawing are reduced; and storing the primers and the probe Mix solution in a dark place.
The invention also aims to provide the application of the multiplex fluorescence quantitative RT-PCR detection kit in the nucleic acid detection of cytomegalovirus, Epstein-Barr virus and herpes simplex virus and the judgment of sample collection state and nucleic acid extraction condition.
The use method of the kit comprises the following steps: a positive control and a negative control are arranged in each fluorescence quantitative detection. The standard was diluted to 1X 10 with TE Buffer (cat. No.: AM9849) from Invitrogen2-1×107copies/ml。
Extraction of sample nucleic acid: 200ul serum samples were taken for extraction. The Nucleic Acid extraction adopts Viral Nucleic Acid extraction Kit II of Geneaid company, the extraction operation is carried out according to the instruction in the Kit, and 10ul of final product sample Nucleic Acid is taken as a subsequent template.
Detection of nucleic acids: 10ul of the extracted nucleic acid of the sample to be tested was used as a template. The total reaction volume was 50. mu.l, where 25. mu.l of the fluorescent quantitative PCR Mix, 2. mu.l of the Taq enzyme Mix, 12. mu.l of the primer probe Mix (20. mu. mol/L, containing four sets of primers and fluorescent probe), 10. mu.l of the template, and the remainder was made up to a final volume of 50. mu.l with water. Detecting on an ABI QuantStudio5 fluorescent quantitative PCR instrument, wherein the reaction program is as follows: reverse transcription at 42 deg.C for 5 min; the hot start was carried out at 95 ℃ for 10s, followed by multiplex fluorescence detection at 95 ℃ for 5s and 55 ℃ for 40s at 55 ℃ for a total of 40 cycles.
And (3) judging an experimental result: firstly, samples are collected to be qualified, and the CT values detected by RNase P, cytomegalovirus, Epstein-Barr virus and herpes simplex virus correspond to corresponding fluorescence labeled probes, and if the samples are collected to be qualified and the nucleic acid extraction is successful, the RNaseP is positive; when CT values of the remaining test samples were 0, 40 or undetermined, the report was negativeAnd (4) sex. ② detecting the C of cytomegalovirus, Epstein-Barr virus and herpes simplex virus of samplesTWhen the value is less than or equal to 35, the report is positive, and the detection sample CTA value of not less than 35 and<40 sample, Recommendations results CTIf the value is less than 38, reporting the virus as positive according to the judgment standard of II. And calculating the virus copy numbers (copies/ml) of the cytomegalovirus, Epstein-Barr virus and herpes simplex virus of the specimen to be detected according to the obtained standard curve.
The research designs primer probes aiming at cytomegalovirus, Epstein-Barr virus and herpes simplex virus; meanwhile, the ribozyme RNase P is used as an internal reference. The single-tube one-step method is adopted for multiplex real-time fluorescence quantitative RT-PCR, cytomegalovirus, Epstein-Barr virus and herpes simplex virus can be detected simultaneously by a single tube, the detection cost is obviously reduced, rich detection information can be obtained, the quality of sample nucleic acid can be judged by introducing internal parameters, and the judgment of a detection result is assisted. In addition, the multiplex real-time fluorescence quantitative RT-PCR also has extremely high specificity and sensitivity.
The invention has the advantages that the invention adopts a high-specificity and high-sensitivity one-step real-time fluorescence quantitative RT-PCR technology, designs highly specific primers and fluorescence labeled probes for cytomegalovirus, Epstein-Barr virus and herpes simplex virus, introduces a conservative reference gene RNase P, and develops a multiplex fluorescence quantitative RT-PCR detection kit for detecting the cytomegalovirus, the Epstein-Barr virus and the herpes simplex virus, judging the sample collection and nucleic acid extraction conditions. The invention can simultaneously detect whether cytomegalovirus, Epstein-Barr virus and herpes simplex virus exist in a serum sample through a PCR reaction, and compared with a single fluorescence quantitative PCR method, the invention is simpler, more convenient and faster, saves cost and has good repeatability. The virus to be detected is subjected to totally-enclosed detection and single-tube amplification real-time quantification, and the blood-borne infectious virus can be effectively and quickly screened according to the copy number of virus infection. The technology can be applied to the rapid screening of the blood-borne infectious virus and the research of epidemiology of the blood-borne infectious virus.
Drawings
FIG. 1 is a schematic structural diagram of the kit, wherein 1 is a fluorescent quantitative PCR Mix tube, 2 is a Taq enzyme Mix tube, 3 is a primer probe Mix tube, 4 is a negative control tube, 5 is a standard tube (5-1 cytomegalovirus, 5-2Epstein-Barr virus, 5-3 herpes simplex virus, 5-4RNase P, 4 tubes in total), and 6 is a positive control tube (6-1 cytomegalovirus, 6-2Epstein-Barr virus, 6-3 herpes simplex virus, 6-4RNase P, 4 tubes in total).
FIG. 2 is an example of the kit for detecting a serum specimen of a cytomegalovirus positive patient.
FIG. 3 is an example of the use of the kit for detecting serum samples from patients who are Epstein-Barr virus positive.
FIG. 4 is an example of the kit for detecting a serum specimen of a patient positive for herpes simplex virus.
FIG. 5 shows the sensitivity of the kit for detecting cytomegalovirus, which is 10 from left to right (1-7)8、107、106、105、104、103、102copies/ml。
FIG. 6 is a schematic diagram of gel electrophoresis of a cytomegalovirus standard real-time fluorescent quantitative RT-PCR product of the kit, wherein Lane Marker is DL2000 Marker, Lane 1 is negative control, and Lane 2-8 are 10 respectively2、103、104、105、106、107、108Real-time fluorescence quantitative RT-PCR products.
FIG. 7 is a real-time fluorescent quantitative RT-PCR standard curve of the cytomegalovirus standard substance of the kit.
FIG. 8 shows the sensitivity of the kit for detecting Epstein-Barr virus, which is 10 from left to right (1-7)8、107、106、105、104、103、102copies/ml。
FIG. 9 is a schematic diagram of gel electrophoresis of real-time fluorescence quantitative RT-PCR products of the Epstein-Barr virus standard of the kit, wherein Lane Marker is DL2000 Marker, Lane 1 is negative control, and Lane 2-8 are respectively 102、103、104、105、106、107、108Real-time fluorescence quantitative RT-PCR products.
FIG. 10 is a real-time fluorescent quantitative RT-PCR standard curve of the Epstein-Barr virus standard substance of the kit.
FIG. 11 shows the sensitivity of the kit for detecting herpes simplex virus, which is 10 from left to right (1-7)8、107、106、105、104、103、102copies/ml。
FIG. 12 is a schematic diagram of the real-time fluorescence quantitative RT-PCR product gel electrophoresis of the herpes simplex virus standard substance of the kit, wherein Lane Marker is DL2000 Marker, Lane 1 is negative control, and Lane 2-8 are respectively 102、103、104、105、106、107、108Real-time fluorescence quantitative RT-PCR products.
FIG. 13 is a real-time fluorescent quantitative RT-PCR standard curve of the herpes simplex virus standard substance of the kit.
FIG. 14 shows the sensitivity of the kit for detecting RNase P, which is 10 from left to right (1-7)8、107、106、105、104、103、102copies/ml。
FIG. 15 is a schematic diagram of gel electrophoresis of real-time fluorescent quantitative RT-PCR products of the RNase P standard substance of the kit, wherein Lane M is DL2000 Marker, Lane 1 is negative control, and Lane 2-8 are respectively 102、103、104、105、106、107、108Real-time fluorescence quantitative RT-PCR products.
FIG. 16 is the real-time fluorescent quantitative RT-PCR standard curve of the RNase P standard substance of the kit.
FIG. 17 shows the results of mixed detection of positive samples of cytomegalovirus, Epstein-Barr virus and herpes simplex virus.
Detailed Description
The invention will now be further elucidated with reference to the drawings and specific examples, which are only illustrative and not limiting of the scope of the invention.
Example 1
A multiplex fluorescence quantitative RT-PCR detection kit for detecting blood-borne infectious viruses comprises quantitative RT-PCR reaction liquid, enzyme Mix liquid, primer probe Mix liquid, standard substances (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P), positive control substances (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P) and negative control substances. The box body is provided with container holes for respectively placing a quantitative RT-PCR reaction liquid tube, an enzyme Mix liquid tube, a primer probe Mix liquid tube, a standard sample tube (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P), a positive control sample tube (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P) and a negative control sample tube.
Referring to fig. 1, the kit package is provided with container holes for placing corresponding kit components: a quantitative RT-PCR reaction liquid tube (reference numeral 1), an enzyme mixed liquid tube (reference numeral 2), a primer probe mixed liquid tube (reference numeral 3), a negative control tube (reference numeral 4), a standard tube (reference numeral 5) (comprising 5-1 cytomegalovirus, 5-2Epstein-Barr virus, 5-3 herpes simplex virus and 5-4RNase P standard substance, 4 tubes in total), and a positive control tube (reference numeral 6) (comprising 6-1 cytomegalovirus, 6-2Epstein-Barr virus, 6-3 herpes simplex virus and 6-4RNase P positive control substance, 4 tubes in total). The kit comprises: quantitative RT-PCR reaction solution, enzyme mixture, primer probe mixture, standard substance (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P), positive control substance (cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P) and negative control substance.
Example 2
1 materials and methods
1.1 serum samples and viral nucleic acids:
clinical samples of cytomegalovirus, Epstein-Barr virus and herpes simplex virus are derived from serum specimens of patients with positive nucleic acid for cytomegalovirus, Epstein-Barr virus and herpes simplex virus in the first hospital affiliated to the medical college of Zhejiang university and other hospitals in Zhejiang province.
In addition, positive specimens of other herpes viruses such as VZV, HHV-6, HHV-7 and HHV-8 are provided by the key laboratory in the diagnosis and treatment of infectious diseases.
1.2 primers and probes
A plurality of gene sequences of cytomegalovirus, Epstein-Barr virus and herpes simplex virus, which cover the country and abroad, are downloaded from an NCBI gene library. The homology comparison is carried out by using DNAman software, and the conserved region of the viral genome is determined. A Primer Express 3.0 software is used for designing a highly specific Primer and a Taqman probe in a conserved region of the Taqman probe, and the sequences of the Primer and the probe are verified by Blast, so that the Taqman probe has better specificity.
The primer and probe sequences were as follows:
upstream primer CMV-F: 5'-CTGGGCATAAGCCATAATCTCATC-3'
Downstream primer CMV-R: 5'-CAGCCTTCCCTAAGACCACC-3'
Specific probe CMV-P5 '-FAM-TGCCGCCATGGCCTGACTGCAGCCA-BHQ 1-3'
Upstream primer EBV-F: 5'-CCAAGAAGGTGGCCCAGAT-3'
Downstream primer EBV-R of 5-TCCAGTGCTTGGGCCTTC-3'
Specific probe EBV-P5 '-VIC-TGAGCCTGACGTGCCCCCGGGAGCG-BHQ 1-3'
Upstream primer HSV-F: 5'-AGAGGGACATCCAGGACTTTG-3'
The downstream primer HSV-R: 5'-CTTGTAATACACCGTCAGGTGG-3'
Specific probe HSV-P5 '-CY 5-ACCGCCGAACTGAGCAGACACCCGC-BHQ 3-3'
The upstream primer RNase P-F: 5'-GATTTGGACCTGCGAGCG-3'
The downstream primer RNase P-R: 5'-GAGCGGCTGTCTCCACAAGT-3'
The specific probe RNase P-P is 5 '-ROX-TTCTGACCTGAAGGCTCTGCGCG-BHQ 2-3'.
Wherein, the probe primer of the RNase P is introduced from J.Clin.Microbiol.2005,43(4): 1851. The above primers and probes were synthesized by Competition Biotechnology (Shanghai) Co., Ltd.
1.3 extraction and standard substance quantitative standard of virus nucleic acid:
extraction of sample nucleic acid: 200ul of serum samples were aspirated into EP tubes for nucleic acid extraction. The Nucleic Acid extraction adopts Viral Nucleic Acid extraction kit II of Geneaid company, and the extraction is carried out according to the kit instruction, and 10ul of extracted Nucleic Acid of a sample to be detected is taken as a template.
Each gene standard fragment was synthesized and ligated to the plasmid Vector pMDTM19-T Simple Vector (TaKaRa Co.). And culturing after transformation. After identification, plasmid DNA was extracted, and the copy number of the DNA was determined by measuring the concentration of the plasmid DNA using a NanoDrop ND-2000Spectrophotometer as a quantitative mother solution for a standard. According to the experiment requirement, diluting the quantitative mother liquor of the standard product to the required highest concentration, and performing tenfold dilution to the lowest concentration, and storing at-20 ℃ for later use.
1.4 optimization of multiplex real-time fluorescent quantitative RT-PCR reaction system and conditions:
detection of nucleic acids: 5ul of extracted nucleic acid of the sample to be tested was taken as a template. The total volume of the reaction was 50. mu.l, wherein 25. mu.l of the quantitative RT-PCR reaction solution, 2. mu.l of the enzyme mixture, 12. mu.l of the primer-probe mixture (20. mu. mol/L, containing four groups of specific primers and corresponding four fluorescent probes), 10. mu.l of the template, and water were added to make up to 50. mu.l. The detection is carried out on an ABI QuantStudio5 fluorescent quantitative PCR instrument (or other PCR instruments with four colors of fluorescence and above), and the reaction parameters are as follows: reverse transcription at 42 deg.C for 5 min; the hot start was carried out at 95 ℃ for 10s, followed by multiplex fluorescence detection at 95 ℃ for 5s and 55 ℃ for 40s at 55 ℃ for a total of 40 cycles.
And (5) judging a result: selecting fluorescence detection channels FAM, VIC, CY5 and ROX, selecting average value of fluorescence signals of 3-15 cycles from fluorescence baseline, and setting threshold value to be just beyond the highest point of negative control product. Firstly, samples are collected, and CT values detected by qualified RNase P, cytomegalovirus, Epstein-Barr virus and herpes simplex virus correspond to corresponding fluorescently-labeled probes. If the sample collection is qualified and the nucleic acid extraction is successful, the RNase P detection is positive; when the CT value of the test sample is 40, 0 and no value, the test sample is reported as negative. And when the CT value of the cytomegalovirus, Epstein-Barr virus and herpes simplex virus of the detected sample is less than or equal to 35, reporting as positive. And thirdly, when the CT value of the detected sample is more than or equal to 35 and less than 40, the sample is recommended to be rechecked, and if the CT value of the rechecked result is less than 38, the sample is reported as positive corresponding virus according to the judgment standard II. And (3) optimizing the system, namely screening the most appropriate concentrations of the primer and the probe by adopting a matrix method in a reaction system taking the positive nucleic acid with the same concentration as a template, wherein the primer concentration is 1-20 mu M, the probe concentration is 1-20 mu M, and the most appropriate primer and probe concentration is selected according to the lowest Ct value and the highest fluorescence intensity increase value (delta Rn).
1.5 multiplex real-time fluorescent quantitation RT-PCR specificity, sensitivity and repeatability tests
Selecting cytomegalovirus, Epstein-Barr virus, herpes simplex virus positive specimens (all from serum which is verified to be positive in nucleic acid by cytomegalovirus, Epstein-Barr virus and herpes simplex virus) and other herpes virus positive specimens such as VZV, HHV-6, HHV-7 and HHV-8, and verifying the specificity of the method by using a multiplex real-time fluorescence quantitative RT-PCR method; for the cytomegalovirus synthetic fragment (10) with calibrated copy number (copies/ml)7copies/ml), Epstein-Barr Virus synthetic fragment (10)7copies/ml), herpes simplex virus synthetic fragment (10)7copies/ml) and RNase P synthetic fragment (10)7copies/ml) were diluted separately and the fluorescence quantitative PCR reaction was performed in parallel experiments to verify the sensitivity of the method. In addition, 3 times of repeated detection are carried out on each positive standard substance with specified concentration, and the standard deviation and the variation coefficient of the Ct value obtained by the experiment are calculated to verify the repeatability of the method.
2 results
2.1 multiplex real-time fluorescent quantitation RT-PCR reaction System and conditions
Detection of nucleic acids: 5ul of extracted nucleic acid of the sample to be tested was taken as a template. The total volume of the reaction was 50. mu.l, wherein 25. mu.l of the quantitative RT-PCR reaction solution, 2. mu.l of the enzyme mixture, 12. mu.l of the primer-probe mixture (20. mu. mol/L, containing four groups of specific primers and corresponding four fluorescent probes), 10. mu.l of the template, and water were added to make up to 50. mu.l. The detection is carried out on an ABI QuantStudio5 fluorescent quantitative PCR instrument (or other PCR instruments with four colors of fluorescence and above), and the reaction parameters are as follows: reverse transcription at 42 deg.C for 5 min; the PCR assay was performed by hot-start at 95 ℃ for 10s, followed by multiplex fluorescent quantitative PCR assay at 55 ℃ for 40 cycles at 95 ℃ for 5s and 55 ℃ for 40 s.
2.2 specificity test
The one-step multiplex fluorescence quantitative RT-PCR method established by the invention has good specificity to cytomegalovirus, Epstein-Barr virus and herpes simplex virus, and can completely detect the clinical positive specimen collected recently. The multiplex primer probe of the invention has no cross reaction with other herpes virus positive samples such as VZV, HHV-6, HHV-7, HHV-8 and the like. The positive results of the cytomegalovirus, Epstein-Barr virus and herpes simplex virus of the serum specimen detected by the method are shown in figure 2, figure 3 and figure 4, wherein the Ct value of the cytomegalovirus is 25.45, the Ct value of the Epstein-Barr virus is 24.23 and the Ct value of the herpes simplex virus is 26.37.
2.3 sensitivity test
Detecting sensitivity of cytomegalovirus, Epstein-Barr virus, herpes simplex virus, and RNase P, and detecting cytomegalovirus (10) with calibrated copy number (copies/ml)7copies/ml), Epstein-Barr Virus synthetic fragment (10)7copies/ml), synthetic fragments of herpes simplex virus (10)7copies/ml) and RNase P synthetic fragment (10)7copies/ml) are respectively diluted in ten times of gradient, the kit is used for detection, and the detection result shows that the detection sensitivity of the method respectively reaches 102、102、102And 102copies/ml. See fig. 5, 8, 11, 14 for results. In addition, 5. mu.l of real-time fluorescence quantitative RT-PCR product was taken, subjected to gel electrophoresis (120V 20min), and the electrophoresis result was detected by a gel imaging system after completion. See fig. 6, 9, 12, 15 for results. Wherein Lane M is DL2000 Marker, Lane 1-6 are respectively segment (10)8copies/ml) real-time fluorescence quantitative RT-PCR products after ten times of gradient dilution, Lane 7 is negative control. The pathogen and internal reference detection results show that the pathogen and internal reference detection can realize single-tube simultaneous detection and differentiation on cytomegalovirus, Epstein-Barr virus and herpes simplex virus positive samples (see figure 17).
2.4 repeatability test
Separately collecting cytomegalovirus synthetic fragments (final concentration is 10)6copies/ml), Epstein-Barr Virus synthetic fragment (10)6copies/ml) synthetic fragment of herpes simplex virus (10)6copies/ml) and RNase P synthetic fragment (10)6copies/ml) diluted 10-fold in 10-fold gradient to 105And 104To 106、105、104The samples were tested 3 times in duplicate and their coefficient of variation and standard deviation were calculated. The results show that the standard deviation of the Ct values detected for different nucleic acid concentrations is 0.03EThe coefficient of variation is less than 1.19% between 0.31, and the repeatability is better (the result is shown in table 1).
TABLE 1 repeatability test for multiplex real-time fluorescent quantitation RT-PCR detection of synthetic nucleic acids
Example 3
The clinical samples mainly collected by adopting the kit to detect the clinical samples mainly come from 200 parts of four detection specimens before the 7 months from 2021 to 5 months. The multiple real-time fluorescent quantitative RT-PCR in the method is adopted to detect the collected specimen, and the result is as follows: the total of 200 parts of four preoperative detection specimens are screened out 2 parts of cytomegalovirus positive specimens, 2 parts of EB virus positive specimens and 0 part of herpes simplex virus positive specimens, and the detection results of all internal reference RNase P genes are positive. The coincidence degree of the detection positive result of the kit and the detection positive result of a commercial kit (Zhijiang biology) reaches 100 percent.
Sequence listing
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<120> multiplex fluorescence quantitative RT-PCR kit for detecting blood-borne infectious viruses
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Claims (5)
1. The multiplex fluorescence quantitative RT-PCR kit for detecting the blood-borne infectious virus is characterized by comprising quantitative RT-PCR reaction liquid, enzyme Mix liquid, primer probe Mix liquid, a standard substance, a positive reference substance and a negative reference substance; the quantitative RT-PCR reaction solution comprises a PCR reaction buffer solution and an enzyme Mix solution, and the primer probe Mix solution comprises four groups of primers and four corresponding probes with different fluorescent labels; the upstream primer and the downstream primer for fluorescent quantitative RT-PCR detection and corresponding specific probe sequences are as follows:
upstream primer CMV-F: 5'-CTGGGCATAAGCCATAATCTCATC-3'
Downstream primer CMV-R: 5'-CAGCCTTCCCTAAGACCACC-3'
Specific probe CMV-P5 '-FAM-TGCCGCCATGGCCTGACTGCAGCCA-BHQ 1-3'
Upstream primer EBV-F: 5'-CCAAGAAGGTGGCCCAGAT-3'
Downstream primer EBV-R of 5-TCCAGTGCTTGGGCCTTC-3'
Specific probe EBV-P5 '-VIC-TGAGCCTGACGTGCCCCCGGGAGCG-BHQ 1-3'
Upstream primer HSV-F: 5'-AGAGGGACATCCAGGACTTTG-3'
The downstream primer HSV-R: 5'-CTTGTAATACACCGTCAGGTGG-3'
Specific probe HSV-P5 '-CY 5-ACCGCCGAACTGAGCAGACACCCGC-BHQ 3-3'
The upstream primer RNase P-F: 5'-GATTTGGACCTGCGAGCG-3'
The downstream primer RNase P-R: 5'-GAGCGGCTGTCTCCACAAGT-3'
Specific probe RNase P-P of 5 '-ROX-TTCTGACCTGAAGGCTCTGCGCG-BHQ 2-3'
The standard substance comprises cytomegalovirus, Epstein-Barr virus, herpes simplex virus and RNase P standard substance, and the gene sequence of a conserved region of the standard substance is as follows:
the cytomegalovirus standard sequence is:
AATCTTAAAGATTTTCTGGGCATAAGACATAATCTCATCAGGAGAGCACTGAGGCAAGTTCTGCAATGCCGCCATGGCCTGACTGCAGCCATTGGTGGTCTTAGGGAAGGCTGAGTTCTTGGTAAAGA
the Epstein-Barr virus standard sequence is:
TTATTTTGAATACCACCAAGAAGGTGGCCCAGATGGTGAGCCTGACGTGCCCCCGGGAGCGATAGAGCAGGGCCCCGCAGATGACCCAGGAGAAGGCCCAAGCACTGGACCCCGGGGTCAGGGT
the herpes simplex virus standard product sequence is as follows:
GCATCACCGACCCGGAGAGGGACATCCAGGACTTTGTCCTCACCGCCGAACTGAGCAGACACCCGCGCGCGTACACCAACAAGCGCCTGGCCCACCTGACGGTGTATTACAAGCTCATGGCCCGCCGC
the sequence of the RNase P standard product is as follows:
ATGGCGGTGTTTGCAGATTTGGACCTGCGAGCGGGTTCTGACCTGAAGGCTCTGCGCGGACTTGTGGAGACAGCCGCTCACCTTGGCTATTCAG。
2. the multiplex quantitative RT-PCR detection kit according to claim 1, wherein the negative control is high temperature autoclaved diethylpyrocarbonate treated water, and the positive control is a positive plasmid sample of cytomegalovirus, Epstein-Barr virus, herpes simplex virus, RNase P.
3. The kit of claim 1, wherein the buffer tube of PCR reaction contains a mixture of magnesium chloride and deoxyribonucleotide triphosphates, and the enzyme Mix contains thermostable TaqDNA polymerase, RNase inhibitor and MMLV reverse transcriptase.
4. The kit for multiplex quantitative fluorescent RT-PCR detection according to claim 1, wherein the tube of the primer probe mixture is a brown tube.
5. The multiplex quantitative RT-PCR assay kit according to claim 1, wherein the kit is stored at-20 ℃.
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| CN114990261A (en) * | 2022-06-01 | 2022-09-02 | 昆明理工大学 | Multiplex qPCR detection reagent for detecting respiratory infectious disease pathogens |
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