CN111286557A - Reagent combination, kit and application for detecting blood transfusion sexually transmitted pathogen - Google Patents
Reagent combination, kit and application for detecting blood transfusion sexually transmitted pathogen Download PDFInfo
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Abstract
The invention discloses application of reagent combination for detecting pathogens in preparing a kit for detecting the quality of blood products, wherein the pathogens are selected from HAV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, HIV,b19, DENV, ZIKV, WNV, Babesia, Trypanosoma cruzi and plasmodium constitute a pathogen group, and belongs to the field of medical inspection. The invention also discloses a corresponding kit and a method for detecting the quality of the blood product. The lowest detection limit of the invention to pathogens is 1.0 x 103The copies/mL has better specificity and repeatability, and provides an important technology for screening and evaluating transfusion sexually transmitted pathogens.
Description
Technical Field
The invention belongs to the field of medical examination, and particularly relates to a reagent combination for detecting a transfusion sexually transmitted pathogen, a kit and application.
Background
According to the regulations on the sanitation and quarantine of special articles for entry and exit, special articles for entry and exit comprise microorganisms, human tissues, biological products, blood and products thereof and the like, the articles may carry pathogens in the processes of production, subpackaging, transportation, use and the like, and some special articles are pathogens, and if effective sanitation and quarantine control is not carried out, the special articles for entry and exit are likely to cause the spread of infectious diseases or other public health hazards. The scientific and effective sanitation quarantine of special entry and exit articles is more and more important and urgent to discuss and research. At present, only environmental protection microorganisms are subjected to special article conformity detection, and no special article risk factor detection project or standard exists, so that the construction of laboratory detection capability enhancement and the capability and level of technical customs enhancement are urgently needed. Among them, blood and blood products have good efficacy in emergency treatment and disease prevention, are widely used in clinic, and have a large proportion of special articles entering and exiting, so that the safety of blood and blood products is ensured, the risk of diseases transmitted by menstrual blood is reduced to the maximum extent, and particularly certain viruses transmitted by blood have high infection rate and serious harm, and are always important concerns of all countries in the world. Transfusion transmitted diseases refer to diseases caused by transfusions of pathogen-containing blood or blood products, and any pathogens that enter the body via the blood route. At present, the diseases transmitted by blood transfusion are heavily abused, which greatly harms human health and is increasingly watched worldwide. The quality and safety of blood products used for transfusion is a major concern to the public. In recent decades, the number of emerging pathogens that have recently affected blood safety has increased year by year, in addition to the major most common pathogens. Although some highly pathogenic pathogens may have a low prevalence or seasonal or geographical limiting factors, there is little correlation with blood safety. However, infection of blood products during transfusion can affect highly vulnerable groups of people, such as newborns, the elderly, or immunocompromised individuals, with fatal consequences.
Therefore, there is a need to establish highly sensitive and highly specific detection methods for these transfusion-transmitted pathogens to assess blood product quality, avoiding the consequences of false positives leading to excessive infections.
Disclosure of Invention
The invention aims to establish a molecular diagnosis and detection system based on a plurality of fluorescent PCR arrays, which is used for detecting Hepatitis A Virus (Hepatitis A Virus, HAV), Hepatitis B Virus (Hepatitis BVirus, HBV), Hepatitis C Virus (Hepatitis C Virus, HCV), Hepatitis E Virus (Hepatitis Evir, HEV), Treponema Pallidum (TP), Human immunodeficiency Virus type I (Human immunodeficiency Virus I, HIV1), Human immunodeficiency Virus type II (Human immunodeficiency Virus II, HIV2), Human T cell Virus type I (Human T lymphotropic Virus I, HTLV1), Human T cell Virus type II (Human T lymphotropic Virus II, HTLV2), Human Cytomegalovirus (Human Cytomegalovirus), Human Cytomegalovirus (Human OVvirus B, Human Cytomegalovirus V19), B19) a total of 19 types of pathogenic microorganisms were screened for Dengue Virus (Dengue Virus, DENV), Zika Virus (ZIKV), West Nile Virus (West Nile River Virus, WNV), Babesia spp (b. micoti), trypanosoma cruzi (t. cruzi), and Plasmodium (Plasmodium spp.).
In order to accomplish the above objects, the present invention provides, in one aspect, use of a combination of reagents for detecting a pathogen in the preparation of a kit for detecting the quality of blood products, wherein the pathogen is selected from at least 2, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, of DENV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, B19, DENV, ZIKV, WNV, babesia, trypanosoma cruzi and plasmodium.
In some embodiments of the invention, the pathogen comprises HBV and HCV. In other embodiments of the invention, the pathogens include HIV1, HIV2, and TP. In still further embodiments of the invention, the pathogens include HTLV1, HTLV2, and HBOV. In still further embodiments of the invention, the pathogen comprises TTV, HCMV and B19. In still further embodiments of the invention, the pathogens include DENV, ZIKV and WNV. In still further embodiments of the invention, the pathogens include babesia, trypanosoma cruzi and plasmodium. In still further embodiments of the invention, the pathogen comprises HAV and HEV.
In some preferred embodiments of the invention, the pathogen comprises HAV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, B19, DENV, ZIKV, WNV, babesia, trypanosoma cruzi and plasmodium, for a total of 19 pathogens.
In a second aspect, the present invention provides a kit for testing the quality of blood products, comprising a combination of reagents for testing a pathogen, wherein the pathogen is selected from at least 2, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, of the group of pathogens consisting of HAV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, B19, DENV, ZIKV, WNV, babesia, trypanosoma cruzi and plasmodium.
In some embodiments of the invention, the pathogen comprises HBV and HCV. In other embodiments of the invention, the pathogens include HIV1, HIV2, and TP. In still further embodiments of the invention, the pathogens include HTLV1, HTLV2, and HBOV. In still further embodiments of the invention, the pathogen comprises TTV, HCMV and B19. In still further embodiments of the invention, the pathogens include DENV, ZIKV and WNV. In still further embodiments of the invention, the pathogens include babesia, trypanosoma cruzi and plasmodium. In still further embodiments of the invention, the pathogen comprises HAV and HEV.
In some preferred embodiments of the invention, the pathogen comprises HAV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, B19, DENV, ZIKV, WNV, babesia, trypanosoma cruzi and plasmodium, for a total of 19 pathogens.
In the present invention, the reagent combination includes a primer pair and a probe for detecting the pathogen.
In some embodiments of the invention, primer pairs and probes for detection of the HAV target their polyprotein genes; detecting the primer pair and the probe of the HBV to target the core protein gene; detecting primer pairs and probes of the HCV that target their 5' UTR genes; detecting ORF2 that the primer pair and the probe of the HEV target the capsid protein gene; detecting that the primer pair and probe of HIV1 target their gag gene; detecting that the primer pair and probe of HIV2 target their gag gene; detecting a primer pair and a probe of the TP to target a lipoprotein antigen Tp47 gene; detecting that primer pairs and probes of the HTLV1 target their Tax gene; detecting that primer pairs and probes of the HTLV2 target their Tax gene; primer pairs and probes for detecting the HBOV target the NS1 gene; primer pairs and probes that detect the TTV target their ORF 1; primer pairs and probes for detecting the HCMV target their UL84 gene; detecting that the primer pair and the probe of the B19 target the NS1 gene; detecting the target of the primer pair and the probe of the ZIKV to the polyprotein gene of the ZIKV; detecting primer pairs and probes of the DENV that target their 3' UTR genes; detecting that the primer pair and probe of the WNV target the NS5 gene; detecting primer pairs and probes of the babesia to target the ITS1 gene; detecting a primer pair and a probe of the trypanosoma cruzi to target a satellite sequence of the trypanosoma cruzi; primer pairs and probes for detecting the plasmodium are targeted to the 18s rRNA gene.
In some embodiments of the invention, the primer pair for detecting HAV comprises an upstream primer having the nucleotide sequence shown by SEQ ID number 1 and an upstream primer having the nucleotide sequence shown by SEQ ID No.2, and the probe has the nucleotide sequence shown by SEQ ID No. 3; the primer pair for detecting the HBV comprises an upstream primer with a nucleotide sequence shown by SEQ ID No.4 and an upstream primer with a nucleotide sequence shown by SEQ ID No.5, and a probe with a nucleotide sequence shown by SEQ ID No. 6; the primer pair for detecting HCV comprises an upstream primer with a nucleotide sequence shown in SEQ ID No.7 and an upstream primer with a nucleotide sequence shown in SEQ ID No.8, and a probe with a nucleotide sequence shown in SEQ ID No. 9; detecting the primer pair of the HEV, wherein the primer pair comprises an upstream primer with a nucleotide sequence shown in SEQ ID No.10 and an upstream primer with a nucleotide sequence shown in SEQ ID No.11, and a probe has a nucleotide sequence shown in SEQ ID No. 12; the primer pair for detecting the HIV1 comprises an upstream primer with a nucleotide sequence shown by SEQ ID number 13 and an upstream primer with a nucleotide sequence shown by SEQ ID No.14, and a probe has a nucleotide sequence shown by SEQ ID No. 15; the primer pair for detecting the HIV2 comprises an upstream primer with a nucleotide sequence shown by SEQ ID number 16 and an upstream primer with a nucleotide sequence shown by SEQ ID No.17, and a probe with a nucleotide sequence shown by SEQ ID No. 18; detecting the TP primer pair comprises an upstream primer with a nucleotide sequence shown in SEQ ID No.19 and an upstream primer with a nucleotide sequence shown in SEQ ID No.20, wherein a probe has a nucleotide sequence shown in SEQ ID No. 21; the primer pair for detecting the HTLV1 comprises an upstream primer with the nucleotide sequence shown in SEQ ID number 22 and an upstream primer with the nucleotide sequence shown in SEQ ID No.23, and a probe has the nucleotide sequence shown in SEQ ID No. 24; the primer pair for detecting the HTLV2 comprises an upstream primer with the nucleotide sequence shown in SEQ ID No.25 and an upstream primer with the nucleotide sequence shown in SEQ ID No.26, and a probe with the nucleotide sequence shown in SEQ ID No. 27; the primer pair for detecting the HBOV comprises an upstream primer with a nucleotide sequence shown by SEQ ID No.28 and an upstream primer with a nucleotide sequence shown by SEQ ID No.29, and a probe with a nucleotide sequence shown by SEQ ID No. 30; the primer pair for detecting the TTV comprises an upstream primer with a nucleotide sequence shown by SEQ ID No.31 and an upstream primer with a nucleotide sequence shown by SEQ ID No.32, and a probe with a nucleotide sequence shown by SEQ ID No. 33; the primer pair for detecting the HCMV comprises an upstream primer with a nucleotide sequence shown by SEQ ID No.34 and an upstream primer with a nucleotide sequence shown by SEQ ID No.35, and a probe with a nucleotide sequence shown by SEQ ID No. 36; detecting that the primer pair of B19 comprises an upstream primer with the nucleotide sequence shown in SEQ ID No.37 and an upstream primer with the nucleotide sequence shown in SEQ ID No.38, and a probe with the nucleotide sequence shown in SEQ ID No. 39; detecting the ZIKV primer pair comprises an upstream primer with a nucleotide sequence shown in SEQ ID No.40 and an upstream primer with a nucleotide sequence shown in SEQ ID No.41, wherein a probe has a nucleotide sequence shown in SEQ ID No. 42; the primer pair for detecting the DENV comprises an upstream primer with a nucleotide sequence shown in SEQ ID No.43 and an upstream primer with a nucleotide sequence shown in SEQ ID No.44, and a probe with a nucleotide sequence shown in SEQ ID No. 45; the primer pair for detecting the WNV comprises an upstream primer with a nucleotide sequence shown by SEQ ID No.46 and an upstream primer with a nucleotide sequence shown by SEQ ID No.47, and a probe with a nucleotide sequence shown by SEQ ID No. 48; the primer pair for detecting the babesia comprises an upstream primer with a nucleotide sequence shown by SEQ ID No.49 and an upstream primer with a nucleotide sequence shown by SEQ ID No. 50, and a probe has a nucleotide sequence shown by SEQ ID No. 51; the primer pair for detecting the trypanosoma cruzi comprises an upstream primer with a nucleotide sequence shown by SEQ ID No.52 and an upstream primer with a nucleotide sequence shown by SEQ ID No.53, and a probe with a nucleotide sequence shown by SEQ ID No. 54; the primer pair for detecting the plasmodium comprises an upstream primer with a nucleotide sequence shown by SEQ ID No.55 and an upstream primer with a nucleotide sequence shown by SEQ ID No.56, and a probe with a nucleotide sequence shown by SEQ ID No. 57.
In the present invention, the kit further comprises a buffer, dNTPs, and a DNA polymerase.
In some embodiments of the invention, the kit further comprises a primer pair and a probe that target an internal reference. In some embodiments of the invention, the internal reference is RNP. In some embodiments of the invention, the targeted reference primer pair comprises an upstream primer having the nucleotide sequence shown in SEQ ID No.58 and an upstream primer having the nucleotide sequence shown in SEQ ID number 59, and the probe has the nucleotide sequence shown in SEQ ID No. 60.
A third aspect of the invention provides a method of testing the quality of a blood product, comprising the steps of:
(1) obtaining a nucleic acid sample of the blood product;
(2) performing qPCR amplification on the nucleic acid sample using the kit of the second aspect of the invention;
(3) and judging the quality of the blood product according to the qPCR detection result.
If the qPCR amplification result corresponding to a certain pathogen is positive, the blood product is polluted by the pathogen and has poor quality, and the blood product cannot be clinically used.
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
the invention establishes a molecular diagnosis detection system based on multiple fluorescent PCR arrays, can simultaneously and effectively detect and screen 19 (types) pathogenic microorganisms of hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, treponema pallidum, human immunodeficiency virus I, human immunodeficiency virus II, human T-cell tropic virus I, human T-cell tropic virus II, human bocavirus, blood transfusion transmission virus, human cytomegalovirus, human parvovirus B19, dengue virus, Zika virus, West Nile virus, Babesia, trypanosoma cruzi and plasmodium, and relates to multiple and complete pathogenic types.
The performance evaluation of the multiplex real-time fluorescence PCR method of the invention shows that the concentration of the plasmid template with the minimum detection limit is 1.0 multiplied by 103copies/mL, has better specificity and repeatability.
The multiple fluorescence PCR array methods established by the invention can be used for qualitative detection of the 19 transfusion transmission pathogens, and provide important technology for screening and evaluating the transfusion transmission pathogens.
Drawings
FIG. 1 shows the validation results of HAV, HBV, HCV, HEV recombinant plasmids.
FIG. 2 shows the validation results of TP, HIV1, HIV2, HTLV1 recombinant plasmids.
FIG. 3 shows the validation results of recombinant plasmids of HTLV2, HBOV, TTV, HCMV.
FIG. 4 shows the results of B19, DENV, ZIKV, WNV recombinant plasmid validation.
FIG. 5 shows the results of the validation of Babesia, Trypanosoma cruzi, Plasmodium, internal standard recombinant plasmid.
FIG. 6 shows the results of multiplex fluorescent PCR amplification of HAV/HBV, HIV1/HIV2/TP, HTLV1/HTLV2/HBOV and TTV/HCMV/B19.
FIG. 7 shows the results of DENV/ZIKV/WNV, Babesia/Trypanosoma cruzi/Plasmodium, HAV/HEV and negative control multiplex fluorescence PCR amplifications.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Examples
1. Materials and methods
1.1 origin of specimen
The plasmids containing the target genes of the transfusion transmitted pathogen adopt an artificial synthesis method and are cloned on the plasmids, and the method comprises the following steps: HAV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, B19, DENV, ZIKV, WNV, Babesia, Trypanosoma cruzi and Plasmodium. The plasmid was delegated to the universal biosynthesis provision of Anhui.
1.2 Main reagents and instruments
OneStep RT-PCR reagent (Jiangsu Shuo Co.), QIAamp Viral RNA Mini Kit nucleic acid extraction reagent (Qiagen), reverse transcriptase SuperScriptTMIII Reverse Transcriptase (ThermoFisher), DNA polymerase PlatinumTMTaq DNA Polymerase (ThermoFisher), dNTP Mix (Promega), ABIQuantStudio7Flex fluorescent PCR instrument (ABI, USA), NanoDrop 2000c Spectrophotometer (Thermoscientific).
1.3 primer and Probe design
According to gene data in GenBank and the design principle of primers, the primers and the probes are designed aiming at 19 (type) pathogen gene specific conserved regions, the 5 'ends of the probes are respectively marked with FAM, VIC, ROX and CY5 fluorescent groups, and the 3' ends of the probes are marked with corresponding quenching groups BHQ1, BHQ2 and BHQ 3. Primers, probes were synthesized and labeled by Baileger Biotech. Primer and probe information are shown in table 1:
TABLE 1 primer and Probe information
1.4 multiplex fluorescent PCR reaction Condition optimization
Setting the annealing extension temperatures of 55 ℃, 58 ℃ and 60 ℃ as the annealing extension temperatures of the reaction for the multiple fluorescence PCR system respectively, completing the fluorescence PCR reaction respectively, and analyzing the influence of the annealing extension temperatures on the multiple fluorescence PCR detection through an amplification curve. 3 concentration gradients are designed for the concentration of the primer and the probe of the multiplex fluorescence PCR reaction system for combined optimization, and the primers and the probes with final concentrations of 100nmol/L, 200nmol/L, 400nmol/L and 600nmol/L are respectively added into a 25 mu L reaction system. The influence of primer and probe concentrations on multiplex fluorescent PCR detection was analyzed by amplification curves. Reaction conditions are as follows: 30min at 50 ℃; 5min at 95 ℃; 10s at 95 ℃, 40s at 60 ℃ and 45 cycles; fluorescence was collected over a 40s period at 60 ℃.
1.5 sensitivity test
Serial 10-fold gradient dilution of recombinant plasmid and optimized multiple fluorescent PCR reaction system to recombinant plasmid (1X 10)1copies/. mu.L), each recombinant plasmid (1X 10)1copies/. mu.L) were diluted 4 portions and the assay was repeated 24 times for each portion for a total of 96 assays, the lowest limit of detection was observed.
1.6 specificity test
On the basis of preliminary analysis and verification of the specificity of each primer and probe by a Blast program and Bioedit, the designed primers and probes are further used for detection, virus standard products of pathogens with the same source or similar symptoms are used as templates, RNase-free water is used as a negative control, and plasmids are used as a positive control for specificity test.
1.7 precision test
Application of 10 pathogens3copies/. mu.L and 101Duplicate detection was performed on copies/μ L standards. Setting 10 parallel samples for each concentration, calculating the average value and standard deviation of CT value of each concentration corresponding to each virus, and calculating the variation systemThe precision of the reaction system was checked.
2. Results
2.1 verification of Positive recombinant plasmids
The recombinant plasmid containing HAV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, B19, DENV, ZIKV, WNV, Babesia, Trypanosoma cruzi and the target fragment of plasmodium is verified by a sequencing method, the sequence result is consistent with the sequence of a pathogen target gene, and the partial sequencing result is shown in figures 1-5.
2.2 validation of primers and probes
In the detection of multiplex real-time fluorescent PCR reaction, HBV/HCV, HIV1/HIV2/TP, HTLV1/HTLV2/HBOV and TTV/HCMV/B19, DENV/ZIKV/WNV, Babesia/Trypanosoma cruzi/plasmodium, HAV/HEV are paired with 102copies/. mu.L and 104The positive control detection rate of copies/. mu.L was 100.0%, see Table 2. Seven sets of primers and probes are described for multiplex real-time fluorescent PCR typing detection of hepatitis B virus, hepatitis C virus, human immunodeficiency virus type I, human immunodeficiency virus type II, treponema pallidum, human T-cell virus type I, human T-cell virus type II, human bocavirus, transfusion transmitted virus, human cytomegalovirus, human parvovirus B19, dengue virus, West Nile virus, Babesia, Trypanosoma cruzi, Plasmodium, hepatitis A virus and hepatitis E virus.
TABLE 2 detection results of primers and probes for each recombinant plasmid
2.3 primer and Probe dosages
The ratio of the final concentration of primers and probe in a 25. mu.l reaction was 600 nmol/L: 400nmol/L, 600 nmol/L: 200nmol/L, 400 nmol/L: 200nmol/L and 400 nmol/L: 100 nmol/L. Positive controls for all concentrations were detectable in groups 1, 2 and 3, and Trypanosoma cruzi in group 4To 102The positive control detection rate of copies/. mu.L is less than 100%, see Table 3. Therefore, the group 3 with a smaller dosage is selected as the optimal dosage of the primers and probes in the reaction system, i.e., the final concentration of the primers is 400nmol/L and the final concentration of the probes is 200 nmol/L.
TABLE 3 detection results of the amounts of primers and probes used for each recombinant plasmid
2.4 annealing temperature optimization
The annealing temperature of the fluorescent PCR reaction process is optimized to three annealing temperatures of 55 ℃, 58 ℃ and 60 ℃. Set 1, set 2 and set 3 pairs 102copies/. mu.L and 104The detection rate of the copies/mu L positive control is 100.0%, and different annealing temperatures have no influence on the detection, which is shown in Table 4. In subsequent experiments, 60 ℃ is selected as the annealing temperature, namely 50 ℃ for 30 min; 5min at 95 ℃; 10s at 95 ℃, 40s at 60 ℃ and 45 cycles; fluorescence was collected over a 40s period at 60 ℃.
TABLE 4 results of detection of the respective recombinant plasmids by annealing temperature
2.5 amounts of the component reagents
In a 25. mu.L reaction system, according to the recommended dosage of OneStep RT-PCR instruction, 7.5. mu.L OneStep RT-PCR amplification reaction solution, 5. mu.L Enzyme mixture, 7.5. mu.L primer probe detection solution, and 5. mu.L template were taken respectively. The reaction condition is 30min at 50 ℃; 5min at 95 ℃; 10s at 95 ℃, 40s at 60 ℃ and 45 cycles; fluorescence was collected over a 40s period at 60 ℃. The results of multiplex fluorescence PCR detection of HBV/HBV, HIV1/HIV2/TP, HTLV1/HTLV2/HBOV and TTV/HCMV/B19 are shown in FIG. 6, and DENV/ZIKV/WNV, Babesia/Trypanosoma cruzi/Plasmodium, HAV/HEV and negative control are shown in FIG. 7.
2.6 sensitivity
After 10-fold gradient dilution with plasmid containing specific fragment, the prepared concentration was 1X 101Fluorescence PCR detection was performed using copies/. mu.L of the standard as a template. Each recombinant plasmid (1X 10)1copies/. mu.L) were diluted 4 portions and the assay was repeated 24 times for each portion for a total number of 96 assays. At 1 × 101The copies/. mu.L standard was used to verify the lowest detection limit of the reaction system. The results are shown in Table 5, and the statistical analysis of the results was carried out at a concentration of 1X 101The detection rates of the copies/mu L standard products are respectively more than 95 percent. Therefore, the sensitivity of the present reaction system was determined to be 1X 101copies/μL。
TABLE 5 sensitivity of multiplex fluorescent PCR arrays to detection of individual recombinant plasmids
2.7 specificity
The primers and probes of 19 (type) pathogens are crossed to carry out real-time fluorescent PCR specificity verification: no cross reaction exists among viruses, only the specific amplification exists between the viruses and the primers, and the amplification results are shown in a table 6. As can be seen from the experimental results, all pathogens are not amplified in the PCR reaction system, so that the established fluorescence PCR method has better specificity.
TABLE 6 specificity of detection of multiplex fluorescent PCR among pathogens
2.8 repeatability
Recombinant plasmids (1X 10) using 19 (types) of pathogens1copies/. mu.L and 1X 103copies/. mu.L) were performed. Setting 10 parallel samples for each concentration, calculating the average value and standard deviation of CT value of each concentration corresponding to each virus, and calculating the coefficient of variationTo check the reproducibility of the reaction system.
The results show that: the coefficient of variation for each system was less than 5%, and the detailed results are shown in Table 7. The results show that the multiplex fluorescent PCR array technology is good in repeatability.
TABLE 7 precision of multiplex fluorescent PCR array for each recombinant plasmid assay
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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Claims (8)
1. Use of a combination of reagents for the detection of a pathogen for the preparation of a kit for the detection of the quality of a blood product, characterized in that the pathogen is selected from at least 2 of the group consisting of HAV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, B19, DENV, ZIKV, WNV, babesia, trypanosoma cruzi and plasmodium.
2. The use according to claim 1, wherein said pathogen comprises HAV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, B19, DENV, ZIKV, WNV, babesia, trypanosoma cruzi and plasmodium.
3. The use of claim 1 or 2, wherein the combination of reagents comprises a primer pair and a probe for detecting the pathogen.
4. A kit for testing the quality of blood products comprising a combination of reagents for testing for a pathogen, wherein the pathogen is selected from at least 2 of the group of pathogens consisting of HAV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, B19, DENV, ZIKV, WNV, babesia, trypanosoma cruzi and plasmodium.
5. The kit of claim 4, wherein said pathogen comprises HAV, HBV, HCV, HEV, TP, HIV1, HIV2, HTLV1, HTLV2, HBOV, TTV, HCMV, B19, DENV, ZIKV, WNV, Babesia, Trypanosoma cruzi, and Plasmodium.
6. The kit of claim 4 or 5, wherein the combination of reagents comprises a primer pair and a probe for detecting the pathogen.
7. The kit of claim 6, wherein the kit further comprises a buffer, dNTPs, and a DNA polymerase.
8. A method of testing the quality of a blood product, comprising the steps of:
(1) obtaining a nucleic acid sample of the blood product;
(2) performing qPCR amplification on said nucleic acid sample using the kit of any of claim 6;
(3) and judging the quality of the blood product according to the qPCR detection result.
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| CN112266978A (en) * | 2020-10-22 | 2021-01-26 | 深圳国际旅行卫生保健中心(深圳海关口岸门诊部) | Primer-probe combination, detection kit and application thereof |
| CN114369649A (en) * | 2022-02-08 | 2022-04-19 | 山东见微生物科技有限公司 | Specific selective amplification and multiplex PCR method and application |
| CN114410847A (en) * | 2022-03-03 | 2022-04-29 | 广东省人民医院 | Legionella pneumophila and dengue fever nucleic acid quantitative combined detection reagent |
| CN115948617A (en) * | 2022-12-23 | 2023-04-11 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting blood borne viruses and application thereof |
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