CN106119417B - Kit and detection method for accurately and quantitatively detecting norovirus type I - Google Patents
Kit and detection method for accurately and quantitatively detecting norovirus type I Download PDFInfo
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Abstract
The invention discloses a kit and a detection method for accurately and quantitatively detecting norovirus type I. In particular to a group of primers and probes for detecting norovirus type I, which have nucleotide sequences shown in sequence tables SEQ ID No.1 to SEQ ID No.3, a kit containing the primers and the probes and a detection method thereof. The invention also provides a detection method for accurately and quantitatively detecting the norovirus type I by utilizing the micro-drop digital PCR technology, which does not need to depend on certified standard substances or other standard substances, has higher accuracy, sensitivity and repeatability and is easy to standardize.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a kit and oligonucleotide for detecting norovirus type I, more specifically to a reverse transcription-micro-droplet digital polymerase chain reaction (RT-ddPCR) rapid quantitative detection kit for detecting norovirus type I.
background
Norovirus (Norovirus) belongs to the family caliciviridae, is a non-enveloped single-stranded positive-strand RNA virus with a genome length of 7.5-7.7kp, and comprises 3 Open Reading Frames (ORFs), wherein ORF1 encodes nonstructural proteins such as nucleotide triphosphates, proteases, and RNA-dependent RNA polymerases (RdRp), ORF2 encodes the major structural protein VP1, and ORF3 encodes the minor structural protein. Norovirus can be classified into 5 genotypes gi-gv based on differences in the sequence information of the RdRP and VP1 genes. Of these, gai and gai are the most common, major viral genotypes that infect humans.
Norovirus is a main food-borne virus causing outbreak of nonbacterial gastroenteritis worldwide, can infect people of all age groups, especially children, and causes symptoms of nausea, vomiting, abdominal pain, even low fever, headache, myalgia, hypodynamia, anorexia and the like of patients. The transmission route is as follows: infection source → feces → water body (food) → human body transmission, and contaminated water and food, especially shellfish, are the main carriers of transmission. Norovirus has strong tolerance to the environment, still has infectivity under the conditions of acidity (pH 2.7), heating (60 ℃) and free chlorine (3.75-6.25 mg/L) concentration in tap water, and has very limited prevention means. Therefore, norovirus in environment and food is detected and analyzed, the development of water environment and shellfish marine product norovirus pollution risk assessment is facilitated, and then the outbreak of food-borne virus diseases is effectively pre-warned, the human health is protected, and the normal running of food trade is ensured.
While norovirus type I is a strict intracellular parasite that cannot reproduce in food products, it is highly infectious, even less than 102The number of copies/mL of virus particles can cause infection. In food samples where norovirus type i contamination may be present, the viral titer is usually at a relatively low level. Therefore, a precise quantitative detection method aiming at the norovirus type I is needed, and effective monitoring of the norovirus type I is realized. At present, the Quantitative detection method of norovirus type i mainly includes Real-time Fluorescent Quantitative PCR (RT-qPCR) based on Polymerase Chain Reaction (PCR) and reverse transcription-loop mediated isothermal amplification (RT-LAMP) method. Both methods require carefully calibrated standard curves and stable norovirus type i standardsAnd moreover, a standard curve needs to be established in each detection, so that the existence of potential subjective factors in result analysis causes the difference of results among multiple detections and the diversity of results among different laboratories, and the standardization and normalization of norovirus type I quantitative detection are influenced.
The Digital PCR (Digital PCR) technology developed in recent years is a brand new method for quantitative detection of nucleic acid. Currently, digital PCR includes: three dPCR systems, namely, a micro reaction Chamber/orifice plate Digital PCR (CD PCR), a Microfluidic Digital PCR (mdPCR) (large-scale integrated Microfluidic chip) and a Droplet Digital PCR (ddPCR). At present, no report related to quantitative detection of norovirus type I by using RT-ddPCR is found at home and abroad.
disclosure of Invention
The invention aims to provide a group of RT-ddPCR primers and probes for detecting norovirus type I, which have strong specificity and high sensitivity.
Another purpose of the invention is to provide a rapid quantitative detection kit for norovirus type I RT-ddPCR, which is simple to operate and accurate in result.
In order to achieve the purpose, the invention adopts the following technical scheme:
The invention designs specific primers and probes by analyzing genome constitution of norovirus type I, selecting a typing gene segment RdRp-VP1, wherein the sequences of the primers and probes for detecting norovirus type I are shown in Table 1, and the probes are labeled with FAM 5 'and labeled with BHQ 3'.
TABLE 1 RT-ddPCR primer and Probe sequences
The invention also provides a kit for accurately and quantitatively detecting the norovirus type I, which comprises the primer and the probe for detecting the norovirus type I. Further, the kit may further comprise materials and reagents required for completion of RT-ddPCR reaction, such as norovirus type I positive control RNA, one-step RT-ddPCR supermix, magnesium acetate, microdroplet oil, microdroplet card, 96-well plate, aluminum foil heat-sealing film, etc., which are preferably packaged separately.
The invention also provides a method for accurately and quantitatively detecting the norovirus type I, namely an RT-ddPCR method, which comprises the following steps:
1. Extracting RNA of a sample to be detected by using a traditional Trizol method or a commercial kit;
2. preparing an RT-ddPCR reaction system, wherein the reaction system comprises primers and probes shown as SEQ ID NO.1 to SEQ ID NO.3 in a sequence table. In a specific embodiment of the present invention, the RT-ddPCR reaction system is shown in Table 2;
TABLE 2 norovirus type I ddPCR reaction System
3. Adding the RT-ddPCR reaction system prepared in the step 2 and the microdroplet generation oil into a microdroplet generation card, and placing the microdroplet generation card into a microdroplet generation instrument to generate microdroplets;
4. carrying out an amplification reaction under the reaction conditions shown in Table 3;
TABLE 3 Noro virus type I RT-ddPCR reaction conditions
note: the temperature rising and falling speed should be less than or equal to 2.5 ℃/s
5. and (6) judging the result.
The amplified 96-well plates were placed in a microdroplet reader (QX200, Bio-Rad, Pleasanton, Calif.) and results read and analyzed using QuantaSoft software. The positive droplets containing amplification products and the negative droplets not containing amplification products will show a difference in fluorescence signal intensity, and a threshold line is set by using the highest point of the fluorescence amplitude of the negative droplet clusters as a boundary. Calculating according to Poisson distribution principle to obtain target fragment content of norovirus type I in RT-ddPCR reaction system, and calculating according to the following formula to obtain accurate quantification of norovirus type I in sample through RNA template sample addition amount (2 mu L) and extracted RNA template volume number.
The invention has the advantages that: 1) absolute quantification of nucleic acid molecules avoids bias caused by differences in PCR amplification efficiency; 2) the evidence standard substance or other standard substances are not required to be relied on; 3) the method has higher accuracy, sensitivity and repeatability, and is easy to standardize; 4) the method is more suitable for detecting nucleic acid with low copy number; 5) since RT-ddPCR is an end-point assay, it is more resistant to inhibitors, and thus can reduce the detection bias caused by the type of substrate.
The invention is further described in the following description with reference to the figures and the detailed description, and any equivalent replacement in the field made in accordance with the present disclosure is within the scope of the present invention.
drawings
FIG. 1 shows the RT-ddPCR detection results of norovirus type I target fragments under different annealing temperature conditions.
FIG. 2 RT-ddPCR kit for detecting norovirus type I specificity analysis results.
FIG. 3 shows the results of the sensitivity analysis of RT-ddPCR kit for detecting norovirus type I.
FIG. 4 shows the result of the repeated analysis of norovirus type I detection by RT-ddPCR kit.
Detailed Description
Example 1 RT-ddPCR primer, Probe design
In order to realize specific detection and absolute quantitative analysis of norovirus type I, a norovirus typing gene segment RdRp-VP1 is selected, sequence analysis and comparison are carried out through an NCBI on-line tool, more than 10 pairs of primer and probe combinations are designed by using Prime Express software V4.0(ABI, Foster City, CA, USA), and 1 set of each primer/probe combination with strong specificity and suitability for RT-ddPCR is finally obtained through screening, wherein the sequences are shown in Table 1. Wherein, the 5 'end of the probe is marked with FAM, and the 3' end is marked with BHQ. The primers/probes were synthesized by Beijing Liuhe Jingmai Nawamao Co.
Example 2 establishment of detection method
(1) RNA extraction: extracting with commercial kit; or extracting RNA by using a traditional Trizol method, which comprises the following specific steps: adding 1mL of Trizol into 100 mu L of sample, shaking for 30s, and standing for 5min at room temperature; ② adding 250 μ L chloroform, shaking vigorously for 30s, standing for 5min at room temperature; centrifuging at 12000g at 4 deg.C for 5 min; transferring the supernatant into a new centrifuge tube, adding 500 mu L of isopropanol, violently shaking and mixing uniformly for 30s, and standing for 5min at room temperature; fourthly, centrifuging for 5min at the temperature of 4 ℃ and at the speed of 5000 g; fifthly, carefully removing the supernatant, washing the precipitate with 1mL of 70% ethanol, then 12000g, centrifuging at 4 ℃ for 5min (sucking the supernatant as far as possible, placing the centrifuge tube on a super clean bench and drying the precipitate); sixthly, adding 50 mu L RNase-free water to dissolve the RNA (heating at 60 ℃ for 10min for better dissolving the viral RNA). The extracted RNA was stored at-80 ℃ for future use.
(2) Prepare 20. mu.L of RT-ddPCR reaction system according to Table 2
(3) Droplet generation
mu.L of ddPCR reaction and 65. mu.L of microdroplet-forming oil were added to 8-well microdroplet-forming cards, respectively, and placed in a microdroplet-forming apparatus (QX200, Bio-Rad, Pleasanton, Calif.) to form microdroplets.
(4) amplification reaction
the resulting water-in-oil droplets (40. mu.L) were slowly transferred to a 96-well plate, sealed and subjected to an amplification reaction on a PCR instrument (Veriti Thermal cycler, Applied BioSystems, Foster City, Calif.) under the conditions shown in Table 3.
(5) Determination of results
the amplified 96-well plates were placed in a microdroplet reader (QX200, Bio-Rad, Pleasanton, Calif.) and results read and analyzed using QuantaSoft software. The positive droplets containing amplification products will exhibit a difference in fluorescence signal intensity from the negative droplets not containing amplification products, and a threshold line can be set bounded by the highest point in the fluorescence amplitude of the cluster of negative droplets. Calculating according to Poisson distribution principle to obtain target fragment content of norovirus type I in RT-ddPCR reaction system, and calculating according to the following formula to obtain accurate quantification of norovirus type I in sample through RNA template sample addition amount (2 mu L) and extracted RNA template volume number.
The invention also optimizes the RT-ddPCR detection conditions, such as annealing temperature, takes the extracted norovirus type I RNA as a template, carries out amplification reaction at the annealing temperatures of 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃ respectively, and compares the detection results of the RT-ddPCR under different conditions. As can be seen from FIG. 1, the amplification efficiency of norovirus type I target gene fragments is low at 50 ℃, and the boundaries between positive droplets and negative droplets are not obvious; the amplification efficiency of the target gene fragment is higher under the conditions of 55 ℃ and 60 ℃, more obvious positive microdroplet clusters can appear, and the fluorescence signal amplified under the condition of 55 ℃ is slightly higher than 60 ℃, so that 55 ℃ is taken as the optimal annealing temperature.
Example 3 kit composition
1. Composition of the kit (stored at-20 ℃ C.)
(1) primers and probes for detecting norovirus type i designed in example 1, SEQ ID nos. 1 to 3, were synthesized by trades ltd, sijoh, beijing;
(2) one-step RT-ddPCR supermix, 25mM magnesium acetate: purchased from BioRad, Inc., USA, cat # 186-3021;
(3) droplet-forming oil: purchased from BioRad, USA, Cat 186-3030;
(4) Droplet generation card: purchased from BioRad corporation, USA, Cat 186-4007;
(5) Aluminum foil heat-sealing film: purchased from BioRad, Inc., USA, Cat # 181-4000;
(6) Twin Tec Semi-skerted 96 well plates: purchased from Eppendorf, germany, cat # 0030128605;
(7) negative control: DEPC water; purchased from Shanghai, cargo number: d1005;
(8) Positive control: norovirus type i RNA standards;
(9) DEPC water: purchased from Shanghai, cargo number: D1005.
2. Preparation of norovirus type I RNA standard substance
the RNA of norovirus type I isA template, a specific fragment of about 68bp is amplified by using a primer SEQ ID No.1-2, and is called as GI (including RT-ddPCR amplification target gene); recombinant plasmid pcDNA II-GI (pcDNA II vector from Invitrogen corporation) was constructed, sequence determination was performed in Shanghai Producer, and homology analysis was performed on the sequence with the gene sequence of norovirus type I in GenBank to achieve 100% homology. Plasmid DNA was extracted and purified using Ribo MAX from PromegaTMThe Large Scale RNA Production system-T7 kit (cat # P1300) was subjected to in vitro transcription (according to the kit instructions), and the in vitro transcription product was digested with DNase to remove the DNA therefrom. The cRNA was further purified using RNeasy MiniElute clear kit (from QIAGEN, cat. No. 74204). The purified cRNA was subjected to sequence determination in Shanghai Biotech, and the results of the sequence determination were subjected to homology analysis with the gene sequence of norovirus type I in GenBank to achieve 100% homology. The cRNA was aliquoted and stored at-80 ℃. The prepared norovirus type I RNA standard substance is subjected to uniformity and stability tests, and all the tests meet related requirements; the prepared standard substance is subjected to fixed value research by adopting a fixed value method in multiple laboratories, and the finally prepared norovirus type I RNA standard substance has the following concentration: 5.43X 107copies/. mu.l. Stored at-80 ℃ as a positive control of the kit
Example 4 detection method specificity and sensitivity evaluation
1. Detection specificity assay
(1) Materials: rotavirus, astrovirus, norovirus type I and norovirus type II RNAs are all provided by the Chinese disease prevention and control center for viral diseases.
(2) the method comprises the following steps: the kit of the invention is used for carrying out RT-ddPCR amplification detection on common diarrhea viruses including rotavirus, astrovirus, norovirus type I and norovirus type II RNA, and observing whether the kit has non-specific reaction.
(3) As a result: rotavirus, astrovirus, norovirus type I and norovirus type II RNAs were amplified using a PCR instrument (Veriti Thermal cycler, Applied BioSystems, Foster City, Calif.), detected using a microdroplet reader (QX200, Bio-Rad, Pleasanton, Calif.), and the results read and analyzed using QuantaSoft software. The result shows that only norovirus type I shows positive microdroplets, and the rest viruses are negative microdroplets, which proves that the method has good specificity. The results are shown in FIG. 2.
2. Assay for detection sensitivity
(1) Materials: the previously prepared norovirus type I RNA standards were diluted in 10-fold gradient to 5.43 copies/. mu.L.
(2) The method comprises the following steps: mu.L of each dilution gradient was taken and RT-ddPCR detection was performed using the kit described in example 3, the lowest limit detectable by analysis of the kit of the invention is shown in Table 2.
(3) As a result: it was found that 5.43 copies/. mu.L of norovirus type I RNA standard substance could be detected using the kit of the present invention. The results are shown in FIG. 3. Therefore, the kit can achieve 5.43 copies/mu L for detecting the norovirus type I, greatly improves the sensitivity of the norovirus type I related detection method, does not depend on a standard product, can realize absolute quantification, and has visual and reliable results.
Example 5 evaluation of the accuracy and reproducibility of the detection method
1. Analysis of detection accuracy
Accuracy (tress) refers to the degree of agreement between the average of a set of test results and an acceptable reference value. According to the European Union and Codex standards, the accuracy criterion is that the measured value should be within + -25% of the reference value within the whole dynamic range of detection.
(1) materials: positive control RNA for norovirus type I detection prepared previously (5.43X 10)7copies/. mu.l) were diluted to 5.43X 10, respectively3copies/μl、5.43×102copies/μl、5.43×101copies/. mu.l and 5.43 copies/. mu.l, indicated as samples U1-U4 (Table 4).
(2) The method comprises the following steps: each sample was tested separately by RT-ddPCR with 7 replicates per sample and 1 blank (DEPC-free water instead of template RNA).
(3) As a result: the RT-ddPCR measurements of norovirus type I RNA were all close to the expected values, with standard deviations of < 15% (Table 4), less than 25% of the criteria, indicating that the method is very accurate.
TABLE 4 accuracy of norovirus type I RT-ddPCR detection
2. Analysis of detection repeatability
(1) the method comprises the following steps: positive control (5.43X 10) of norovirus type I RNA prepared previously was used7copies/. mu.l) to 5.43X 103copies/. mu.l, RT-ddPCR assay was performed, setting 8 replicates, and 1 blank (DEPC-free water instead of template RNA).
(2) As a result: as can be seen from FIG. 4, the coefficient of variation CV of the assay for the positive control of norovirus type I RNA at a specific concentration is less than 25%, which proves that the method has good repeatability when used for quantitative detection of norovirus type I.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.
Claims (2)
1. A kit for accurately and quantitatively detecting norovirus type i, wherein the kit comprises: primers and probes for detecting norovirus type I RNA, norovirus type I positive control RNA, one-step RT-ddPCR supermix, magnesium acetate, droplet generation oil, droplet generation cards, 96-well plates and aluminum foil heat sealing membranes;
The primer for detecting the norovirus type I RNA is a nucleotide sequence shown by SEQ ID No. 1-SEQ ID No.2, the probe for detecting the norovirus type I RNA is a nucleotide sequence shown by SEQ ID No.3, the 5 'end of the probe is marked with FAM, and the 3' end of the probe is marked with BHQ.
2. a method for accurately quantifying norovirus type i for non-diagnostic purposes, comprising:
(1) Extracting RNA of a sample to be detected;
(2) Preparing an RT-ddPCR reaction system, wherein the reaction system comprises primers shown in SEQ ID No. 1-SEQ ID No.2 and used for detecting norovirus type I RNA, a probe shown in SEQ ID No.3 and used for detecting norovirus type I RNA, FAM is marked at the 5 'end of the probe, and BHQ is marked at the 3' end of the probe;
(3) Adding the RT-ddPCR reaction system prepared in the step (2) and the droplet generation oil into a droplet generation card, and placing the card in a droplet generation instrument to generate droplets;
(4) Carrying out an amplification reaction;
(5) and (4) judging a result: calculating the copy number of the norovirus type I RNA according to the Poisson distribution principle, and further calculating the norovirus type I content in the sample to be detected by the following formula:
Norovirus type I content = in sampleThe number of volumes of the RNA template,
Wherein the sample addition amount of the RNA template is 2 mu L.
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| CN107988425A (en) * | 2017-11-06 | 2018-05-04 | 北京出入境检验检疫局检验检疫技术中心 | A kind of I type norovirus detection of nucleic acids standard substances of G and its preparation method and application |
| CN108220401A (en) * | 2018-03-16 | 2018-06-29 | 张家港出入境检验检疫局检验检疫综合技术中心 | A kind of campylobacter jejuni recombinant plasmid standard items quantitative detecting method based on droplet type digital pcr |
| CN108676920B (en) * | 2018-07-07 | 2021-07-20 | 广东省实验动物监测所 | Primer and kit for rapidly detecting mouse norovirus and RT-RPA method thereof |
| CN113025758A (en) * | 2021-04-07 | 2021-06-25 | 拱北海关技术中心 | Primer, probe and kit for detecting GI type norovirus of aquatic product |
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| CN103131798A (en) * | 2013-02-25 | 2013-06-05 | 湖北朗德医疗科技有限公司 | Norovirus real-time fluorescent RT-PCR detection kit and application thereof |
| CN105555972A (en) * | 2013-07-25 | 2016-05-04 | 伯乐生命医学产品有限公司 | Genetic assays |
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| CN103131798A (en) * | 2013-02-25 | 2013-06-05 | 湖北朗德医疗科技有限公司 | Norovirus real-time fluorescent RT-PCR detection kit and application thereof |
| CN105555972A (en) * | 2013-07-25 | 2016-05-04 | 伯乐生命医学产品有限公司 | Genetic assays |
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