CN107400734B - Loop-mediated isothermal amplification Zika virus detection kit - Google Patents
Loop-mediated isothermal amplification Zika virus detection kit Download PDFInfo
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Abstract
The invention provides a loop-mediated isothermal amplification (LAMP) primer, a related kit and a method for detecting Zika virus (also called Zika virus, ZIKV) by using the LAMP primer. The primers can be used for specifically and loop-mediated isothermal amplification of the nucleic acid of the Zika virus. The method has the advantages of good specificity, high sensitivity, good repeatability, simple operation, convenience and rapidness, can be well applied to identifying Zika virus, and is suitable for clinical and laboratory detection.
Description
Technical Field
The invention belongs to the technical field of molecular biology and nucleic acid detection. In particular, the invention relates to a loop-mediated isothermal amplification-based Zika virus detection kit.
Background
Zika virus (ZIKV) is an arbovirus that is transmitted by mosquitoes, and the host is undefined and circulates mainly in wild primates and mosquitoes inhabiting trees, such as aedes africans. This virus was first discovered in 1947 by chance through the yellow fever monitoring network in rhesus monkeys of the bush-kanka forest of lindera indica, and subsequently in 1952 in the population of lindera and tanzania. This viral activity has been relatively occult, with sporadic cases of Zika virus infection only in African, American, Asia and Pacific regions around the equator. The first outbreak was the yabuna island which occurred in the western pacific microcolonia islands in western pacific, the miloney island, and the larger epidemic, which occurred in the french poli west in the oceans in 2013-2014, infected approximately 32000 people. Aedes also transmit three additional viruses in the flaviviridae family, including dengue, chikungunya, and yellow fever, and are also prevalent mainly in tropical and subtropical regions. Several decades ago, african researchers noted that the copy card virus epidemic spread by aedes wonderfully followed the chikungunya virus epidemic spread by aedes. Similar laws began in 2013, with Zika virus following when the chikungunya virus propagated from west to east.
In the prior art, the detection method of Zika mainly comprises virus culture and separation, enzyme-linked immunoassay and fluorescent quantitative PCR detection technology.
The virus culture technique uses the collected sample to culture with cells, and determines whether the sample is infected by virus by observing cytopathic effect or detecting the amplification of virus by other methods. Although the method is the gold standard for detecting the virus infection once, only virus particles with infection capacity in a sample can be detected, and the sensitivity is insufficient. And the operation procedure is complex, the biological safety level requirements are met on experimental sites and equipment, the detection process is long (at least more than one week), the method cannot be used for field detection, and the detection results of different viruses are different.
The enzyme labeling detection based on the antibody, direct and indirect fluorescence detection and colloidal gold rapid detection all utilize the specific combination between the antigen and the antibody, and the visual detection of the pathogen is realized through the antibody coupled with the chromogenic group. The antibody detection does not depend on large-scale equipment, and the detection result can be obtained within 30 minutes. However, the antibody detection has lower sensitivity than the molecular detection because of no signal amplification process, and the result is easy to have false positive and false negative. Due to the long preparation period of the new antibody, the method is difficult to be used for detecting the new virus, and the detection capability of the new virus subtype generated by antigen drift is questionable. The World Health Organization (WHO) study showed that enzyme-linked immunosorbent assay is difficult to distinguish Zika virus from dengue virus.
The nucleic acid molecule detection of the pathogen is that the nucleic acid molecule of the pathogen is amplified by polymerase, then the reaction process or the product is monitored by methods of fluorescence, electrophoresis and the like, and whether the pathogen appears or not is judged according to whether the reaction is carried out or not or whether the product appears or not. However, PCR-based methods all require devices such as thermal cyclers, require detection sites, and require special training for quantitative PCR, which limits the application of PCR-related technologies in rapid detection.
Therefore, there is a strong need in the art to develop a rapid, simple and efficient Zika virus detection method and reagent.
Disclosure of Invention
The invention aims to provide a method and a kit for detecting the Zika virus based on loop-mediated isothermal amplification.
In a first aspect of the present invention, there is provided a primer set for detecting Zika virus, comprising primers capable of specifically binding to the NS4B gene of Zika virus and for amplifying a specific amplification product corresponding to said gene.
In another preferred embodiment, the primer set comprises one or more primer pairs selected from the group consisting of:
a first primer pair:
f3 primer: the sequence is shown as SEQ ID NO. 1;
b3 primer: the sequence is shown as SEQ ID NO. 2;
a second primer pair:
FIP primer: the sequence is shown as SEQ ID NO. 3;
the BIP primer is as follows: the sequence is shown as SEQ ID NO. 4;
a third primer pair:
LF primer: the sequence is shown as SEQ ID NO. 5;
an LB primer: the sequence is shown as SEQ ID NO. 6.
In another preferred embodiment, the primer set is used for loop-mediated isothermal amplification (LAMP) of Zika virus.
In a second aspect of the present invention, there is provided a PCR amplification system, said system comprising: a buffer system for amplification and a primer set according to the first aspect of the invention located in said system.
In another preferred embodiment, the ratio of the molar amounts of the first primer pair, the second primer pair and the third primer pair in the amplification system is (0.8-1.2): (6-10): (4-6), preferably 1: (7-9): (3-5), more preferably 1:8: 4.
In another preferred embodiment, said amplification comprises loop-mediated isothermal amplification.
In another preferred embodiment, the buffer system for amplification comprises: buffer, amplidase, dNTP, and RNase inhibitor.
In another preferred embodiment, the buffer system for amplification further comprises a dye for detection.
In a third aspect of the present invention, there is provided a kit for detecting Zika virus, comprising (i) a container, and (ii) a primer set according to the first aspect of the present invention in the container.
In another preferred embodiment, the kit comprises:
a first container, and a first primer pair located in the first container;
a second container, and a second primer pair located in the second container; and/or
A third container, and a third primer pair located in the third container;
the first container, the second container and the third container can be independent or the same container (including two or three containers being the same container).
In another preferred embodiment, the first container, the second container and the third container are the same container; and said first, second and third primer pairs are mixed together to obtain a primer mixture.
In another preferred embodiment, the concentration of the single primer of the first primer pair in the primer mixture is 0.1-0.3. mu.M, preferably 0.2. mu.M.
In another preferred embodiment, the concentration of the single primer of the second primer pair in the primer mixture is 1.4-1.8. mu.M, preferably 1.5-1.6. mu.M.
In another preferred embodiment, the concentration of the single primer of the third primer pair in the primer mixture is 0.6-1.0. mu.M, preferably 0.7-0.9. mu.M.
In another preferred embodiment, the kit further comprises reagents for amplification, including an amplification enzyme, a buffer, dntps, an rnase inhibitor, and the like; RNA extraction reagents, and dyes for detection.
In another preferred embodiment, the amplification enzymes include Bst2.0DNA polymerase and hot start reverse transcriptase.
In another preferred embodiment, the kit further comprises a detection standard of Zika virus.
Wherein, the buffer solution comprises: thermolpol RB (isotermal Amplification Buffer), magnesium sulfate, and Betaine (Betaine).
In another preferred embodiment, the dye for detection is selected from the group consisting of: hydroxynaphthol blue dye (HNB), Green Fluorescent Nucleic Acid dye SYTO 9(SYTO 9Green Fluorescent Nucleic Acid Stain), Calcein dye (Calcein), or a combination thereof.
In another preferred embodiment, the kit further comprises instructions for use.
In a fourth aspect of the present invention, there is provided a loop-mediated isothermal amplification-based method comprising the steps of:
(a) the primer set of the first aspect of the invention is used for loop-mediated isothermal amplification of a test sample.
In another preferred embodiment, the method is a non-diagnostic method.
In another preferred embodiment, the method is an in vitro method.
In a fifth aspect of the present invention, there is provided a method for detecting Zika virus, comprising the steps of:
(a) amplifying a test sample using the primer set of the first aspect of the invention;
(b) detecting the amplification to determine the presence or absence of Zika virus in the test sample.
In another preferred embodiment, the sample to be tested is a nucleic acid extract, preferably an RNA extract.
In another preferred embodiment, in step (b), if a specific amplification product is produced, it indicates the presence of Zika virus in the test sample; if no specific amplification product is produced, it indicates that the Zika virus is not present in the test sample.
In another preferred embodiment, in step (a), the amplification is loop-mediated isothermal amplification.
In another preferred embodiment, the reaction temperature of the loop-mediated isothermal amplification is 60-65 ℃, preferably 61-63 ℃.
In another preferred embodiment, in step (a), the detection sample is subjected to loop-mediated isothermal amplification using the amplification system according to the second aspect of the present invention, thereby obtaining a reaction mixture containing amplification products.
In another preferred embodiment, in step (b), the detection is performed by visual inspection, and the loop-mediated isothermal amplification time in step (a) is 30min to 120min, preferably 50min to 80 min.
In another preferred embodiment, in step (b), the detection is performed by visual inspection, and an HNB dye, or a Calcein dye, is added to the reaction mixture before visual inspection.
In another preferred embodiment, HNB dye is added to the reaction mixture and if the reaction mixture turns sky blue, amplification is positive and the presence of zika virus in the sample is detected; if the reaction mixture turns to light purple, amplification is negative, and the absence of Zika virus in the test sample is detected.
In another preferred embodiment, Calcein dye is added to the reaction mixture, and if the reaction mixture turns green, amplification is positive, and the presence of Zika virus in the sample is detected; if the reaction mixture turns orange, amplification is negative, and the absence of Zika virus in the sample is detected.
In another preferred embodiment, in step (b), the detection is performed by a real-time quantitative PCR instrument, and the loop-mediated isothermal amplification in step (a) comprises 50-70 cycles, preferably 50-60 cycles.
In another preferred embodiment, the cycle comprises the steps of:
(i) keeping the temperature at 60-65 ℃ for 20-40 seconds, preferably 30 seconds;
(ii) fluorescence is collected at 60-65 deg.C for 20-40 seconds, preferably 30 seconds.
In another preferred embodiment, the loop-mediated isothermal amplification further comprises a step of isothermal reaction at 60-65 ℃ for 1-3min, preferably 1-2min, before the cycling.
In another preferred embodiment, the detection is performed by a real-time quantitative PCR instrument, and the amplification system contains a green fluorescent nucleic acid dye SYTO 9.
In another preferred embodiment, after the loop-mediated isothermal amplification reaction is finished, if an amplification curve exists, amplification is positive, and Zika virus exists in a detection sample; if there is no amplification curve, it indicates that the amplification is negative, and the absence of Zika virus in the sample is detected.
In another preferred embodiment, the method is a Zika virus pre-detection method.
In another preferred embodiment, the method is a non-diagnostic method.
In another preferred embodiment, the method is an in vitro method.
In a sixth aspect of the present invention, there is provided a use of the primer set according to the first aspect of the present invention for preparing a kit for detecting Zika virus.
The invention also provides a method for identifying Zika virus, which comprises the following steps:
taking RNA of a sample to be detected as a template, and amplifying by using a specific amplification primer; if the specific amplification occurs, the fact that the sample to be detected contains the Zika virus is indicated;
wherein, the specific amplification primer comprises:
primer pair 1:1 and 2 SEQ ID NO;
and (3) primer pair 2: 3 and 4;
and (3) primer pair: 5 and 6 SEQ ID NO;
in another preferred embodiment, the amplification is loop-mediated isothermal amplification.
In another preferred embodiment, the loop-mediated isothermal amplification conditions are:
(1) reacting at 62 + -2 deg.C (preferably 62 + -1 deg.C) for 2 + -1 min (preferably 1 + -1 min);
(2) collecting fluorescence for 62 +/-2 ℃ (preferably 62 +/-1 ℃) for 30 seconds and 62 +/-2 ℃ (preferably 62 +/-1 ℃) for 30 seconds; the first step and the second step are repeated for 60 +/-10 times (preferably 55 +/-5 times).
In another preferred embodiment, the loop-mediated isothermal amplification system comprises: primer pair 1-3 mixture, loop-mediated isothermal amplification reaction buffer, Bst2.0DNA polymerase (Bst2.0DNA polymerase, Bst2.0), hot start Reverse Transcriptase (WarmStart RTx Reverse Transcriptase, WSRTx), hydroxynaphthol blue dye (Hydroxy naphthol blue, HNB), Green Fluorescent Nucleic Acid dye SYTO 9(SYTO 9Green Fluorescent Nucleic Acid Stain), Calcein dye (Calcein), RNase inhibitor (RNase inhibitor).
Wherein, the buffer solution for the loop-mediated isothermal amplification reaction comprises: dNTP, Thermolpol RB (Isothermal Amplification Buffer), magnesium sulfate, Betaine (Betaine).
In another preferred embodiment, (a) the dye is hydroxynaphthol blue dye (HNB) by visual inspection; after the loop-mediated isothermal amplification reaction is finished, if sky blue exists in the amplification product, the amplification is positive; the presence of light purple color in the amplification product indicates negative amplification. (b) Visually detecting, wherein said dye is Calcein dye (Calcein); after the loop-mediated isothermal amplification reaction is finished, if green exists in the amplification product, the amplification is positive; if the amplified product has orange color, the amplification is negative. (c) Real-time quantitative monitoring using green fluorescent nucleic acid dye SYTO9 as dye; after the loop-mediated isothermal amplification reaction is finished, if an amplification curve exists, the amplification is positive; the absence of an amplification curve indicates negative amplification.
In another preferred embodiment, the method for identifying Zika virus is a non-disease diagnostic method.
In another preferred embodiment, the primers are used in the form of a mixture for amplification:
the final concentrations of SEQ ID NO 1 and SEQ ID NO 2 in the primer mixture were both 0.2. mu.M;
the final concentrations of SEQ ID NO 3 and SEQ ID NO 4 in the primer mixture were both 1.6. mu.M;
the final concentrations of SEQ ID NO 5 and SEQ ID NO 6 in the primer mixture were both 0.8. mu.M;
the present invention also provides a buffer for loop-mediated isothermal amplification, the buffer comprising: dNTP, Thermolpol RB (Isothermal Amplification Buffer), magnesium sulfate, betaine.
In another preferred embodiment, the final concentration of each reagent in the buffer solution in the reaction system is: dNTP (1.0-1.6mM), Thermolpol RB (1X), magnesium sulfate (6-10mM), betaine (0-0.8M).
The invention also provides a kit for identifying Zika virus, which comprises: the kit comprises (i) a container, and (ii) a primer combination of the first aspect of the invention in the container.
In another preferred embodiment, the kit further comprises:
buffer solution for loop-mediated isothermal amplification or independently subpackaging dNTP, Thermolpol RB and magnesium sulfate; a test standard comprising Zika virus; RNA extraction reagent; bst2.0DNA polymerase; WSRTx reverse transcriptase; an RNase inhibitor;
dye: HNB dye (final concentration is 120 mu M) or calcein dye (calcein and manganese chloride are used in a concentration ratio of 1:100, and the final concentration is 500 mu M: 5mM respectively) is used for visual detection; real-time monitoring was performed using SYTO9 dye (final concentration 0.4. mu.M).
Instructions for use in a method for identifying Zika virus.
In another preferred embodiment, the kit is used for identifying Zika virus from a sample to be tested.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the results of an orthogonal assay of Real-time quantitative PCR detection LAMP reaction conditions.
FIG. 2 shows the sensitivity of Real-time quantitative PCR detection LAMP.
FIG. 3 shows the specificity of Real-time quantitative PCR detection LAMP.
Fig. 4 shows a graph of the results of visual monitoring of the course of the reaction of Zika LAMP. Wherein, the upper row is dyed by HNB dye, the positive is sky blue, and the negative is purple; the middle row and the lower row are dyed by calcein dye, which respectively shows results under visible light and naked eyes, and the calcein is green in positive, yellow in negative (naked eyes and visible light), bright in positive and dark in negative (ultraviolet).
FIG. 5 shows a real-time monitoring of the reaction process of Zika LAMP.
Fig. 6 shows a graph of the results of visual monitoring of Zika LAMP for HNB dye in clinical samples.
Fig. 7 shows a real-time monitoring of Zika LAMP on a simulated clinical sample.
Fig. 8 shows Zika LAMP clinical sample detection maps.
FIG. 9 shows the results of RT-QPCR (electrophoresis) validation of Zika LAMP.
FIG. 10 shows a real-time monitoring plot of the sensitivity of different Zika LAMP primer combinations.
Detailed Description
The present inventors have made extensive and intensive studies and experiments to disclose, for the first time, a loop-mediated isothermal amplification (LAMP) detection method for Zika virus (ZIKV). Through comparative screening, a primer with good specificity to the Zika virus is found, and the primer does not perform specific amplification on nucleic acid containing Zika virus. The method of the invention can be well applied to the identification of Zika virus components and has good reproducibility and sensitivity.
The inventor finds a sequence segment which is relatively conserved in a plurality of Zika virus genomes through analyzing a plurality of Zika virus genome sequences, and screens primers based on the sequence segment to obtain a primer which can specifically identify Zika virus, wherein the primer specifically amplifies the Zika virus RNA, but not specifically amplifies nucleic acids of other viruses.
LAMP (Loop-mediated isothermal amplification) is an isothermal, continuous, rapid, highly specific and visually detectable nucleic acid amplification method which is first reported in 2000 and is formed by continuous improvement thereafter. In view of the difficulty in finding a proper and broad-spectrum amplification primer and the difficulty in realizing result judgment according to color change, no RT-LAMP detection product for detecting Zika virus exists at home and abroad at present.
Compared with the PCR method, the LAMP does not need a thermal cycler (PCR instrument), and because a large amount of by-product white magnesium pyrophosphate precipitates are generated in the LAMP reaction, the result of the amplified product can be judged by visual observation or a turbidimeter; therefore, LAMP is suitable for rapid detection in the field, field in wartime or in laboratories with poor conditions. The LAMP technology is increasingly widely applied in the fields of scientific research of nucleic acid, identification of pathogenic microorganisms, detection of transgenic food and the like due to the advantages of rapidness, accuracy, simple and convenient operation and the like. In general, the LAMP method is a simple, rapid and highly specific gene amplification method, does not need special reagents and instruments, and can establish a detection system with low total cost.
The invention adopts a loop-mediated isothermal amplification (LAMP) rapid detection technology, designs three pairs of primers aiming at eight sections of target sequences, realizes the cyclic amplification of the target sections by using a neck ring structure which is formed by a strand displacement polymerase and an amplification product and can be combined with the primers, and the final product is a series of long chains with different lengths and target sequences. The amplification efficiency of this amplification method is 100 times that of PCR. Meanwhile, a large amount of pyrophosphate which is a byproduct of the reaction is accumulated, and the degree of the reaction can be monitored through the amount of white precipitate formed by the pyrophosphate and magnesium ions in the reaction system and can be used for judging the positive and negative results.
Accordingly, the present invention provides a primer, which is a LAMP amplification primer, comprising: primer pair 1:1 and 2 SEQ ID NO; and (3) primer pair 2: 3 and 4; and (3) primer pair: SEQ ID NO 5 and SEQ ID NO 6.
The primers disclosed by the invention are used for carrying out LAMP reaction, and whether a sample to be detected contains Zika virus or not can be rapidly judged through direct observation by naked eyes or detection by a nephelometer. Preferably, HNB is used as a dye in the LAMP reaction system, and after the loop-mediated isothermal amplification reaction is finished, sky blue exists in an amplification product, so that amplification is positive; the amplification product is light purple, which indicates that the amplification is negative and can be obviously observed by naked eyes. Preferably, Calcein is used as a dye in the LAMP reaction system, and green color exists in an amplification product after the loop-mediated isothermal amplification reaction is finished, so that amplification is positive; if orange color exists in the amplification product, the amplification is negative, and the orange color can be obviously observed by naked eyes. Preferably, SYTO9 can be used as a dye for Real-time quantitative monitoring, and after the loop-mediated isothermal amplification reaction is finished, an amplification curve indicates positive amplification; the absence of an amplification curve indicates negative amplification.
The invention also provides a method for identifying a Zika virus, comprising: taking RNA of a sample to be detected as a template, and amplifying by using a primer for specifically amplifying the Zika virus; if the specific amplification occurs, the Zika virus is contained in the sample to be detected. More preferably, based on the specific primers provided by the invention and suitable for identifying Zika virus, the method comprises the following steps: taking RNA of a sample to be detected as a template, and amplifying by using mixed primers of the primer pair 1-the primer pair 3; if the specific amplification occurs, the Zika virus is contained in the sample to be detected.
Methods for obtaining RNA from a sample to be tested are well known to those skilled in the art, and may be, for example, the conventional phenol/chloroform/isoamyl alcohol method, or may be some commercially available RNA extraction kit, which is well known to those skilled in the art.
The invention also relates to a kit for identifying the Zika virus, wherein the kit contains LAMP amplification primers aiming at the Zika virus.
In addition, the kit may also contain other reagents for identifying Zika virus, such as (but not limited to): a test standard comprising Zika virus; a buffer for loop-mediated isothermal amplification; a test standard comprising Zika virus; RNA extraction reagent; bs2.0 polymerase; WarmStart RTx reverse transcriptase; HNB; calcein and SYTO9 dyes; instructions for use of the method for identifying Zika virus.
In addition, the kit can also contain instructions for identifying the Zika virus and standard operating procedures.
The kit can realize the purposes of rapid detection and batch detection of Zika virus.
Zika virus
As used herein, the terms "Zika virus" and "Zika virus" are used interchangeably.
Zika virus (ZIKV) belongs to Flaviviridae, single-stranded positive-strand RNA virus, 20nm in diameter, is an arbovirus transmitted by mosquitoes, has an undefined host, and circulates mainly in wild primates and mosquitoes inhabiting trees, such as Aedes africans.
Zika virus is divided into 2 subtypes of African type and Asian type. The method can be divided into the following categories according to the source of the infected host: human Zika virus, mosquito Zika virus, and monkey Zika virus (i.e., human Zika virus infected, mosquito Zika virus infected, and monkey Zika virus infected).
The latency period (time from exposure to symptomatic) of Zika virus disease is unknown and can be several days. Of the Zika virus infected individuals, only about 20% exhibit mild symptoms, with typical symptoms including low fever with acute onset, maculopapules, joint pain (mostly involving the small joints of the hands and feet), conjunctivitis, and other symptoms including myalgia, headache, orbital pain and weakness. Additional rare symptoms include abdominal pain, nausea, vomiting, ulceration of the mucous membranes and itching of the skin. Symptoms are usually mild, lasting less than a week, and severe conditions requiring hospitalization are not common. In 2013 and 2015 during the cases of farina poli and brazika epidemic respectively, it was reported that the zika virus disease may cause neurological and autoimmune system complications.
Many young neonates were found in the fulminant epidemic of zika in brazil 2015 (born neonates were more than two standard deviations below the mean compared to matched children of the same sex and gestational age). In total, 4000 pregnant women infected with Zika virus were reported to have delivered microcephaly between 5 months and 2016 and 1 month in 2015, which was a 20-fold increase in the proportion of microcephaly in the past year. Cranial CT and cranial ultrasound in 35 cases of microcephaly neonates suggest the presence of diffuse brain tissue calcification, mainly in the lateral ventricles, the parenchyma, and the thalamic and basilar regions. Ventricular atrophy caused by cortical and subcortical atrophy is also seen. Articular contractures appear in a small portion of infants, suggesting involvement of the peripheral and central nervous systems. The investigation of the Zika epidemic situation shows that more and more evidences indicate that the Zika virus is related to the microcephaly. However, more investigations still need to be done before explaining the relationship between infantile microcephaly and Zika virus.
The main advantages of the invention are:
(1) the primer capable of specifically identifying the Zika virus is disclosed for the first time, the primer is good in specificity, and specific amplification can be realized for the Zika virus. In addition, the primer has good reproducibility and stable and reliable results.
(2) The primers or the detection kit containing the primers can be used for rapidly detecting the Zika viruses in large batch, rapidly and accurately distinguishing the Zika viruses from samples to be detected, and is small in required sample amount and simple to operate.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
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. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
I. Versatile materials and methods
Method for extracting virus nucleic acid
The Zika viral RNA fragments used in the examples were synthesized by in vitro reverse transcription by the Shanghai Pasteur institute of Chinese academy of sciences. Nucleic acids in other non-Zika virus samples were extracted using the QIAamp Viral RNA Mini Kit (Qiagen) RNA extraction Kit and eluted with RNase-free water, and the eluate was used as a reaction template.
LAMP reaction buffer solution
mu.L of 100mM MgSO43.5. mu.L of 10mM dNTPs, 2.5. mu.L of 10 × Thermolpol RB, 7. mu.L of RNase-free water.
Wherein 10 × Thermolpol RB is Bst2.0DNA polymerase buffer solution of NEB company, prepared from 200mM Tris-HCl, 100mM (NH)4)2SO4，500mM KCl，20mM MgSO 41% Triton X-20, pH8.8(25 ℃).
Enzyme for reaction
mu.L of Bst2.0 polymerase (8U/. mu.L), 0.5. mu.L of hot start Reverse Transcriptase (WarmStart RTx Reverse Transcriptase) and 0.5. mu.L of RNase inhibitor (RNase inhibitor) were added to each reaction at a rate of 8U/. mu.L, 15U/. mu.L.
Nucleic acid dyes
Zika virus
In all the examples except example 6, the Zika virus used in the other examples was a Zika virus cultured in vitro by Pasteur's cells.
RT-LAMP reaction
The basic system of RT-LAMP reaction is shown in Table 1.
TABLE 1 Zika RT-LAMP reaction basic System
The reaction steps are as follows:
(a) visual detection: the color change was directly observed at 62 ℃ for 60 min.
The dye is HNB or Calcein.
(b) Real-time fluorescence quantitative detection is as follows:
at 62 ℃, 120s, 1 cycle,
collecting fluorescence at 62 deg.C for 60s for 60 cycles
The dye was SYTO9, and the channel for fluorescence acquisition was set as SYBR Green I channel.
Example II
Example 1
Primer design and screening
Obtaining the whole genome sequences of all Zika viruses in GenBank, carrying out multiple sequence alignment and sequence analysis to search a conserved region, wherein the sequence 7621bp-7813bp segment of the Zika virus (taking KU740184.1 as an example) has higher conservation and is suitable for being used as a primer design region. Intercepting the region from the comparison result, and carrying out primer design after re-comparison.
The designed primers are screened by using the established RT-LAMP detection system to obtain the primers meeting the requirements, 6 primers are obtained by screening and shown in the table 2, and the 6 primers are prepared into A, B, C3 primer pair combinations with different final concentrations according to the table 3.
TABLE 2 Zika Virus RT-LAMP specific primer sequences
TABLE 3 Zika Virus ABC3 primer pairs
Example 2
RNA in vitro transcription
In the in vitro transcription example, the Zika virus plasmid template (pGH-new vector) is synthesized by Shanghai Jieli bioengineering Co., Ltd, a region with a sequence of 7621bp-7813bp (taking KU740184.1 as an example) is amplified by using a specific primer, an amplification product is subjected to electrophoresis analysis by using 1% agarose gel, a target band is cut and recovered after the correct size is determined, and a purified DNA product can be used as the template for in vitro transcription.
In vitro transcription Using the Promega "RiboMAX Large Scale RNA Production System-T7" kit, the reaction was carried out using the protocol provided by the reagent manufacturer. In vitro transcription RNA products were measured for concentration and purity using Nanodrop, converting units to copies/. mu.L. To avoid repeated freezing and thawing of RNA, appropriate amount of the extract is diluted to 1 × 1010copies/. mu.L were aliquoted and stored at-80 ℃.
Example 3
System building and optimization
By using Zika virus RNA fragment as a template and the system in Table 1 as a basic system, three horizontal memorability orthogonal tests are set by four factors by optimizing the final concentration of Tween (0%, 10%, 30%), dNTP (1.0, 1.4, 1.6mM), betaine (0, 0.4, 0.8M) and magnesium ions (6, 8, 10mM) in the reaction system, and 9 condition combinations are designed as shown in Table 4. The Real-time fluorescent quantitative detection system uses SYTO9 as a dye, and a channel for collecting fluorescence is set as SYBR Green I.
The reaction method comprises the following steps:
at 62 ℃, 120s, 1 cycle,
collecting fluorescence at 62 deg.C for 60s for 60 cycles
The channel for fluorescence acquisition was set to SYBR Green I using SYTO9 dye.
TABLE 4 Zika Virus System optimization Condition combinations
The real-Time fluorescence quantitative detection results of LAMP amplification under different reaction conditions in the system are shown in FIG. 1, the change process of a fluorescence amplification curve in the reaction process is analyzed, the reaction is carried out at the fastest speed, and the condition combination with the minimum Cycle Time value (CT value) is used as the optimal combination.
Example 4
Sensitivity and specificity assessment
Using RNase-free H2O respectively carrying out gradient dilution on RNA templates obtained by in vitro transcription of the Zika virus to obtain the RNA templates with the concentration of 1 × 10 in sequence6copies/μL，1×105copies/μL，1×104copies/μL，1×103copies/μL，1×102copies/μL，1×101copies/. mu.L and 1X 100The RNA template of copi es/. mu.L was detected using the optimal system in example 3. The detection reaction steps are as follows:
at 62 ℃, 120s, 1 cycle,
collecting fluorescence at 62 deg.C for 60s for 60 cycles
The channel for fluorescence acquisition was set to SYBR Green I using SYTO9 dye.
The sensitivity results are shown in FIG. 2, and the appearance of a "log curve" indicates that Zika virus amplified at this concentration. Wherein: the amplification curve is 1X 10 from left to right6copies/μL，1×105copies/μL，1×104copies/μL，1×103copies/μL，1×102copies/. mu.L and 1X 101copies/μL。
The concentration is 1X 100No amplification curve was observed for copies/. mu.L RNA template and negative control, indicating that the lowest detectable level of the LAMP system was 1X 101The copy/mu L, the sample loading is 5 mu L, so the lowest detection line of the method is 5 multiplied by 102copies/μL。
The Zika viral RNA fragments used in the examples were synthesized by in vitro reverse transcription by the Shanghai Pasteur institute of Chinese academy of sciences. Other negative control virus standard strains include: 4 subtypes of Dengue virus (Dengue-1, Dengue-2, Dengue-3, Dengue-4) and several common respiratory viruses RSV A (VR-26), RSVB (VR-1580), Influenza A (VR-99), Influenza B (VR-789), PIV-3(VR-93), Adenovirus (VR-930), Rhinovirus (VR-1162), HCoV-229E (VR-740) and HCoV-OC43(VR-1558) (all provided by the Shanghai Pasteur research, available from ATCC).
The detection of the above viruses was carried out by using the optimal system in example 3 and the method of step (b) in example 1, and the results are shown in fig. 3 and table 5, and the experimental results show that the Zika virus primer set designed by the LAMP system has high specificity.
As a result, the specificity of the designed LAMP primer group of the Zika virus is good, and the detection is negative for non-Zika virus.
TABLE 5 specificity results for Zika LAMP
Example 5
Visual and real-time monitoring of reaction processes
Respectively at a concentration of 5 μ L of 1X 106copies/μL，1×105copies/μL，1×104copies/μL，1×103copies/μL，1×102copies/μL，1×101copies/μL，1×100The effectiveness of the primer set was determined by using copies/. mu.L Zika virus RNA and water (negative control) as templates and reacting on a PCR instrument and a quantitative fluorescence PCR instrument, respectively, using the following reaction systems.
Using the best system of example 3, the detection method for visual and real-time monitoring of the reaction was as follows:
(1) the reaction was visually checked as follows:
visual inspection was carried out by thermostating at 62 ℃ for 60 minutes, followed by visual inspection and scanning for photographs. The use of HNB dyes and Calcein dyes;
(2) the fluorescent quantitative real-time detection reaction steps are as follows:
at 62 ℃, 120s, 1 cycle,
collecting fluorescence at 62 deg.C for 60s for 60 cycles
The channel for fluorescence acquisition was set to SYBR Green I using SYTO9 dye.
The visual detection results and the fluorescence quantitative real-time detection results are shown in FIG. 4 and FIG. 5, and it can be seen that 3 methods are performed after 60 minutes of LAMP reactionThe results are consistent: the concentration is 1X 105copies/μL，1×104copies/μL，1×103copies/μL，1×102copies/μL，1×101Zika virus RNA of copies/μ L is positive; and the concentration is 1X 100The results were negative for copies/. mu.L Zika viral RNA and water (negative control). Therefore, the detection result of the LAMP system is stable, the method has good repeatability and the accuracy is high.
Example 6
Detection of clinical samples
Because domestic Zika virus clinical samples are very limited, a 7621bp-7813bp segment (taking KU740184.1 as an example) of Zika virus is synthesized by adopting an artificial gene synthesis mode and is reversely transcribed into an RNA segment to be used as a Zika virus positive sample. Nasal swab samples of the respiratory tract of 16 cold children were collected from the southern flying hospital, Shanghai. 2 μ L of the suspension was added at a concentration of 1X 108copies/. mu.L of artificially synthesized Zika virus RNA fragments were added to 2mL nasal swab specimens, only 7 of 16 specimens were added with Zika virus RNA fragments, and the remaining 9 were added with water, and mixed well for nucleic acid extraction. Viral RNA in the sample leachate was extracted with QIAamp Viral RNA Mini Kit (Qiagen) RNA extraction Kit and eluted with RNase-free water. The eluent can be used as a reaction template.
The results are shown in fig. 6 and 7, and the visual detection results (HNB dye) are consistent with the fluorescent quantitative real-time detection (SYTO9 dye) 2 methods: of the 16 nasal swab samples, 7 were Zika virus positive and 9 were Zika virus negative. The result shows that the LAMP system and the LAMP method have high accuracy and good detection effect.
Urine from 1 patient infected with Zika virus was used as a positive sample for Zika virus. Urine of 2 persons not infected with Zika virus was used as a negative sample for Zika virus. mu.L of each of the 3 kinds of urine was extracted with QIAamp Viral RNA Mini Kit (Qiagen) RNA extraction Kit, and eluted with RNase-free water. The eluate was used as a reaction template, and the detection result of Zika LAMP was also verified by RT-QPCR (plus electrophoresis) method.
LAMP results are shown in FIG. 8, in which Zika virus amplification occurred in urine of Zika virus patients, but Zika virus amplification did not occur in the other 2 Zika virus negative samples. As shown in FIG. 9, the Zika LAMP results were consistent with the RT-QPCR (electrophoresis) results. The results show that the Zika LAMP system and the method have the advantages of high sensitivity, high accuracy and good detection effect.
Comparative example
Before obtaining the primer combination of the present application, the applicant performed a large amount of primer screening work against Zika virus. One set of primers is used as an example for explanation.
In a similar manner to example 1, a LAMP amplification primer set (a) for 9731bp-9923bp segments (for example KU 740184.1) of Zika virus NS5 gene was designed, and the specific primers are shown in Table 6:
TABLE 6 Zika Virus RT-LAMP primer sequences (comparative examples)
1X 10 detection of the primer combinations shown in Table 6 and the primer combinations shown in Table 2 of example 1 (primer combinations of the present application) were performed respectively3copies/. mu.L of human Zika virus, 3 replicates per group.
As a result, as shown in fig. 10, the detection using the primer combinations shown in table 2 was significantly superior to the detection using the primers shown in table 6.
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.
Claims (20)
1. A primer set for detecting zika virus, comprising primers capable of specifically binding to NS4B gene of zika virus and for amplifying a specific amplification product corresponding to said gene;
and, the primer set includes the following three primer pairs:
a first primer pair:
f3 primer: the sequence is shown as SEQ ID NO. 1;
b3 primer: the sequence is shown as SEQ ID NO. 2;
a second primer pair:
FIP primer: the sequence is shown as SEQ ID NO. 3;
the BIP primer is as follows: the sequence is shown as SEQ ID NO. 4;
a third primer pair:
LF primer: the sequence is shown as SEQ ID NO. 5;
an LB primer: the sequence is shown as SEQ ID NO. 6.
2. The primer set of claim 1, wherein the primer set is used for loop-mediated isothermal amplification (LAMP) of Zika virus.
3. A PCR amplification system, comprising: a buffer system for amplification and a primer set of claim 1 disposed in said system;
wherein, the molar weight ratio of the first primer pair, the second primer pair and the third primer pair in the amplification system is (0.8-1.2): (6-10): (4-6).
4. The amplification system of claim 3, wherein the molar ratio is 1: (7-9): (3-5).
5. An amplification system according to claim 3, wherein the buffer system for amplification comprises: buffer, amplidase, dNTP, and RNase inhibitor.
6. The amplification system of claim 5 wherein the buffer system for amplification further comprises a dye for detection.
7. A kit for detecting zika virus comprising (i) a container, and (ii) the primer set of claim 1 in said container.
8. The kit of claim 7, wherein the kit comprises:
a first container, and a first primer pair located in the first container;
a second container, and a second primer pair located in the second container; and
a third container, and a third primer pair located in the third container;
wherein, the first container, the second container and the third container can be independent or the same container.
9. The kit of claim 8, wherein the first container, the second container, and the third container are the same container; and said first, second and third primer pairs are mixed together to obtain a primer mixture.
10. The kit of claim 9, wherein the primer mixture has a single primer concentration of the first primer pair of 0.1 to 0.3 μ M;
the concentration of a single primer of the second primer pair in the primer mixture is 1.4-1.8 mu M; and
the concentration of the single primer of the third primer pair in the primer mixture is 0.6-1.0 mu M.
11. The kit of claim 9, wherein the primer mixture has a single primer concentration of the first primer pair of 0.2 μ Μ;
the concentration of a single primer of the second primer pair in the primer mixture is 1.5-1.6 mu M; and
the concentration of the single primer of the third primer pair in the primer mixture is 0.7-0.9 mu M.
12. The kit of claim 7, further comprising reagents for amplification, RNA extraction reagents, and dyes for detection.
13. The kit of claim 12, wherein the dye for detection is selected from the group consisting of: a hydroxynaphthol blue dye HNB, a green fluorescent nucleic acid dye SYTO9, a calcein dye, or a combination thereof.
14. A loop-mediated isothermal amplification-based method, comprising the steps of:
(a) performing loop-mediated isothermal amplification on a test sample by using the primer set of claim 1;
and, the method is a non-diagnostic method.
15. A method for detecting Zika virus, comprising the steps of:
(a) performing loop-mediated isothermal amplification on a test sample by using the primer set of claim 1;
(b) detecting the amplification to determine the presence or absence of Zika virus in the test sample;
and, the method is a non-diagnostic method.
16. The method of claim 15, wherein in step (b), the presence of Zika virus in the test sample is indicated if a specific amplification product is produced; if no specific amplification product is produced, it indicates that the Zika virus is not present in the test sample.
17. The method of claim 15, wherein in step (b), the detection is performed by visual inspection and the loop-mediated isothermal amplification time in step (a) is 30min to 120 min.
18. The method of claim 17, wherein the loop-mediated isothermal amplification time in step (a) is 50min to 80 min.
19. The method of claim 15, wherein in step (b), the detecting is performed by a real-time quantitative PCR instrument, and wherein the loop-mediated isothermal amplification in step (a) comprises 50-70 cycles.
20. Use of the primer set according to claim 1 for preparing a kit for detecting Zika virus.
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Address after: Building 2, No. 225, Chongqing South Road, Huangpu District, Shanghai, 200025 Patentee after: Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences Address before: No. 411, Hefei Road, Huangpu District, Shanghai 200025 Patentee before: INSTITUT PASTEUR OF SHANGHAI, CHINESE ACADEMY OF SCIENCES |