CN113025726A - Primer, probe, kit and method for visual rapid detection of schistosoma japonicum nucleic acid by LFD-RPA - Google Patents
Primer, probe, kit and method for visual rapid detection of schistosoma japonicum nucleic acid by LFD-RPA Download PDFInfo
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Abstract
The invention discloses a primer, a probe, a kit and a method for LFD-RPA visual rapid detection of schistosoma japonicum nucleic acid, wherein the sequence of the primer is shown as SEQ ID NO. 1-2, and the sequence of the probe is shown as SEQ ID NO. 3. The invention designs RPA primer and probe by taking schistosoma japonicum G55A (SjG55A) gene as target sequence, and adopts RPA amplification technology combined with lateral flow chromatography test paper strip method to realize sensitive and rapid visual detection of schistosoma japonicum nucleic acid, the detection limit of schistosoma japonicum genome DNA can reach 10fg, and the method is hopeful to realize the identification and detection with schistosoma mansoni, 1 positive oncomelania in 1500 negative oncomelania can be detected by the method, the operation is simple and rapid, no special instrument is needed, the reaction temperature is close to room temperature, the result can be observed by naked eyes, and the method is favorable for realizing the detection and monitoring of laboratory and on-site schistosome intermediate host.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer, a probe, a reaction system, a kit and a detection method for LFD-RPA visual rapid detection of schistosoma japonicum nucleic acid.
Background
Schistosomiasis is an ancient helminthiasis disease, mainly distributed in tropical and subtropical zones. It is estimated that schistosomiasis is prevalent in 78 countries and regions worldwide, about 8 million people are at risk of infection, 4.4 million people are infected with flukes, about 28 thousand deaths per year occur, and the damage to human is extremely large. The schistosoma japonicum infecting human body mainly comprises 6 species of schistosoma japonicum (S.japonicum), schistosoma mansoni (S.mansoni), schistosoma meikongi (S.mekongi), schistosoma interjaponicum (S.intercalarium), schistosoma japonicum (S.haematobium) and schistosoma malayi (S.malayensis). China is epidemic in only schistosomiasis japonica. After 70 years of prevention and treatment, the prevention and treatment work of schistosomiasis in China currently achieves remarkable achievement, the propagation control standard is achieved in 2015 in China, and the infection rate and the infection degree of residents are both at a lower level. However, epidemic factors of the schistosomiasis in China such as Hunan, Hubei, Jiangxi, Anhui and Jiangsu still exist, and the consolidation of the blood defense result is still greatly challenged by natural disasters such as flood and the like and the north-south water transfer engineering. Under the urgent need of rapid and effective detection means, the nucleic acid amplification technology represented by Polymerase Chain Reaction (PCR) is rapidly developed, and the purpose is to detect specific schistosome nucleic acid fragments with different infection degrees and fixed and unchanged in different periods after infection. The traditional nucleic acid detection method comprises conventional PCR, nested PCR (nested PCR), real-time fluorescent Quantitative PCR (Quantitative real-time PCR) and the like, but because the technologies depend on expensive instruments, need high-temperature circulation, have high cost, long time consumption and the like, most of the technologies are limited in laboratories with excellent conditions, and are difficult to be widely applied to field and field detection.
Recombinase Polymerase Amplification (RPA) is a brand-new nucleic acid isothermal amplification technology developed by Piepenburg et al in 2006, and is reported in the detection of schistosomiasis and other parasitic diseases. The method is based on the principle of a T4 bacteriophage in-vivo nucleic acid replication mechanism, adopts a novel nucleic acid isothermal amplification technology involving multiple proteins and enzymes, can amplify trace nucleic acid to a detectable level within 20min at 37-42 ℃, has high sensitivity and specificity, is simple to operate, and can realize rapid detection of nucleic acid under non-laboratory conditions. Due to the high sensitivity, the intermediate host in the stage of infection of the mother metacercaria, the daughter metacercaria and the cercaria can be detected, and the method is superior to the traditional detection mode. The technology is not only suitable for the DNA template, but also can realize the amplification of RNA. In addition, the amplification product can be detected by agarose gel electrophoresis, and can also be visualized by combining with a plurality of methods such as a lateral flow chromatography test strip, a nucleic acid dye and the like, but the nucleic acid dye has an inhibiting effect on nucleic acid amplification. Piepenburg et al established a lateral flow assay (LF-RPA) test strip-RPA detection method which can directly and visually observe the final amplification result, and is suitable for on-site rapid detection. In a word, the RPA technology has the advantages of simple primer design, short time consumption, low temperature requirement, low cost, no limitation of detection conditions and fields and the like, is considered to be a new nucleic acid detection technology which is expected to replace PCR in the future, and is a new trend of the future molecular biology detection development.
Disclosure of Invention
The invention aims to solve the technical problem of providing a visual rapid detection method for schistosoma japonicum nucleic acid, which has the characteristics of high sensitivity, rapid detection, simplicity, portability, no need of expensive instruments, capability of visually observing a detection result, and suitability for early diagnosis, identification and screening of schistosomiasis in laboratories and on-site.
In order to solve the technical problems, the invention designs and provides the following technical scheme according to the schistosoma japonicum SjG55A subcloned gene sequence with the accession number of AF 412221.1.
In a first aspect, the invention provides a pair of specific RPA primers for LFD-RPA visual rapid detection of schistosoma japonicum nucleic acid, wherein the nucleotide sequence of the RPA primers is as follows:
an upstream primer: 5'-CCAATAAGGGCCGCTGACAGTTTATAGAGA-3', respectively; (shown as SEQ ID NO. 1);
a downstream primer: 5 '-Biotin-CAACTACCAACGGTCAGTAGCTTCATGAGC-3'; (shown as SEQ ID NO. 2).
As a preferred example, the 5' end of the downstream primer (RPA-PR) in the primer is modified by biotinylation.
The specific RPA primer provided by the invention is longer than a PCR primer, and the optimal length is 30-35 bp; the 5' end avoids poly G (guanine), and can strive for C (cytosine); c, G at the 3' end is beneficial to the stable combination of polymerase, and the amplification performance of the primer is improved; GC content is 40-60%.
In a second aspect, the invention provides a probe for LFD-RPA visual detection of schistosoma japonicum nucleic acid, wherein the probe sequence is specifically bound between primer amplification sequences shown as SEQ ID No.4, the length of the probe sequence is 46-52nt (49 nt in the invention 3), the 5' end of the probe is fluorescein or digoxin modification (fluorescein and FAM in the invention), the middle position of 30-35 nt (30 nt in the invention) away from the 5' end is marked with a dspacer (tetrahydrofuran and THF) as a recognition site of exonuclease, and the 3' end of the probe is phosphorylation modification. THF is generally about 15 bases (19 nt in the present invention) from the 3' end.
Specifically, the invention provides an RPA-nfo probe for LFD-RPA visual detection of schistosoma japonicum nucleic acid, wherein the nucleotide sequence of the probe is as follows:
5'-FAM-CACCACACACTCACCATAATGTTTGAAGAA(THF) TAATAAGTAAATATTGC-C3 Spacer-3'. (shown as SEQ ID NO. 3).
Specifically, in the nucleotide sequence of the probe, the 5 'end is a fluorescein modification (FAM), the 31 st base is a tetrahydrofuran residue (THF), and the 3' end is a phosphorylation modification (C3-spacer).
The invention also provides a combination of the primer and the probe. The specific amplified fragment of the primer and the probe is a sequence between 152 and 386bp of a G55A subclone gene sequence (Genbank No. AF412221.1) of schistosoma japonicum similar non-long-end retrotransposon SjR2, the size is 235bp, and the amplified sequence is as follows:
CCAATAAGGGCCGCTGACAGTTTATAGAGACTTAAATAAATAAATAAAATACACCACACACTCACCATAATGTTTGAAGAATTAATAAGTAAATATTGCCAAGTTCTAAAGAATGATGTTTCTGAAGCCCAGTCTTGTTTAGTTTAGTTCAGTCCATCACAAACAATACCTTCAGGTTAGAGGTGATCAAACTAAAATATGACAAGCTCATGAAGCTACTGACCGTTGGTAGTTG (shown in SEQ ID NO. 4).
The invention also provides application of the combination of the primer and the probe in preparing a kit for LFD-RPA visual rapid detection of schistosoma japonicum nucleic acid.
In a third aspect, the invention provides a kit for LFD-RPA visual rapid detection of schistosoma japonicum nucleic acid, which comprises: an RPA primer pair shown as SEQ ID NO. 1-2 and an RPA-nfo probe shown as SEQ ID NO. 3. Preferably, the kit further comprises RPA amplification reaction system reagents. The RPA amplification reaction system reagent refers to all reagent components in the RPA amplification reaction system except a primer pair, a probe and a template. The RPA amplification reaction system reagent comprises: RPA reaction buffer solution, RPA amplification reagent, magnesium acetate and double distilled water. Wherein, the RPA amplification reagent refers to a reagent which depends on the action of recombinase, single-strand binding protein and DNA polymerase to realize the amplification of target genes. The components of the RPA amplification reagent comprise: phage recombinase, DNA polymerase, single-stranded DNA binding protein, dNTP and exonuclease.
Preferably, the kit further comprises a lateral flow chromatography test strip (LFD).
Preferably, the kit may further comprise a negative control. In one embodiment, the negative control is double distilled water.
Preferably, the kit may further comprise a positive control. In one embodiment, the positive control is schistosoma japonicum genomic DNA.
In a fourth aspect, the invention discloses a method for visually detecting schistosoma japonicum nucleic acid by using LFD-RPA with a non-diagnostic purpose, which comprises the following steps:
1) extracting total DNA in a sample to be detected;
2) amplification of RPA: mixing an RPA primer pair shown as SEQ ID NO. 1-2, an RPA-nfo probe shown as SEQ ID NO.3, an RPA reaction buffer solution, a total DNA template and magnesium acetate in a reaction tube containing an RPA amplification reagent to prepare an RPA reaction system, setting the reaction temperature to be 20-50 ℃ and the reaction time to be 5-30 min, and carrying out RPA amplification; setting double distilled water as negative control and schistosoma japonicum genome DNA as positive control;
3) LFD detection of RPA amplification product: detecting the RPA amplification product by using a lateral flow chromatography test strip (LFD); and diluting the amplification product, dropwise adding the diluted amplification product on a sample pad of the lateral flow chromatography test strip, vertically inserting the sample pad of the lateral flow chromatography test strip into a test strip buffer solution, and reacting for 5-10 min at room temperature. Observing the result by naked eyes, and when the test strip detection line and the quality control line are both colored (purple bands appear), indicating that the RPA amplification product is positive and the sample contains schistosome DNA; if only the quality control line is developed, the RPA has no target amplification product, and the sample has no bloodsucker DNA; if the quality control line is not colored, the experimental result is invalid.
As a specific example, the RPA amplification reagent component comprises: phage recombinase, DNA polymerase, single-stranded DNA binding protein, dNTP and exonuclease.
As a specific example, the reaction Buffer is a Rehydration Buffer.
As a specific example, the lateral flow chromatography test strip is a Milena Genline hybrid-1 test strip.
As a specific example, the RAP reaction system is typically a 50ul reaction system: adding 29.5 mu l of Rehydration Buffer, 2-2.5 mu l of 10 mu M upstream primer, 2-2.5 mu l of 10 mu M downstream primer, 2-2.5 mu l of 10 mu M magnesium acetate and 1-5 mu l of DNA template into the RPA component-containing freeze-dried powder, and supplementing the mixture to 50 mu l by double distilled water.
Preferably, the preferred conditions for RPA amplification are: the reaction temperature was 39 ℃ and the reaction time was 20 min.
Preferably, the preferred conditions for LFD detection of the RPA amplification product are: diluting the amplification product by 100 times, dripping 10 mu l of the amplification product on a lateral flow chromatography test strip sample pad, vertically inserting the test strip sample pad into 100 mu l of test strip buffer solution, reacting for 5min at room temperature, and observing the result with naked eyes.
The method provided by the invention can be applied to rapid screening and classification of samples from different sources in scientific research and teaching. From the candidate samples, samples (such as intermediate hosts) of the Schistosoma japonicum infection were screened and confirmed as required for further study.
In order to realize the sensitive, rapid and simple detection of the schistosoma japonicum katsurada nucleic acid, the invention selects the G55A subclone sequence of SjR2 gene through a large amount of preliminary screening work, designs a novel RPA primer and a probe, establishes the visual rapid sensitive detection method of the schistosoma japonicum katsurada nucleic acid by combining the RPA technology and a lateral flow chromatography test strip method, and performs preliminary evaluation on the sensitivity, the specificity and the early detection feasibility of the circulating nucleic acid of the schistosoma japonicum katsurada nucleic acid. In the invention, a fluorescence-labeled probe and a biotin-labeled primer are introduced to generate a large amount of double-stranded DNA products with one end labeled with FAM and the other end labeled with biotin in the RPA reaction process. When the RPA reaction liquid is dripped into a gold-labeled test strip and moves along with the test strip chromatography, an amplification product marked with FAM and biotin, a nanogold coupling antibody and a ligand on a detection line form a compound to enable nanogold to be aggregated and developed, and the detection line appears. The quality control line on the test strip is coated with secondary antibody, and can be combined with free FAM probe and nano gold particles marked by FAM antibody for color development, so that the quality control line appears to indicate the effectiveness of the result.
The invention takes the G55A subclone sequence of schistosoma japonicum similar non-long-end retrotransposon SjR2 as a target sequence, designs a novel RPA amplification primer and an RPA-nfo probe, combines recombinase polymerase amplification with a lateral flow test strip, and establishes a method for the visual rapid detection of schistosome nucleic acid by LFD-RPA. The LFD-RPA method disclosed by the invention can detect the positive strip of the schistosome by naked eyes after reacting for 5min at 20-45 ℃, can detect 10fg of the genome DNA of the schistosoma japonicum adult schistosome at the lowest under the optimal reaction condition of 39 ℃ and 20min, and has higher sensitivity than an RPA gel running detection method (the detection limit is 100fg) and a PCR method (the detection limit is 1pg) which are automatically established by taking G55A as a target. The method of the invention basically has no cross reaction when detecting the genomic DNA of the schistosoma japonicum, the clonorchis sinensis, the paragonimus westermani and the schistosoma mansoni, and prompts that the method can be used for the detection of the schistosoma japonicum and the differential diagnosis of other flukes.
The invention can detect SjG55A fragments in DNA samples of only 1 positive oncomelania in 1500 negative oncomelania at the lowest success, shows the feasibility and excellent performance of the method for detecting trace quantity schistosome DNA in intermediate hosts, and has the identification value of the intermediate hosts.
In conclusion, the beneficial effects of the invention are as follows: the LFD-RPA detection method and the kit provided by the research are simple to operate, do not need special instruments and equipment (such as a fluorescence detector, an electrophoresis apparatus, an electrochemical DNA sensor and the like), have wide reaction temperature range, are close to room temperature in common temperature, are quick in reaction, can observe results by naked eyes, and have strong schistosomiasis prevention and treatment field application potential. The method can detect the schistosoma japonicum nucleic acid fragment formed by mixing the positive and negative oncomelania, has detection value and is expected to be used for detecting the schistosoma japonicum infected intermediate host.
Drawings
FIG. 1 is a color development result chart of a test strip optimized for the reaction temperature of LFD-RPA detection of Schistosoma japonicum genomic DNA in example 2. Wherein, 1-7: the left reaction temperature is 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃ respectively; the right side is control sterilized double distilled water.
FIG. 2 is a color development result chart of the test strip optimized in reaction time for LFD-RPA detection of Schistosoma japonicum genomic DNA in example 2. Wherein, 1-7: the reaction time on the left side is respectively 0min, 5min, 10min, 15min, 20min, 25min and 30 min; the right side is sterilized double distilled water.
FIG. 3 is a color development result chart of the test strip for the specific evaluation of LFD-RPA detection of Schistosoma japonicum genomic DNA in example 3. Wherein, 1-10: respectively are Japanese blood fluke, Schistosoma mansoni, Egyptian blood fluke, positive oncomelania, negative oncomelania, positive double-umbilical snail, negative double-umbilical snail, clonorchis sinensis, fasciola gigantica and paragonimus westermani genome DNA; 11: sterilizing double distilled water.
FIG. 4 is a color development result chart of the test strip for evaluating the sensitivity of LFD-RPA detection plasmid DNA in example 3. Wherein, 1-7: the concentration of the genomic DNA was 105、104、103、10210, 1, 0.1 copies/. mu.l; 8: sterilizing double distilled water.
FIG. 5 is a color development result chart of the test strip for the sensitivity evaluation of LFD-RPA detection of Schistosoma japonicum genomic DNA in example 3. Wherein, 1-7: the concentration of the genome DNA is 1ng/ul, 100 pg/ul, 10 pg/ul, 1 pg/ul, 100 fg/ul, 10 fg/ul, 1fg/ul and 0.1fg/ul respectively; 8: sterilizing double distilled water.
FIG. 6 is a color development result chart of the test strip for evaluating the effect of LFD-RPA detection of mixed oncomelania DNA in example 4. Wherein, 1: schistosoma japonicum adult genome DNA; 2-9: the mixing ratio of the oncomelania DNA is 1:10, 1:50, 1:100, 1:500, 1:1000, 1:1500, 1:2000, 1: 2500; 10: sterilizing double distilled water.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1: preparation of template DNA, primer and probe
1) Preparation of template DNA: using tissue DNA extraction kit (
Blood&Tissue kit, purchased from Qiagen) extracts genomic DNA of adult schistosoma japonicum, adult schistosoma mansoni, eggs of schistosoma japonicum, paragonimus westermani, adult clonorchis sinensis, mixed oncomelania, and field oncomelania.
2) Designing a primer and a probe: the G55A gene fragment is used as a target sequence, the Primer 5 software is adopted to combine with BLAST to design an RPA Primer and a probe, and the length of an amplification product is 235 bp. All primers and probe DNAs were synthesized by Shanghai Biotech, Inc.
The upstream primer (RPA-PF) is: 5'-CCAATAAGGGCCGCTGACAGTTTATAGAGA-3', respectively;
the downstream primer (RPA-PR) is: 5'-Biotin-CAACTACCAACGGTCAGTAGCTTCATGAGC-3'
Probe (RPA-nfo probe): 5' -FAM-CACCACACACTCACCATAATGTTTGAAGAA(THF) TAATAAGTAAATATTGC-C3 Spacer); wherein, the 5' end is fluorescein modification (FAM); [ THF ] is a tetrahydrofuran (tetrahydrofuran) residue, which is a recognition site for exonuclease; 3' is a phosphorylation modification (C3-spacer) to block the extension of DNA strand.
Example 2: establishment and condition optimization of LFD-RPA method for detecting schistosoma japonicum genome DNA
1) The LFD-RPA detection method is established as follows:
reference to
In the nfo kit specification, 2.1. mu.l each of the upstream and downstream primers (10. mu. mol/L) designed in example 1, 0.6. mu.l of the probe (10. mu. mol/L) designed in example 1, 29.5. mu.l of the reaction buffer, and ddH2O12.2. mu.l, template DNA 1. mu.l, MgAc2Preparing 2.5 mul into a 50 mul mixed system, adding an RPA reaction tube to dissolve the freeze-dried powder, fully mixing uniformly, reacting in a water bath kettle, and taking double distilled water as negative control. The reaction temperature was set at 7 gradients: 20. 25, 30, 35, 40, 45 and 50 ℃, the reaction amplification time was set at 7 gradients: 0. 5, 10, 15, 20, 25 and 30min, with the other conditions being the same, to determine the optimal RPA reaction temperature and time.
The RPA amplification products were detected using a Milena Genline HybriDetect 1 lateral flow chromatography test strip. Diluting the amplification product by 100 times, dripping 10 mu l of the amplification product on a test strip sample pad, vertically inserting the test strip sample pad into 100 mu l of test strip buffer solution, reacting for 5min at room temperature, and observing the result with naked eyes.
When the test strip detection line and the quality control line both have red strips, the RPA amplification product is indicated to be positive, and if only the quality control line is colored, the RPA is indicated to have no target amplification product; if the quality control line is not colored, the experimental result is invalid.
2) LFD-RPA detection of schistosoma japonicum reaction temperature and time condition optimization results:
under the condition of reaction for 20min, LFD-RPA shows strips on quality control lines under the set gradient temperature conditions of 25, 30, 35, 40 and 45, but does not show strips at 50 ℃, wherein the strips of the detection lines are obvious within the range of 30-45 ℃, and the strips of the detection lines are deepest within the range of 35-45 ℃ (figure 1), which shows that the temperature of 30-45 ℃ is a suitable reaction temperature range of the RPA, and the temperature of 35-45 ℃ is the optimal reaction temperature, so that 39 ℃ recommended by an RPA kit is selected as the amplification temperature of a subsequent experiment.
Amplifying for 5min at the amplification temperature of 39 ℃ to reach the level of a detectable product; within 5-30 min, the positive bands gradually increased with the increase of reaction time, and the positive bands almost reach saturation within 20min (FIG. 2). In order to meet the detection efficiency of the low-concentration template, improve the timeliness and avoid the generation of non-specific amplification reaction, 20min is selected as the reaction time for subsequent performance evaluation.
Example 3: evaluation of sensitivity and specificity of LFD-RPA for detecting schistosoma japonicum
Selecting 39 deg.C, 20min as RPA amplification condition, setting Japanese blood fluke genome DNA template to have plasmid concentration of 105、104、103、102LFD-RPA detection sensitivity was evaluated by setting negative controls to 7 concentration gradients of 10, 1, 0.1 copies/. mu.l, etc.
LFD-RPA detection sensitivity is evaluated by selecting 39 ℃ and 20min as RPA amplification conditions, setting Japanese blood fluke genome DNA templates to 7 concentration gradients of 1ng/ul, 100 pg/ul, 10 pg/ul, 1 pg/ul, 100 fg/ul, 10 fg/ul, 1fg/ul and the like, and setting negative controls.
And 1ng of genomic DNA of schistosoma mansoni, schistosoma japonicum, paragonimus westermani, clonorchis sinensis, positive oncomelania, negative oncomelania, positive amphibian snail and negative amphibian snail is taken as a template, and the specificity of LFD-RPA detection is evaluated.
The results show that: in the evaluation of plasmid sensitivity (FIG. 4), 105、104、103、102Obvious detection line bands were visible for both copies/μ l groups,and the color of the detection line gradually deepens along with the increase of the amount of the template, and the lowest detection limit of LFD-RPA on the schistosoma japonicum plasmid DNA can reach 102Copies/. mu.l. In adult DNA detection (figure 5), except for double distilled water and 1 fg/. mu.l group, 1 ng/. mu.l, 100 pg/. mu.l, 10 pg/. mu.l, 1 pg/. mu.l, 100 fg/. mu.l and 10 fg/. mu.l groups can all see obvious detection line bands, and along with the increase of the template amount, the detection line color gradually deepens, and the lowest detection limit of LFD-RPA on the genomic DNA of the schistosoma japonicum can reach 10 fg. The result of the specificity evaluation (figure 3) shows that LFD-RPA detects that negative oncomelania, schistosoma mansoni, positive amphibian snail, negative amphibian snail, Egyptian schistosome, paragonimiasis westermani, clonorchis sinensis and fasciola gigantica do not have cross reaction and have specificity. Detecting red strips of the schistosoma japonicum and the positive oncomelania; the G55A sequence and the primer adopted by the invention are prompted to have the specificity of the schistosoma japonicum and are expected to be used for the detection of the schistosoma japonicum and the oncomelania intermedia.
Example 4: evaluation of Effect of LFD-RPA detection on Mixed Oncomelania Hupensis Gredler
And (3) carrying out LFD-RPA detection on the DNA of the simulated mixed oncomelania by adopting optimized experimental conditions, and investigating the feasibility and the performance of the method for detecting the schistosoma japonicum nucleic acid in the intermediate host oncomelania.
Detecting samples of positive oncomelania and negative oncomelania in different proportions, wherein the mixing proportion of oncomelania DNA is 1:10, 1:50, 1:100, 1:500, 1:1000, 1:1500, 1:2000 and 1: 2500. mixing samples according to the proportion, extracting DNA, and evaluating the detection value of the positive intermediate host of the detection technology.
The results show that, as shown in FIG. 6, LFD-RPA detection is performed at a mixing ratio of 1: after 1500, red strips do not appear on the test strip, when the red strips are mixed in a ratio of 1:10, 1:50, 1:100, 1:500, 1:1000 and 1:1500, the red strips can be seen by naked eyes, positive detection lines can be seen on the test strip, the color depth of the strips becomes lighter along with the increase of the dilution ratio of the positive oncomelania DNA, and the negative oncomelania group has no detection line. The result shows the feasibility and excellent performance of the invention used for detecting the trace positive schistosome nucleic acid in oncomelania in the intermediate host.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
<110> Chinese disease prevention and control center for prevention and control of parasitic diseases institute (national center for research on tropical diseases)
Primer, probe, kit and method for visual rapid detection of schistosoma japonicum nucleic acid by <120> LFD-RPA
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<213> Schistosoma japonicum SjG55A (SjG55A)
<400> 4
ccaataaggg ccgctgacag tttatagaga cttaaataaa taaataaaat acaccacaca 60
ctcaccataa tgtttgaaga attaataagt aaatattgcc aagttctaaa gaatgatgtt 120
tctgaagccc agtcttgttt agtttagttc agtccatcac aaacaatacc ttcaggttag 180
aggtgatcaa actaaaatat gacaagctca tgaagctact gaccgttggt agttg 235
Claims (13)
1. A pair of primers for visual and rapid detection of schistosome nucleic acid by LFD-RPA is characterized in that the nucleotide sequence of the primers is as follows:
an upstream primer: 5'-CCAATAAGGGCCGCTGACAGTTTATAGAGA-3', respectively;
a downstream primer: 5 '-Biotin-CAACTACCAACGGTCAGTAGCTTCATGAGC-3'.
2. The primer of claim 1, wherein the 5' end of the downstream primer is biotinylated, the specific fragment amplified by the pair of primers is a partial sequence cloned from G55A of schistosoma japonicum non-long-terminal retrotransposon SjR2 gene, the size is 235bp, and the amplification sequence is:
CCAATAAGGGCCGCTGACAGTTTATAGAGACTTAAATAAATAAATAAAATACACCACACACTCACCATAATGTTTGAAGAATTAATAAGTAAATATTGCCAAGTTCTAAAGAATGATGTTTCTGAAGCCCAGTCTTGTTTAGTTTAGTTCAGTCCATCACAAACAATACCTTCAGGTTAGAGGTGATCAAACTAAAATATGACAAGCTCATGAAGCTACTGACCGTTGGTAGTTG。
3. the LFD-RPA probe for visually detecting schistosoma japonicum nucleic acid is characterized in that a probe sequence is specifically combined between the following amplification sequences:
CCAATAAGGGCCGCTGACAGTTTATAGAGACTTAAATAAATAAATAAAATACACCACACACTCACCATAATGTTTGAAGAATTAATAAGTAAATATTGCCAAGTTCTAAAGAATGATGTTTCTGAAGCCCAGTCTTGTTTAGTTTAGTTCAGTCCATCACAAACAATACCTTCAGGTTAGAGGTGATCAAACTAAAATATGACAAGCTCATGAAGCTACTGACCGTTGGTAGTTG；
the length of the probe sequence is 46-52nt, the 5' end of the probe is modified by fluorescein or digoxin, a dspacer is marked at the middle position which is 30-35 nt away from the 5' end and serves as a recognition site of exonuclease, and the 3' end of the probe is modified by phosphorylation.
4. A probe for LFD-RPA visual detection of schistosoma japonicum nucleic acid is characterized in that the nucleotide sequence of the probe is as follows:
5'-FAM-CACCACACACTCACCATAATGTTTGAAGAA(THF)TAATAAGTAAATATTGC-C3Spacer-3'。
5. the combination of the primer and the probe for LFD-RPA visual rapid detection of schistosoma japonicum nucleic acid is characterized in that the nucleotide sequence of the primer is as follows:
an upstream primer: 5'-CCAATAAGGGCCGCTGACAGTTTATAGAGA-3', respectively;
a downstream primer: 5 '-Biotin-CAACTACCAACGGTCAGTAGCTTCATGAGC-3';
the nucleotide sequence of the probe is as follows:
5'-FAM-CACCACACACTCACCATAATGTTTGAAGAA(THF)TAATAAGTAAATATTGC-C3Spacer-3'。
6. the use of the combination of the primer and the probe according to claim 5 for preparing a kit for LFD-RPA visual rapid detection of schistosome nucleic acid.
7. A kit for LFD-RPA visual rapid detection of schistosoma japonicum nucleic acid is characterized in that the kit comprises: a primer pair shown as SEQ ID NO. 1-2 and a probe shown as SEQ ID NO. 3.
8. The kit of claim 7, wherein the kit further comprises RPA amplification reaction system reagents; the RPA amplification reaction system reagent comprises: RPA reaction buffer solution, RPA amplification reagent, magnesium acetate and double distilled water; wherein the RPA amplification reagent comprises the following components: phage recombinase, DNA polymerase, single-stranded DNA binding protein, dNTP and exonuclease.
9. The kit of claim 7, wherein the kit further comprises a lateral flow assay strip.
10. A method for visually detecting schistosoma japonicum nucleic acid by using LFD-RPA for non-diagnosis purposes is characterized by comprising the following steps:
1) extracting total DNA in a sample to be detected;
2) amplification of RPA: mixing an RPA primer pair shown as SEQ ID NO. 1-2, an RPA-nfo probe shown as SEQ ID NO.3, an RPA reaction buffer solution, a total DNA template and magnesium acetate in a reaction tube containing an RPA amplification reagent to prepare an RPA reaction system, setting the reaction temperature to be 20-50 ℃ and the reaction time to be 5-30 min, and carrying out RPA amplification; double distilled water is used as a negative control, and schistosoma japonicum genomic DNA is used as a positive control;
3) LFD detection of RPA amplification product: and (3) detecting the RPA amplification product by adopting a lateral flow chromatography test strip, diluting the amplification product, dripping the diluted amplification product on a sample pad of the lateral flow chromatography test strip, vertically inserting the sample pad into a test strip buffer solution, and reacting for 5-10 min at room temperature.
11. The method of claim 10, wherein in step 2), the reaction system of RPA is 50ul, comprising: 29.5 mul of Rehydration Buffer, 2-2.5 mul of 10 mul of upstream primer, 2-2.5 mul of 10 mul of downstream primer, 2-2.5 mul of 10 mul of magnesium acetate, 1-5 mul of DNA template, and making up to 50 mul with double distilled water.
12. The method according to claim 10, wherein in the step 2), the conditions for RPA amplification are as follows: the reaction temperature was 39 ℃ and the reaction time was 20 min.
13. The method according to claim 10, wherein in step 3), the conditions for LFD detection are: diluting the amplification product by 100 times, dripping 10 mu l of the amplification product on a sample pad, vertically inserting the sample pad into 100 mu l of test strip buffer solution, reacting for 5min at room temperature, and observing the result with naked eyes.
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