CN111321233A - Fluorescent RAA primer, probe and detection method for detecting schistosoma japonicum - Google Patents
Fluorescent RAA primer, probe and detection method for detecting schistosoma japonicum Download PDFInfo
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Abstract
The invention discloses a fluorescent RAA primer, a probe and a detection method for detecting schistosoma japonicum, belonging to the field of in vitro nucleic acid detection, wherein the nucleotide sequence information of a forward primer of a primer pair is shown as SEQ ID NO.1, and the nucleotide sequence information of a reverse primer is shown as SEQ ID NO. 2; the probe is an oligonucleotide probe, and the sequence information is shown in SEQ ID NO. 3; a kit for detecting schistosoma japonicum based on RAA fluorescence comprises an RAA basic fluorescence universal reaction reagent, a reaction buffer solution, a negative quality control product, a positive quality control product, a primer pair and a probe; the detection method can realize single-tube rapid detection of the schistosoma japonicum, totally-enclosed reaction, real-time monitoring of fluorescence data, no need of subsequent processing, avoidance of pollution and guarantee of the reliability of the detection result; the kit does not depend on expensive instruments such as PCR and the like, can detect even at the normal temperature of 39 ℃, can obtain a diagnosis result within 20 minutes, and greatly shortens the detection time; can detect the schistosome and the ovum quickly and qualitatively.
Description
Technical Field
The invention belongs to the field of in vitro nucleic acid detection, and particularly relates to a fluorescent RAA primer, a probe and a detection method for detecting schistosoma japonicum.
Background
Schistosoma japonicum (Schistosoma japonicum Katsurad, 1904), a species of Schistosoma japonicum, also known as Schistosoma japonicum. Schistosomiasis is an endemic parasitic disease caused by the parasitism of schistosoma japonicum in the human body. There are three main types of schistosomes parasitic on the human body: i.e., schistosoma japonicum, which is prevalent in the northern part of Africa, schistosoma mansoni, which is prevalent in Latin America and in the middle part of Africa, and schistosoma japonicum, which is prevalent in Asia. In addition, the schistosoma japonicum and meigong schistosoma japonicum can parasitize human bodies. In China, only schistosomiasis japonica is prevalent, so schistosomiasis japonica is often referred to as schistosomiasis japonica for short.
Schistosomiasis is a parasitic disease which is commonly suffered by human and animals, and is one of neglected tropical diseases which are listed as extremely easy to reproduce and reappear by WHO. With the gradual progress of preventing and treating schistosomiasis in China, the development of preventing and treating the schistosomiasis in a low epidemic situation depends on a sensitive and efficient monitoring and early warning system.
Schistosoma cercaria, schistosomulum and ovum cause mechanical damage to the host and cause complex immunopathological reactions. Cercaria causes dermatitis when they penetrate the skin, which occurs only in people who have been infected with cercaria, and is an immediate and delayed type of allergic response. Cercaria dermatitis has a certain promotion effect on the damage of the trichinella in the skin, and is the response of acquired immunity of a host to reinfection. When the baby insects move in vivo, vasculitis, capillary embolism and rupture, local cell infiltration and punctate bleeding are caused to organs, mainly lung, through which the baby insects pass. Patients may manifest as cough, hemoptysis, fever, eosinophilia, and the like.
Diagnosis is the central link of schistosomiasis control work. The diagnosis of each link of schistosomiasis control work such as the determination of chemotherapy objects, the evaluation of chemotherapy effect, the plan of prevention and treatment activities, implementation, the evaluation of prevention and treatment effect and the like needs to provide necessary information and basis. Although various etiology and immunology technologies are applied to the detection of schistosoma japonicum infection at present, the method is not ideal in the aspects of early diagnosis and detection sensitivity and specificity.
The traditional detection method mainly carries out morphological examination, has strong subjectivity and needs a certain experience of experimenters, so the control of the disease course and epidemic situation is often delayed. The molecular biological diagnosis method can detect the infection of the worm eggs in the symptom period, is quick and sensitive, and is currently applied to the detection of the schistosome. At present, the schistosome detection methods established at home and abroad comprise a real-time PCR detection method, a semi-nested PCR detection method, an LAMP detection method and the like, but the PCR method needs a complicated instrument and a laboratory with fine equipment, the LAMP method has complicated primers and needs to be designed by special software, and amplification products obtained by the LAMP method are fragments with different sizes, cannot be directly cloned and sequenced and can only be used for judging the existence of target genes.
Disclosure of Invention
The invention aims to: the fluorescent RAA primer, the probe and the detection method for detecting the schistosoma japonicum are provided, so that the defects that a PCR method needs complex instruments and a laboratory with fine equipment, and a LAMP method has complex primers, can only be used for judging the existence of a target gene and cannot be directly cloned and sequenced are overcome.
The technical scheme adopted by the invention is as follows:
a primer pair and a probe for detecting schistosoma japonicum based on RAA fluorescence are characterized in that: the nucleotide sequence information of the forward primer of the primer pair is shown as SEQ ID NO.1, 5'-CATTGTGTGAGCAGCCAGGAAGTGACAATC-3', and the nucleotide sequence information of the reverse primer is shown as SEQ ID NO.2, 5'-CTATATTAGAGGCGTGAGGTTATACAGTTA-3'; the probe is an oligonucleotide probe, and the sequence information is shown as SEQ ID No.3, and 5' -CATAGGAGGTCATCTTGTTCAAGGTCAAG/FAM-Dt// THF// BHQ-dT/CACCATCAACTCTTA// phospate/-3 ', wherein FAM-dT represents T base modified carboxyfluorescein, THF represents C base modified tetrahydrofuran group and replaces C base, BHQ-dT represents T base modified black hole quenching group, and phospate represents 3' for phosphorylation termination blocking.
Oligonucleotide probes: 5'-CATAGGAGGTCATCTTGTTCAAGGTCAAG (FAM-dT) (THF) (BHQ-dT) CACCATCAACTCTTA(phosphate) -3';
wherein: FAM: 6-Carboxyfluoroscein; THF: tetrahydrofuran; BHQ is black holequercer; 3' phosphate to block interaction.
The technical scheme of the invention is that a specific primer and a fluorescent RAA probe are designed aiming at a specific conserved region of a schistosome SjG28 gene as a target region.
A kit for detecting schistosoma japonicum based on RAA fluorescence comprises an RAA basic fluorescence universal reaction reagent, a reaction buffer solution, a negative quality control product, a positive quality control product, a primer pair and a probe, wherein the volume ratio of the forward primer to the reverse primer to the probe is 3.5:3.5: 1.
A detection method for detecting schistosoma japonicum based on RAA fluorescence comprises the following steps:
(a) extracting DNA of a sample to be detected;
(b) preparing an RAA reaction system, wherein 50 parts of the total system is calculated according to the parts by volume, the RAA reaction system comprises 25 parts of reaction buffer solution, 2.1 parts of forward primer and reverse primer respectively, 0.6 part of probe and 16.7 parts of double distilled water, 46.5 parts of mixed solution is added into a fluorescence basic reaction unit, 2.5 parts of magnesium acetate is added, and 1 part of sample DNA template to be detected is added;
(c) performing RAA reaction by taking DNA of a sample to be detected as a template through amplification of an RAA reaction system under the constant temperature amplification condition of 39 ℃ for 20 min;
(d) and (4) result judgment standard: if the negative control has no amplification curve and the positive control has an amplification curve, the experimental data is valid, otherwise, the experimental result is regarded as invalid;
and (3) describing and judging results: and judging the sample to be negative if no amplification curve exists, and judging the sample to be positive if an amplification curve exists.
More preferably, (d) a result determination criterion: negative control: FAM channel has no amplification curve and no Tt value; positive control: the FAM channel has an amplification curve, Tt values are less than or equal to 8min, the two requirements need to be met in the same experiment, otherwise, the experiment is invalid and needs to be carried out again.
And (3) describing and judging results: when all FAM channels have amplification curves and the quality control is normal, the positive schistosome nucleic acid can be judged; when the FAM channel has No amplification curve and the Tt value shows Undet or No Tt, and the quality control is normal, the schistosoma nucleic acid can be judged to be negative.
Preferably, the sample to be detected in the step (a) comprises blood, tissue, cells or feces, the DNA is separated for standby after being extracted, and the blood, tissue or cell genome DNA extraction kit and the feces genome DNA extraction kit are frozen at the temperature of-80 ℃ and purchased from Tiangen Biochemical technology Co., Ltd.
Preferably, the concentration of the forward primer, the reverse primer and the probe in step (b) is 10 pmol/uL.
The application of the primer pair and the probe or the kit in detecting the schistosoma japonicum.
In the technical scheme of the application: in the RAA reaction process, schistosome RNA completes the melting of double chains by using recombinase to replace PCR high-temperature denaturation, the solved double chains are combined by single-chain binding protein to prevent DNA chain renaturation, then polymerase completes the chain extension, the target product is synthesized by using DNA as a template, and the reaction is carried out for 20 minutes at 39 ℃. In addition, the RAA technology can also complete the amplification of multiple primers, and a set of RAA multiple fluorescence real-time detection system can be formed by matching with a fluorometer, namely, different target genes are detected in the same reaction by using fluorescent labels with different colors, which cannot be compared with other constant-temperature nucleic acid amplification technologies or nested PCR technologies. Moreover, the RAA technology does not depend on the convenience and the convenience of a PCR instrument, the detection time is obviously shortened compared with a PCR method or fluorescence PCR, the sensitivity is equivalent to or even higher than that of the fluorescence PCR, the method is a revolutionary technical breakthrough, and the application field of the nucleic acid amplification technology can be fully expanded.
In the technical scheme of the application, in a sequence table, the sequence of a forward primer is as follows: 5'-cattgtgtgagcagccaggaagtgacaatc-3' (30 bp); sequence of the reverse primer: 5'-ctatattagaggcgtgaggttatacagtta-3' (30 bp); the unmodified probe sequence was: cataggaggtcatcttgttcaaggtcaagtctcaccatcaactctta (47bp), the modified sequence is the oligonucleotide probe: 5'-CATAGGAGGTCATCTTGTTCAAGGTCAAG (FAM-dT) (THF) (BHQ-dT) CACCATCAACTCTTA(phosphate) -3'.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. according to the invention, the schistosoma japonicum can be rapidly detected by a single tube, the reaction is totally closed, the fluorescence data is monitored in real time, the subsequent processing is not needed, the pollution is avoided, and the reliability of the detection result is ensured;
2. the kit does not depend on expensive instruments such as PCR and the like, can detect even at the normal temperature of 39 ℃, can obtain a diagnosis result within 20 minutes, and greatly shortens the detection time;
3. the detection method can be used for quickly and qualitatively detecting the schistosome and the ovum;
4. the probe-method fluorescent RAA technology has the specific advantages of stronger specificity and higher sensitivity, completely meets the requirements of quick diagnosis and whole-process monitoring of epidemic situations, and strives for time for early diagnosis and early treatment of epidemic situations, reduction of fatality rate and control of epidemic situations.
Drawings
FIG. 1 is a diagram showing the detection results of the lowest detection line of the primers and probes of the present invention;
FIG. 2 is a diagram showing the results of specific detection of the primers and probes of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A primer pair and a probe for detecting schistosoma japonicum based on RAA fluorescence are disclosed, wherein the nucleotide sequence information of a forward primer of the primer pair is shown as SEQ ID NO.1, 5'-CATTGTGTGAGCAGCCAGGAAGTGACAATC-3', and the nucleotide sequence information of a reverse primer is shown as SEQ ID NO.2, 5'-CTATATTAGAGGCGTGAGGTTATACAGTTA-3'; the probe is an oligonucleotide probe, and the sequence information is shown as SEQ ID NO.3, and 5' -CATAGGAGGTCATCTTGTTCAAGGTCAAG/FAM-Dt// THF// BHQ-dT/CACCATCAACTCTTA// phospate/-3 ', wherein FAM-dT represents T base modified carboxyfluorescein, THF represents C base modified tetrahydrofuran group and replaces C base, BHQ-dT represents T base modified black hole quenching group, and phospate represents 3' for phosphorylation termination blocking; FAM: 6-Carboxyfluoroscein; THF: tetrahydrofuran; BHQ is black hole queue; 3' phosphate to blocking.
Example 2
A kit for detecting schistosoma japonicum based on RAA fluorescence comprises an RAA basic fluorescence universal reaction reagent, a reaction buffer solution, a negative quality control product, a positive quality control product, a primer pair and a probe, wherein the volume ratio of the forward primer to the reverse primer to the probe is 3.5:3.5: 1.
Example 3
A detection method for detecting schistosoma japonicum based on RAA fluorescence comprises the following steps:
(a) extracting DNA of a sample to be detected, wherein the sample to be detected is a blood sample, and subpackaging for later use after DNA extraction and freezing and storing at-80 ℃;
(b) preparing an RAA reaction system, counting 50 mu L of the total system according to volume parts, including 25 mu L of reaction buffer solution, 2.1 mu L of forward primer and reverse primer respectively, 0.6 mu L of probe and 16.7 mu L of double distilled water, mixing uniformly, adding 46.5 mu L of the mixed solution into a reaction tube with dry powder, mixing uniformly again, adding 2.5 mu L of magnesium acetate into the reaction tube, mixing uniformly, adding 1 mu L of sample DNA template to be detected, wherein the concentrations of the forward primer, the reverse primer and the probe are all 10 pmol/mu L, and the concentration of the magnesium acetate is 280mM (mmol/L);
(c) placing the reaction tube in an F1620 fluorescent gene detector (Jiangsu Qitian gene biotechnology Co., Ltd.), namely, performing RAA reaction by taking DNA of a sample to be detected, which is extracted by amplification of an RAA reaction system, as a template, and performing constant temperature amplification for 20min at 39 ℃;
(d) and (4) result judgment standard: negative control: FAM channel has no amplification curve and no Tt value; positive control: the FAM channel has an amplification curve, Tt values are less than or equal to 8min, the two requirements need to be met in the same experiment, otherwise, the experiment is invalid and needs to be carried out again;
and (3) describing and judging results: when all FAM channels have amplification curves and the quality control is normal, the positive schistosome nucleic acid can be judged; when the FAM channel has No amplification curve and the Tt value shows Undet or No Tt, and the quality control is normal, the schistosoma nucleic acid can be judged to be negative.
Test example 1
As shown in FIG. 1, the experiment verified that the detection method had the lowest detection limit, which was 1.0 × 107copies/mL、 1.0×106copies/mL、1.0×105copies/mL、1.0×104copies/mL、1.0×103copies/mL、102The positive reference substance such as copies/mL is used as the reference substance with detection limit, and the experiment shows that the detection limit of the detection method reaches 102copies/mL. ▲ line represents the concentration of 1.0 × 107copies/mL positive control, line △ representing concentration 1.0 × 106copies/mL positive control, t-ray at a concentration of 1.0 × 105copies/mL positive control, line ▽ representing concentration 1.0 × 104copies/mL positive control, line ◆ representing concentration 1.0 × 103copies/mL positive control, line ◇ representing concentration 1.0 × 102copies/mL positive control, line ● represents negative control.
In FIG. 1, 107Copy, 106Copy, 105Copy, 104Copy, 103Copy, 102Copy, 102Copies each indicated a concentration of 1.0 × 107copies/mL、1.0×106copies/mL、1.0×105copies/mL、1.0×104copies/mL、1.0×103copies/mL、102The positive quality control substance amplification curve of copies/mL, the negative quality control substance, the abscissa represents the reaction time, and the ordinate mv represents the fluorescence value.
The detection limit of the detection method is 10copies/mL as illustrated in FIG. 1.
Test example 2
As shown in figure 2, the fluorescence reaction detection and RAA basic amplification are respectively carried out on adult schistosoma mansoni, adult ascariasis lumbricoides and ancylostoma duodenale by the method, the fluorescence reaction amplification result shows that only schistosoma japonicum detects a corresponding specific amplification curve, other worms have no corresponding amplification and no cross reaction, the experimental result refers to figure 2, line ▲ represents schistosoma japonicum, line XX represents adult schistosoma mansoni, line ◆ represents ascariasis lumbricoides lumbricus, and line ● represents ancylostoma duodenale.
In FIG. 2, the abscissa represents the reaction time and the ordinate mv represents the fluorescence value. None of the 3 specific references had an amplification curve, indicating that the specificity of the detection method is good.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Sichuan international travel health care center (Chengdu customs port outpatient department)
<120> fluorescent RAA primer, probe and detection method for detecting schistosoma japonicum
<130>AJ2059311
<141>2020-04-10
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
cattgtgtga gcagccagga agtgacaatc 30
<210>2
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
ctatattaga ggcgtgaggt tatacagtta 30
<210>3
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
cataggaggt catcttgttc aaggtcaagt ctcaccatca actctta 47
Claims (6)
1. A primer pair and a probe for detecting schistosoma japonicum based on RAA fluorescence are characterized in that: the nucleotide sequence information of the forward primer of the primer pair is shown as SEQ ID NO.1, 5'-CATTGTGTGAGCAGCCAGGAAGTGACAATC-3', and the nucleotide sequence information of the reverse primer is shown as SEQ ID NO.2, 5'-CTATATTAGAGGCGTGAGGTTATACAGTTA-3'; the probe is an oligonucleotide probe, the sequence information is shown as SEQ ID NO.3, and the sequence information is 5' -CATAGGAGGTCATCTTGTTCAAGGTCAAG/FAM-dT// THF// BHQ-dT/CACCATCAACTCTTA// phosphate/-3', wherein FAM-dT represents T base modified carboxyfluorescein, THF represents C base modified tetrahydrofuran group and replaces C base, BHQ-dT represents T base modified black hole quenching group, and phosphate represents 3' for phosphorylation termination blocking.
2. A kit for detecting schistosoma japonicum based on RAA fluorescence is characterized in that: comprises RAA basic fluorescence universal reaction reagent, reaction buffer solution, negative quality control substances and positive quality control substances, and also comprises the primer pair and the probe of claim 1.
3. A detection method for detecting schistosoma japonicum based on RAA fluorescence is characterized by comprising the following steps:
(a) extracting DNA of a sample to be detected;
(b) preparing an RAA reaction system, wherein 50 parts of the total system is calculated according to the parts by volume, the RAA reaction system comprises 25 parts of reaction buffer solution, 2.1 parts of forward primer and reverse primer respectively, 0.6 part of probe and 16.7 parts of double distilled water, 46.5 parts of mixed solution is added into a fluorescence basic reaction unit, 2.5 parts of magnesium acetate is added, and 1 part of sample DNA template to be detected is added;
(c) performing RAA reaction by taking DNA of a sample to be detected as a template through amplification of an RAA reaction system under the constant temperature amplification condition of 39 ℃ for 20 min;
(d) and (4) result judgment standard: if the negative control has no amplification curve and the positive control has an amplification curve, the experimental data is valid, otherwise, the experimental result is regarded as invalid;
and (3) describing and judging results: and judging the sample to be negative if no amplification curve exists, and judging the sample to be positive if an amplification curve exists.
4. The RAA fluorescence-based assay for schistosoma japonicum according to claim 3, wherein the sample to be tested in step (a) comprises blood, tissue, cell or feces, and the DNA is extracted and stored in a refrigerated storage at-80 ℃.
5. The RAA fluorescence-based assay for schistosoma japonicum according to claim 3, wherein the concentration of the forward primer, the reverse primer and the probe in step (b) is 10 pmol/uL.
6. Use of the primer set and probe of claim 1 or the kit of claim 2 for the detection of schistosoma japonicum.
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| CN111961737A (en) * | 2020-09-23 | 2020-11-20 | 江苏省血吸虫病防治研究所 | Primer group, probe, kit and detection method for detecting Schistosoma japonicum intermediate host oncomelania |
| CN112063726A (en) * | 2020-09-21 | 2020-12-11 | 杭州市疾病预防控制中心 | Primer, probe and detection method for detecting oseltamivir drug resistance mutation |
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Application publication date: 20200623 |