CN104673901B - For detecting the purposes and kit of target nucleic acid - Google Patents

Abstract

The present invention relates to uses and kits for detecting target nucleic acids. Specifically, the present invention relates to the use of a combination of at least one capture probe and at least one detection probe in the preparation of a kit for detecting target nucleic acids, wherein the capture probes are complementary bound to the surface of the capture carrier; The capture probe and the detection probe each bind complementary to the target nucleic acid.

Description

Uses and kits for detecting target nucleic acids

technical field

The present invention relates to uses and kits for detecting target nucleic acids. Specifically, the present invention relates to a use and a kit for high-throughput and sensitive detection of Plasmodium (Genus Plasmodium).

Background technique

Malaria is a vector-borne infectious disease caused by Plasmodium. It is clinically characterized by periodic chills, fever, headache, sweating, anemia, and splenomegaly. It is widely prevalent in tropical, subtropical and even temperate regions. The transmission of Plasmodium depends on the intermediate host Anopheles. When a female Anopheles infected with Plasmodium bites a human, the sporozoites in her saliva enter the peripheral blood of the human body along with the saliva, and then enter the liver. The sporozoites develop and proliferate in the liver. When the schizonts mature, the parasitic hepatocytes rupture and release the merozoites into the peripheral blood circulation. Some merozoites are phagocytized by phagocytes, and the other part invades red blood cells and develops and proliferates in them[1]. After invasion, merozoites first form ring bodies, pass through large trophozoites, immature schizonts, and finally form mature schizonts containing a certain number of merozoites, which eventually cause red blood cells to rupture and release merozoites. The newly released merozoites can invade other erythrocytes again, and after the whole replication process is repeated several times, some merozoites no longer proliferate after invading erythrocytes, but develop into female and male gametophytes. Gametocytes can infect Anopheles mosquitoes when they bite the human body, and further develop in their stomachs, and gametocytes cannot develop in humans.

There are five species of Plasmodium that can cause malaria in humans: Plasmodium falciparum (P.falciparum), Plasmodium vivax (P.vivax), Plasmodium ovale (P.ovale), Plasmodium malariae (P.malariae) Plasmodium knowlesi. Among them, malaria caused by Plasmodium falciparum has the highest fatality rate, mostly in Africa. Malaria caused by P. vivax is less lethal than P. falciparum, but it is more widespread and recurrent [2]. The infection rates of the other three Plasmodium species were significantly lower than those of the first two [3].

Globally, in the past 150 years, great progress has been made in the prevention and control of malaria, and more than half of the countries and regions have eradicated malaria. Nearly half of the countries and regions in the remaining malaria-endemic areas have begun to eradicate malaria [4]. As of 2012, there are still 104 countries and regions in the malaria endemic area (of which 5 countries are about to complete the eradication work)[3]. However, in recent years, due to the reduction of malaria control in some areas, the financial crisis, the increase of pathogen drug resistance, and the increase of population mobility, the eradication of malaria is facing more and more difficulties[5]. According to the report of the World Health Organization [3], as of 2011, there are still 3.3 billion people worldwide facing the threat of malaria infection.

In view of the clear transmission route of malaria, clear treatment methods, and mature prevention and control strategies, the World Health Organization believes that all countries and regions should take the complete eradication of malaria as the ultimate goal[3], and recommends that countries and regions that have reached the conditions for malaria eradication vigorously To carry out malaria eradication campaigns, measures should be taken to prevent the influx of new malaria infection sources in areas where malaria has been eradicated.

In my country, the malaria epidemic situation and prevention and control work have undergone great changes in the past few decades. The "2006-2015 National Malaria Prevention and Control Plan" issued by the Ministry of Health of my country in 2006 pointed out that malaria is an important vector-borne infectious disease that seriously endangers the health and life safety of our people and affects social and economic development. Over the past 50 years, my country's malaria prevention and control work has achieved remarkable results: as of the release of the plan, the number of malaria cases has been reduced from more than 20 million in the early 1970s to hundreds of thousands, significantly reducing the scope of severely endemic areas Falciparum malaria has been eliminated in all provinces except Yunnan and Hainan. However, due to the complex epidemic factors of malaria, the characteristics of rapid transmission and easy recurrence, and the weakening of the prevention and control work in some areas in recent years, insufficient funding, and the impact of floating population and epidemic situation in some neighboring countries on my country's border areas, in 2000 Since then, the malaria epidemic in my country has rebounded, and outbreaks have occurred in some areas [6]. As of the release of the plan in 2006, there were 21 provinces, autonomous regions, and municipalities directly under the central government that had malaria transmission. Among them, the malaria epidemic in Yunnan and Hainan was still relatively serious, and the five provinces of Jiangsu, Shandong, Henan, Anhui, and Hubei in the central region were endemic for malaria. It has not been effectively controlled, and many provinces are threatened by imported falciparum malaria, and the situation of prevention and control is still very severe. After the release of the 2006-2015 National Malaria Prevention and Control Plan, the central and local governments at all levels increased their support and investment in malaria prevention and control, effectively curbing the rebound of the epidemic in some areas. By 2010, among the 24 malaria-endemic provinces, autonomous regions, and municipalities directly under the Central Government, the incidence of malaria in 95% of counties (cities, districts) had dropped to less than 1/10,000, and only 87 counties (cities, districts) had malaria incidence More than one ten thousandth [7,8].

According to this latest situation, in order to effectively protect the health of the general public, promote the coordinated development of economy and society, and respond to the global malaria eradication initiative proposed by the United Nations High-Level Meeting on Millennium Development Goals, the Chinese government has decided to comprehensively carry out malaria elimination work in 2010 , to achieve the goal of eliminating malaria nationwide by 2020 [7].

Changes in the global malaria control situation and my country's latest malaria prevention and control strategy have put forward new requirements for the detection method of malaria parasites [9]: in areas with low malaria prevalence or close to eradication, malaria parasite infection is often asymptomatic , and the patient's blood plasmodium content may be very low [10,11], which requires a diagnostic method with extremely high sensitivity. In addition, in order to achieve malaria eradication, the detection strategy of Plasmodium should be changed from passive waiting for patients to hospital diagnosis to active screening of all potential malaria patients in the local area, including potential patients without clinical symptoms. Because asymptomatic malaria patients are a very serious source of malaria infection [12-14]. According to the latest report of the World Health Organization, my country does not yet have the disease surveillance capabilities needed to eradicate malaria [3]. Therefore, in order to better complete the global malaria prevention and control and eradication work, and better meet the needs of the malaria eradication strategy formulated by the Chinese government, in the screening and monitoring of malaria, we urgently need a Characteristic Plasmodium detection method: it has the ability to process large batches of samples to meet the needs of large-scale screening; it has extremely high sensitivity to meet the needs of detecting asymptomatic infected persons and blood sample pooling; it has extremely high specificity and Guide the Center for Disease Control and Prevention to establish relevant models and guide physicians to formulate treatment plans reasonably.

At present, the main malaria diagnosis methods include: relying on symptom diagnosis, microscopic examination, RDT rapid diagnostic test, detection technology based on nucleic acid amplification, etc. details as follows:

Diagnosis by symptoms. In some areas, the clinical diagnosis of malaria is based only on the patient's symptoms. Clinical symptoms of malaria include fever, vomiting, dizziness, headache, chills, lethargy, and difficulty breathing. However, the specificity of these symptoms is not high, and it is easy to be confused with the symptoms of other diseases. Therefore, diagnosis based on symptoms may lead to misdiagnosis of non-malarial patients in malaria-endemic areas, and may lead to misdiagnosis of malaria patients in non-malaria-endemic areas[ 15].

Microscopic examination. Thick and thin blood film staining microscopy is currently the most commonly used method. In this method, thick and thin blood films are made by taking peripheral blood, and the malaria parasites are found by microscopic examination after staining with Ji's or Wright's stain. Giemsa thin section staining steps are roughly: air drying, methanol fixation, Giemsa staining, rinsing and drying, 100x oil lens observation [16]. Because the presence or absence and quantity of parasites can be seen intuitively, microscopy is still recognized by the academic community as the gold standard for clinical malaria diagnosis, and is recommended by the World Health Organization as two methods for diagnosing Plasmodium infection in malaria-endemic areas. One of the methods [3]. However, this method requires more manpower and time, and cannot be used for rapid diagnosis of a large number of samples. Not only that, this method of microscopic inspection has high professional requirements for inspectors, and is easily affected by subjective factors. Different inspectors may also have large differences in the results of the same sample inspection [17]. This leads to large errors in the quantification of Plasmodium by microscopy and thus affects the choice of treatment options. Another big disadvantage of this method is its low sensitivity. The lower limit of detection of malaria samples by professional microscope examiners is about 50,000 Plasmodium per milliliter of blood sample, while the detection limit of molecular biology methods can be 2500 lower than that of microscope inspection. times [18].

RDT rapid diagnostic test. RDT rapid diagnostic test uses rapid diagnostic test strips to detect trace amounts of blood. It is based on immunochromatographic technology and the detection object is the antigen of Plasmodium. According to the different target antigens of RDT diagnostic methods, RDT methods can be divided into four categories[19], these four target antigens are: histidine-rich protein 2 (HRP2), a water-soluble protein, only produced in malignant Plasmodium; Plasmodium lactate dehydrogenase (pLDH), an enzyme present in all Plasmodium parasites; Plasmodium aldolase, an enzyme present in four Plasmodium species (Plasmodium falciparum, Plasmodium vivax Plasmodium, Plasmodium malariae, Plasmodium ovale) enzyme; the fourth antigen is the specific antigen of Plasmodium vivax, the information of this antigen has not been published, but the specificity of this antigen has been proved by experiments in the literature [20]. Compared with other diagnostic methods, the RDT rapid diagnostic test is more accurate, reliable, fast, and economical, and is more suitable for emergencies or situations where there are no professional malaria diagnostic personnel. In addition, this method does not require electricity, microscopes and other equipment, and can play an important role in remote areas. The World Health Organization also recommends RDT Plasmodium detection technology as one of the two methods for confirming Plasmodium infection in malaria-endemic areas [3]. In a survey of malaria prevalence in the border areas between my country and Myanmar, the RDT method showed a sensitivity and specificity comparable to that of microscopy in the diagnosis of malaria [21]. However, the RDT diagnostic method has limited effectiveness in identifying Plasmodium species. Most kits can only distinguish Plasmodium falciparum, and cannot accurately judge other Plasmodium species[22]. In addition, kits targeting histidine-rich protein 2 (HRP2) are prone to false positive results in recovered malaria patients because the antigen persists after treatment [23], Such characteristics greatly limit the application of such kits in evaluating curative effect and prognosis. Kits based on the detection of Plasmodium lactate dehydrogenase (pLDH) may appear negative earlier than microscopic examination in the process of evaluating the therapeutic effect; however, after the disease relapses, such products lag behind the positive time of microscopic examination [ 19].

Detection technology based on nucleic acid target amplification. In order to solve a series of problems in the "gold standard" of malaria diagnosis, a large number of new molecular biology methods have been applied to the detection of malaria parasites. Most of these methods select the conserved region of the 18S ribosomal RNA gene of Plasmodium as the molecular target, and achieve highly sensitive and specific detection of Plasmodium in blood by amplifying the target.

PCR technology is the most common detection technology based on nucleic acid target amplification. For clinical diagnosis, fluorescent quantitative PCR is more suitable than traditional PCR, because it replaces traditional agarose gel electrophoresis with the detection of fluorescent signals, which is not only faster, more accurate, and more sensitive, but also can effectively reduce the gap between amplified products. False positives due to cross-contamination. This method uses a pair of primers to amplify the 18S ribosomal RNA genes of four kinds of Plasmodium (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale) simultaneously, and then cooperates with the genes designed for different species of Plasmodium. The hydrolysis probe can realize rapid and high-sensitivity detection and typing of Plasmodium[24]. The consistency rate between fluorescent quantitative PCR and microscopic examination reached 86%, and the rest of the inconsistent cases were verified by clinical follow-up, other molecular biological methods and nucleic acid sequencing, and the detection results were consistent with fluorescent quantitative PCR [24]. It can be seen that the fluorescent quantitative PCR has higher sensitivity and specificity than the traditional smear microscopy method.

In addition to the fluorescent quantitative PCR method, nested PCR is also widely used. This PCR can detect not only the malaria parasite in the blood, but also the malaria parasite in the patient's saliva or urine. Nested PCR was performed using DNA extracted from patient saliva or urine using specially designed primers targeting the Plasmodium 18S ribosomal RNA gene. The results showed that compared with microscopic examination, the sensitivity of nested PCR test using saliva and urine was 74.1% (P.falciparum), 84% (P.vivax) and 44.4% (P.falciparum), 34.0% (P.vivax), with a specificity of 100% [25]. Given that drawing blood samples is one of the necessary steps for microscopy and most molecular biology methods, and blood drawing is dangerous and cannot be performed under certain conditions (field, infants), this new nested PCR The emergence of the method provides a safer, painless and convenient non-invasive malaria diagnosis method for doctors and patients. In addition to being used for immediate diagnosis, nested PCR can also be used for retrospective diagnosis by detecting the Plasmodium 18S ribosomal RNA gene in serum [26]. Retrospective malaria diagnosis is of great significance for estimating the local malaria prevalence. In retrospective diagnosis, the only samples that can be used are often serum, and the content of Plasmodium DNA in serum is lower than that in whole blood, which is not conducive to the application of conventional fluorescent quantitative PCR. Diagnosis [26]. Nested PCR has a sensitivity of 87% and a specificity of 100% for the detection of Plasmodium DNA extracted from serum, and can successfully detect Plasmodium in serum samples frozen for up to 2.5 years [26].

In addition to PCR, loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) technology is also a very important in vitro nucleic acid amplification technology. The active Bst DNA polymerase and a set of specific primers are used to rapidly amplify the target DNA sequence, and 10 ⁹ to 10 ¹⁰ copies of the target DNA sequence can be synthesized in about 1 hour. Loop-mediated isothermal amplification technology has the characteristics of simplicity, rapidity, high-efficiency amplification and high specificity. In recent years, this technique has been widely used in the rapid molecular detection of various pathogens [27]. In terms of malaria diagnosis, this method has high sensitivity and specificity [28], and is very suitable for the detection of malaria parasites in the field. When microscopic examination was used as the standard, the sensitivity and specificity of loop-mediated isothermal amplification for the detection of falciparum malaria infection were 96.7% and 91.7%, respectively; The detection sensitivity and specificity of infection can reach 98.9% and 100%, respectively [29].

In addition to detection methods that target the 18S ribosomal RNA gene (DNA) of Plasmodium, there are also some nucleic acid amplification detection methods that target the corresponding 18S ribosomal RNA, such as nucleic acid sequence-based amplification (Nucleic acid sequence-based amplification, NASBA). The nucleic acid sequence-dependent amplification method is mainly used for the amplification, detection and sequencing of RNA. The principle of this method is roughly divided into two steps: the first step is to add primers and purified RNA samples at 65°C to open the secondary structure of RNA molecules; the second step is to add reverse transcriptase, T7 RNA polymerase and RNase H at 37°C , and achieve target amplification by constant temperature reaction at 37°C[30]. The advantage of the nucleic acid sequence-dependent amplification method is that it is easy to operate, does not require special instruments, and does not require temperature cycles, and after improvement, the fluorescent quantitative nucleic acid sequence-dependent amplification method can directly give Test results [31], the method is extremely sensitive to the detection of RNA extracted from the whole blood of malaria patients, as long as there is 0.1 Plasmodium in each detection reaction (50 microliters of blood sample), a positive signal can be obtained [31].

The above molecular detection methods all need to purify the nucleic acid from the sample first, and then perform the detection. The extraction and purification of nucleic acid will greatly affect the performance of subsequent detection reactions [24,29,31], and it is also time-consuming and labor-intensive when processing large quantities of clinical samples, which can easily cause cross-contamination between samples. Such problems make most of the detection methods involving nucleic acid extraction and purification unable to meet the requirements of the latest malaria prevention and control in my country and even the world [32]. Although fluorescent quantitative PCR and loop-mediated isothermal amplification methods have been reported in recent reports to omit the nucleic acid extraction step and detect Plasmodium directly from whole blood[29,33], the direct detection of whole blood makes this The sensitivity of the two methods was significantly weakened: when using purified DNA for detection, fluorescent quantitative PCR detected 74 cases of Plasmodium infection, but when the same batch of samples was tested directly using whole blood, the number of positive blood samples dropped to 69 cases, 5 cases of positive samples were misjudged as negative[33]; when the purified DNA was tested, the lower limit of detection of Plasmodium by loop-mediated isothermal amplification technology could reach 1-10 Plasmodium per microliter of blood sample , but when whole blood was used, the lower limit of detection of the method became 40 parasites per microliter. It can be seen that these methods are still difficult to omit the step of extracting sample nucleic acid under the premise of ensuring the quality of detection.

In the absence of a Plasmodium detection method with high-throughput, high-specificity, and high-sensitivity requirements at the same time, malaria prevention and control and eradication efforts have caused at least three obstacles: First, due to the lack of reliable and high-throughput Diagnostic methods, it is difficult for medical institutions to make accurate and timely estimates of the local malaria prevalence, which will lead clinicians in some areas to tend to use antimalarial drugs for patients with negative malaria diagnosis[34]. In the most developed areas, less than 1% of patients treated with antimalarial drugs are actually infected with Plasmodium [35]. What's more serious is that while anti-malarial drugs are abused, some malaria parasite-infected patients fail to receive malaria treatment due to misdiagnosis. The result is a situation where malaria patients are not effectively treated and antimalarial drugs are overused in non-plasmodium-infected patients [36]. Such a situation is very unfavorable for the application of relatively expensive antimalarial drugs, such as artemisinin-based antimalarial drugs. The excellent curative effect makes this kind of drug play an important role in the prevention and control of malaria. However, due to its high cost, if it is abused, it will waste a lot of limited resources in malaria prevention and control work, and it is also easier to induce drug-resistant malaria parasites. [37,38] appears. Second, in the absence of reliable malaria census results, the epidemiological model of the disease can only be estimated based on the situation of patients admitted to medical institutions, which may lead to the failure of malaria eradication and resurgence programs due to estimation bias. Misassessment of associated risks, costs, benefits, and feasibility ultimately affects the implementation of malaria eradication policies [5,39,40]. Third, in the absence of a reliable high-throughput malaria screening method, it is difficult to monitor the large floating population at risk of malaria infection, which is likely to include asymptomatic malaria patients. It can easily lead to re-emergence of malaria in areas where malaria has been successfully eradicated [32].

In summary, it can be seen that the current malaria control and eradication efforts urgently need a high-sensitivity, high-throughput screening method for Plasmodium parasites.

In order to ensure high-throughput, high-specificity, and high-sensitivity detection of the source of the disease, and to control the detection cost within the range acceptable to most regions, we designed a new method.

Contents of the invention

The present inventors have developed a new detection method, which does not require nucleic acid extraction, directly lyses the sample, hybridizes and captures the target DNA, and after the sample is cleaved, at least one capture probe (CP) fixes the target nucleic acid on the capture carrier, such as A 96-well plate modified with a specific probe, at the same time (or subsequently), at least 2 other detection probes (DP) are also combined with the target RNA. The binding site of DP is located between CP, and there is no gap between the binding site of all probes and RNA target. After all probes are bound to the target, the detection probe (DP) is ligated into a single nucleic acid using nucleic acid ligase. see picture 1. The nucleic acid produced by the ligation is then detected by fluorescent quantitative PCR or the like.

In the method of the present invention, a part of the capture probe binds to the target nucleic acid, and the other part binds to the probe immobilized on the plate. The probes immobilized on the plate combine with the capture probes through complementary base pairing.

According to the method of the present invention, wherein the plate is a solid support, the probes are immobilized on the surface of the solid support, and the immobilization methods are well known in the art. The probe plate can be purchased directly from Diacurate (http://www.diacurate.com/), Cat. No. 10009.

The combination of the probes on the immobilized plate and the capture probes depends on the complementary base pairing of DNA, which is reversible under the incubation conditions of this method, and only the hybrid strands that conform to the nucleic acid binding mode of the experimental design can be immobilized (captured) probe-target-detection probe).

In one aspect, the invention provides a method for detecting target nucleic acid in a subject's biological sample, the method comprising the following steps:

a) Provide a sample containing the nucleic acid to be detected;

b) optionally, releasing the nucleic acid from the test sample of step a);

c) incubating the sample obtained in step b) with at least 1 capture probe and at least 1 detection probe under conditions sufficient to hybridize complementary bases, wherein the capture probe is bound to the surface of the capture carrier; the The capture probe and the detection probe are respectively complementary to the adjacent parts of the target nucleic acid;

d) Detecting the presence or absence of one nucleic acid linked by the detection probe.

According to the method of the present invention, wherein the detection probe is one detection probe, the 3' and 5' ends of the detection probe are complementary to the target nucleic acid respectively to form a circular detection probe.

Further, according to the method of the present invention, wherein the detection probes are at least 2 detection probes, the at least 2 detection probes are complementary to consecutive target nucleic acid sites, and connected by ligase to form 1 detection probe Detection probe.

In another aspect, the present invention provides the use of a combination of at least one capture probe and at least one detection probe in the preparation of a kit for detecting a target nucleic acid, wherein the capture probes are complementary bound to the surface of the capture carrier; The capture probe and the detection probe respectively bind complementary to the target nucleic acid.

Preferably, according to the use of the present invention, wherein the detection probe is one detection probe, the 3' and 5' ends of the detection probe are complementary to the target nucleic acid respectively to form a circular detection probe.

Preferably, according to the use of the present invention, wherein the detection probes are at least 2 detection probes, the at least 2 detection probes are complementary to consecutive target nucleic acid sites, and are connected by ligase to form 1 detection probe.

Preferably, according to the use of the present invention, there are 1-10, preferably 2-8, more preferably 2-6, more preferably 2-4, more preferably 2 capture probes. There are 2-10, preferably 2-9, more preferably 2-8, more preferably 2-5, more preferably 3 detection probes.

According to the use of the present invention, one end of the capture probe is complementary to the oligonucleotide sequence covalently bonded to the surface of the capture carrier, and the other end is complementary to the target nucleic acid through a complementary base.

Further, according to the use of the present invention, whether there is a target nucleic acid is determined by detecting whether the detection probe is connected; wherein, the connection of the detection probe refers to the formation of two or more probes under the action of ligase A probe, or a self-circular ligation of a probe.

Further, according to the use of the present invention, wherein the capture carrier is a solid support.

Further, according to the use of the present invention, wherein the capture carrier is a 96-well plate.

Further, according to the use of the present invention, wherein the target nucleic acid is Plasmodium 18S ribosomal RNA.

Further, according to the use of the present invention, wherein the sample used for detection is whole blood or dried blood film.

On the other hand, according to the purposes of the present invention, wherein the Plasmodium is selected from Plasmodium falciparum (Plasmodium falciparum), Plasmodium vivax (Plasmodium vivax), Plasmodium malariae (Plasmodium malariae), Plasmodium ovale (Plasmodium ovale) and Plasmodium knowlesi.

On the other hand, the present invention provides a kind of kit for detecting target nucleic acid, it comprises:

a) at least 1 capture probe and at least 1 detection probe, wherein the capture probe is complementary to the surface of the capture carrier; the capture probe and the detection probe are respectively complementary to the target nucleic acid;

b) Capture vector.

Further, according to the kit of the present invention, wherein the target nucleic acid is Plasmodium 18S ribosomal RNA.

Description of drawings

Figure 1: Schematic representation of the method of the present invention. DP, detection probe; CP, capture probe. After the sample is lysed, the target RNA is immobilized on the fixed surface by two capture probes (CP), and the other three detection probes (DP) also bind to the target RNA. The binding site of the DP is located between the CPs, and there is no gap between the binding site of all 5 probes and the RNA target. After all probes are bound to the target, the 3 detection probes (DP) are ligated into one nucleic acid using nucleic acid ligase.

Figure 2: Using the Plasmodium falciparum samples cultivated in the laboratory, the samples were serially diluted 3 times in healthy human blood, and the concentration of Plasmodium was from 70/μl to 0.0107/μl. All samples were duplicated. All samples consistent with the dissolution curve of the positive control sample were judged as positive, otherwise they were judged as negative, and samples with a Ct value greater than 40 were also judged as negative.

Figures 3A and 3B: Schematic diagrams of the method of the present invention. In Figure 3A, there is one detection probe, the two ends of which bind complementary to the target sequence to form a circle. Fig. 3B, two detection probes, one of which is complementary to the target sequence at both ends to form a circle.

Detailed ways

In the following embodiments, the inventors disclosed the detection of Plasmodium 18sRNA using the newly established detection method of the inventors, and compared it with the standard fluorescent quantitative PCR method.

The principle of detection technology of the present invention

The principle is to design 3 capture probes (captureprobe, CP) near the target site of the Plasmodium 18S ribosomal RNA gene. The 3 capture probes respectively include the sequence Seq ID No1-3. AP complementary paired sequence) and 3 detection probes (detection probe, DP, 3 detection probes respectively include the sequence Seq ID No4-6, in actual use at the 5' end of Seq ID No: 4, Seq ID No: 6 The 3' end of the detection probe is additionally added with the sequence corresponding to the primer used in the detection), wherein the capture probe and the detection probe are complementary to the adjacent parts of the target nucleic acid, and the complementary binding sites of the three detection probes are located at 3 There is no base gap between the complementary binding sites of the capture probes and the binding sites of all six probes to the target nucleic acid. After all probes are bound to the target, the 3 detection probes are ligated into one nucleic acid using nucleic acid ligase. see picture 1. The nucleic acid generated by the ligation is then detected by fluorescent quantitative PCR (see Figure 1 for a schematic diagram of the principle).

In this embodiment, continuous detection probes are used to recognize the breaks between the ligases, and only those that are completely paired can be connected. Subsequent detection only needs to detect the connected detection probes (several detection probes connected into one ). The probes in the prior art do not have this step of specific screening and tend to produce strong background signals, and the specificity is not as good as the new method.

probe used

probe name sequence SEQ ID NO capture sequence TCTAAgAATTTCACCTCTgACATCTg SEQ ID NO: 1 capture sequence gCAgTTgTTCgTCTCCAgAAAA SEQ ID NO: 2 capture sequence TCggCATAgTTTATggTTAAgATTA SEQ ID NO: 3 detection sequence TGGAAGTATTTTAGACAAATGCTTTC SEQ ID NO: 4 detection sequence CCCTTAACTTTCgTTCTTgATTAA SEQ ID NO: 5 detection sequence CGACGGTATCTGATCGTCTTCACT SEQ ID NO: 6

Example:

Experimental procedure

1. Whole blood (20% of the total system), or dried blood slices (no more than 20 3mm diameter/300μl total system), 33% lysis buffer, 1% probe mixture, 2% protease, (purchased from Diacurate company ) fill up # with enzyme-free water. Shake to mix, incubate at 56°C for 30 minutes (shake while incubating, or shake once every 10 minutes).

2. Transfer the above lysate to a 96-well capture plate, 100 μl/well, seal the plate with tin foil, and incubate overnight at 55°C (12-18h).

3. Dilute the washing buffer (1000x, purchased from Diacurate Company) to 1x with enzyme-free water. The above-mentioned 96-well plate was drained, and 150 μl/well of 1x washing buffer was added, repeated three times, and centrifuged at 600 g for 30 seconds after being drained for the last time.

4. Mix #98% ligase buffer and 2% ligase (purchased from Diacurate Company) on ice, transfer to the above-mentioned 96-well plate, 50 μl/well, seal the plate with tin foil, and incubate at 37° C. for 30 minutes. Then blot dry with a pipettor.

5. Mix #50% Mastermix, 4% primers, make up with enzyme-free water, transfer to the above-mentioned 96-well plate, 25 μl/well.

6. Fluorescent quantitative PCR: 95°C for 30s, 45 cycles [95°C for 5s, 60°C for 20s]. Use the default conditions of the instrument for melting curve analysis.

result

Methodology Validation

Plasmodium falciparum samples of different concentrations were obtained by serially diluting the Plasmodium falciparum cultured in the laboratory with a 3-fold concentration in healthy human blood, and tested separately, and the detection limit of this method was 0.01 Plasmodium/μl , the signal has a good linear relationship with the concentration of Plasmodium (R2=0.99645, Figure 2).

Clinical Sample Validation

3,358 clinical dried blood samples were tested and 14 positive samples were found, 4 of which were asymptomatic primary school students. The positive results were verified by the standard fluorescent quantitative PCR method, and the results were consistent (see the table below, only the samples detected as positive by any method are listed. The numbers in the second and third columns are the Ct values of the fluorescent quantitative PCR).

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Claims (10)

1. Use of a combination of at least 1 capture probe and at least 3 detection probes in the preparation of a test kit for detecting target nucleic acids, wherein the capture probes are complementary to the capture carrier surface; the capture probes and The detection probes are respectively complementary to the target nucleic acid, wherein there is no space between the binding sites of the probes and the target nucleic acid, and are ligated by ligase to form one probe. 2. The use according to claim 1, wherein one end of the capture probe is complementary to the oligonucleotide sequence covalently bonded to the surface of the capture carrier, and the other end is complementary to the target nucleic acid through a complementary base. 3. The use according to claim 1 or 2, wherein the presence or absence of the target nucleic acid is determined by detecting whether the detection probe is ligated. 4. The use according to claim 1 or 2, wherein the capture carrier is a solid support. 5. The use according to claim 4, wherein the capture carrier is a 96-well plate. 6. The use according to claim 1 or 2, wherein the target nucleic acid is Plasmodium 18S ribosomal RNA. 7. The use according to claim 6, wherein the sample used for detection is whole blood or dried blood film. 8. The use according to claim 6, wherein the malaria parasite is selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale ) and Plasmodium knowlesi. 9. A test kit for detecting target nucleic acid, comprising: a) At least 1 capture probe and at least 3 detection probes, wherein the capture probes are complementary bound to the surface of the capture carrier; the capture probes and detection probes are complementary to the target nucleic acid respectively, wherein the probes There is no gap between the binding site of the needle and the target nucleic acid, and it is connected by ligase to form a probe; b) Capture carrier. 10. The kit for detecting a target nucleic acid according to claim 9, wherein the target nucleic acid is Plasmodium 18S ribosomal RNA.