Multiplex digital PCR (polymerase chain reaction) detection method for simultaneously detecting and typing plasmodium and application

Abstract

The invention relates to a multiple digital PCR (polymerase chain reaction) detection method for simultaneously detecting and typing plasmodium and application. The invention discloses a multiplex digital PCR (polymerase chain reaction) method for simultaneously detecting and typing plasmodium and application. According to the digital PCR primer, the probe, the kit and the detection method for detecting and typing the plasmodium, four types of plasmodium can be rapidly, accurately and ultrasensitively detected and typed. Through detection and typing of plasmodium, malaria patients can be accurately and efficiently treated, disease transmission is prevented, and blind medication or wrong medication is avoided. The plasmodium can be accurately detected and typed, the method is very important for treatment strategies of patients, and due to the fact that treatment schemes of each pathogen plasmodium are different, the situation that drugs are used improperly can be effectively reduced through typing information. Detection and prevention of input malaria are key work of malaria prevention and control.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to primers, probes, methods and kits for multiplex digital PCR for simultaneously detecting and typing malarial parasites. Background Art

[0002] There are more than 200 known species of malarial parasites, but there are four main species that can naturally infect humans: Plasmodium falciparum (pf), Plasmodium vivax (pv), Plasmodium malariae (pm), and Plasmodium ovale (po). The severity and clinical symptoms of the disease caused by different species are not uniform. Combination therapy drugs based on artemisinin are usually the most effective method for treating falciparum malaria. Chloroquine is recommended for the treatment of Plasmodium vivax infection. Primaquine is used to prevent recurrence of Plasmodium vivax and Plasmodium ovale infections.

[0003] Comprehensive laboratory testing methods are the first line of defense against malaria recurrence. Commonly used laboratory testing methods for malaria include microscopy, rapid diagnostic tests (RDTs) targeting Plasmodium antigens, and nucleic acid tests based on polymerase chain reaction (PCR). Reading thick and thin blood smears under a microscope is the recognized gold standard for malaria diagnosis. Although this method can save costs, it is highly dependent on experience and time-consuming, and it is easy to miss when the density of Plasmodium is low. Currently, RDT is the most widely used rapid test method. However, due to the limited sensitivity and specificity of immunochromatographic technology and the presence of P. falciparum parasites with Pfhrp2 / 3 gene deletion, the reliability of RDT diagnosis is questionable in low-parasitemia infections with non-P. falciparum species and P. falciparum parasites with Pfhrp2 / 3 gene deletion. Therefore, molecular diagnosis can be used as a more sensitive tool for detecting parasitic malaria infections, especially for low-density, asymptomatic cases. Digital PCR (dPCR) technology has high sensitivity and repeatability, and can perform multiple tests to distinguish between parasite species. It can give an accurate copy number of Plasmodium while typing, providing doctors with more pathogen information that helps with precise treatment. Compared with traditional qPCR detection methods, dPCR can better tolerate PCR inhibitors and provide superior consistency between different clinical laboratories. It can overcome the contamination problem of nested PCR and perform absolute quantification better than qPCR. This detection method is particularly suitable for the current situation of complete and comprehensive elimination of malaria. Digital PCR detection can also be used to dynamically monitor the effectiveness of drug treatment. dPCR detection is a fast, convenient and accurate molecular diagnostic method for laboratory diagnosis, typing and quantification of Plasmodium. Summary of the invention

[0004] The purpose of the present application is to provide multiplex PCR primer pairs, probes, microfluidic cartridges, kits, detection systems and detection methods for specific detection of 4 types of malaria parasites, which can quickly, accurately and ultra-high sensitivity and quantitatively detect and type 4 types of malaria parasites that cause human malaria. The PCR primer pairs, probes, microfluidic cartridges, kits, detection systems and detection methods can be used for any PCR detection system, including but not limited to conventional PCR, RL-PCR, RNA-PCR, fluorescent quantitative PCR, digital PCR, PCR-enzyme-linked immunosorbent assay (PCR-ELISA), nested PCR-high resolution melting analysis (nPCR-HRM), etc., preferably for digital PCR. For example, the purpose of the present application is to provide PCR primer pairs, probes, microfluidic cartridges, kits, detection systems and detection methods for specific detection of 4 types of malaria parasites.

[0005] In order to achieve the above objectives, this application adopts the following technical solutions:

[0006] The present application relates to a PCR primer and probe combination for detecting and typing malarial parasite species, wherein the combination comprises a pair of universal primers for amplifying four malarial parasite target sequences and four probes for typing four malarial parasite specific sequences, four different fluorescent groups are used to label the four probes respectively, and the type of fluorescent group carried by each of the four probes is different, so as to achieve the purpose of simultaneously detecting four target sequences by using four fluorescence in one well of a chip, and typing and quantifying malarial parasites.

[0007] In a specific embodiment, the four primers and probes include the following: a universal primer targeting a specific 18S ribosomal RNA gene sequence, the primer comprising an upstream primer as shown in SEQ ID NO: 1, and a downstream primer as shown in SEQ ID NO: 2; a probe for specifically detecting a Plasmodium falciparum sequence, the probe comprising a probe as shown in SEQ ID NO: 3; a probe for specifically detecting a Plasmodium vivax sequence, the probe comprising a probe as shown in SEQ ID NO: 4; a probe for specifically detecting a Plasmodium ovale sequence, the probe comprising a probe as shown in SEQ ID NO: 5; a probe for specifically detecting a Plasmodium malariae sequence, the probe comprising a probe as shown in SEQ ID NO: 6.

[0008] In a specific embodiment, the target sequence is the 18S ribosomal RNA gene sequence of the four Plasmodium species that cause malaria.

[0009] In a specific embodiment, the four Plasmodium-specific and conserved fragments are one or more Plasmodium fragments selected from the following: Plasmodium falciparum PF-specific fragment, Plasmodium vivax PV-specific fragment, Plasmodium malariae PM-specific fragment, Plasmodium ovale PO-specific fragment, and Plasmodium housekeeping gene conserved fragment.

[0010] In a specific embodiment, the 5' end of the probe sequence is labeled with a fluorescent reporter group, and the 3' end of the probe sequence is labeled with a fluorescent quencher group; when the probes are in the same reaction system, the spectral ranges of different types of fluorescent reporter groups are different.

[0011] In a specific embodiment, the fluorescent group is selected from FAM, HEX, ROX, CY5, CY5.5, JOE, TET, TAMRA, BHQ1, BHQ2, BHQ3, and / or Eclipse.

[0012] The combination of the present application for detecting, typing and quantifying four Plasmodium-specific sequences is a combination used in digital PCR (dPCR) reaction.

[0013] The present application also relates to a digital PCR reaction premix for specifically detecting Plasmodium species, wherein the digital PCR reaction premix comprises the above-mentioned PCR primer and probe combination for detecting and typing Plasmodium species of the present application.

[0014] In a specific embodiment, the digital PCR reaction premix further comprises one, two, three, four, five, six, seven or eight components selected from the following: hot start Taq enzyme, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, NH4 + , and Mg 2+ ion.

[0015] The present application also relates to a digital PCR microfluidic cartridge, wherein the digital PCR microfluidic cartridge comprises the above-mentioned digital PCR reaction premix for specifically detecting Plasmodium species of the present application.

[0016] In a specific embodiment, the microfluidic cartridge comprises at least one hole or at least two holes.

[0017] The present application also relates to a kit for detecting and typing Plasmodium species, wherein the kit comprises a digital PCR reaction premix for specifically detecting Plasmodium species described above.

[0018] In a specific embodiment, the kit further comprises a positive quality control product, a negative quality control product, and / or a droplet generating oil.

[0019] In a specific embodiment, the kit further comprises the digital PCR microfluidic cartridge of the present application.

[0020] The present application also relates to a method for detecting Plasmodium species for non-disease diagnosis purposes, the method comprising the following steps:

[0021] (1) Release nucleic acid from the sample to be tested;

[0022] (2) preparing a digital PCR amplification mixture, which contains the nucleic acid of the sample to be tested provided in step (1), the PCR primer and probe combination for detecting and typing Plasmodium species described in the present application, and / or the digital PCR reaction premix for specifically detecting Plasmodium species described in the present application, and distilled water;

[0023] (3) preparing droplets in a digital PCR microfluidic cartridge and performing a PCR amplification reaction;

[0024] (4) Use a digital PCR microfluidic card reader to read the fluorescence signal after the droplet PCR amplification reaction.

[0025] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features described in detail below can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described in detail.

[0026] The beneficial effect achieved by the present application is to provide a primer probe set for detecting 4 pathogenic malarial parasite 18S ribosomal RNA gene sequences, wherein the 5' end of the probe sequence of the present application is labeled with a fluorescent group of different colors, and the target sequence of each target gene is efficiently amplified by digital PCR, so as to achieve absolute quantification of each target sequence to be detected, and directly give the number of copies per microliter (CFU / microliter) of each target sequence in the reaction system. In addition, the present application also provides a method for detecting malarial parasite species for the purpose of non-disease diagnosis using the primer and probe set of the present application method. For example, four malarial parasites can be detected using the combination of the present application, and the sensitivity and specificity of the method of the present application are 98% and 100%, respectively, which exceeds the prior art; and the minimum detection limit is 0.8cps / reaction, which is much lower than the minimum detection limit of the existing qPCR technology for detecting malarial parasite samples.

[0027] In particular, 1) a digital PCR detection kit is used to directly detect nucleic acids in 200 μL of whole blood; 2) a digital PCR primer probe set with multiple fluorescent labeling technologies is used to achieve single-hole detection and typing of four Plasmodium species; 3) detection targets include but are not limited to the Plasmodium 18S ribosomal RNA gene.

[0028] In one embodiment, the sample to be tested is selected from whole blood, serum, plasma, throat swab, sputum, urine, feces, cerebrospinal fluid, body fluid or ascites.

[0029] In one specific embodiment, the samples involved in the test include 39 whole blood samples collected from patients diagnosed with malaria by microscopic examination at Huashan Hospital in Shanghai. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0031] Figure 1 AD in the figure are the detection results of four different types of Plasmodium using the kit of the present application in Example 1.

[0032] Figure 2 AD in the figure are partial results of testing 39 clinical samples from Huashan Hospital using the primer probe set of the present application in Example 2.

[0033] Figure 3 AD in the figure is the test result of the clinical samples with mixed malaria infection in Example 2.

[0034] Figure 4A-4D Specificity test results. DETAILED DESCRIPTION

[0035] The specific implementation methods of the present application are described in detail below, but it should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples without specifying specific experimental conditions are usually operated under conventional conditions or under conditions recommended by the manufacturer.

[0036] Unless explicitly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising" etc. will be understood to include the stated elements or components but not to exclude other elements or components.

[0037] Digital PCR (dPCR) is a new absolute quantitative PCR technology that mainly involves limiting dilution of PCR reactants, followed by PCR amplification in different reaction chambers, and finally deriving the starting copy number or concentration of the target molecule based on the Poisson distribution principle and the number and ratio of positive droplets. Since there are multiple primers and probes in the multiplex digital PCR reaction system, the requirements for the primer-probe combination in the detection system are very high.

[0038] In one aspect, the present application provides a PCR primer and probe combination for detecting and typing Plasmodium species, wherein the combination includes a pair of universal primers for amplifying four Plasmodium target sequences and four probes for typing four Plasmodium specific sequences, and the four probes are respectively labeled with four different fluorescent groups, and the type of fluorescent group carried by each of the four probes is different, thereby achieving the purpose of simultaneously detecting four specific sequences using four fluorescences in one well of the chip to type and quantify Plasmodium.

[0039] In some embodiments, the target sequence is the 18S ribosomal RNA gene sequence of Plasmodium.

[0040] Preferably, the target sequence includes four Plasmodium 18S ribosomal RNA gene sequences.

[0041] The combination of the present application can effectively and specifically and efficiently detect, type and quantify four pathogenic malarial parasites through the different fluorescent colors of the four-plex probes.

[0042] In one embodiment of the present application, the above primer probe set is used to achieve simultaneous detection of four Plasmodium species in one well using four-color fluorescence by digital PCR, and the detection range covers the four most common Plasmodium species that cause malaria in humans.

[0043] The primers and probes of the present application can be applied to any PCR detection system for detecting target genes, including but not limited to conventional PCR, RL-PCR, RNA-PCR, fluorescent quantitative PCR, digital PCR, PCR-enzyme-linked immunosorbent assay (PCR-ELISA), nested PCR-high resolution melting analysis (nPCR-HRM), etc. It is preferred to use the primers and probes involved in the present application for fluorescent quantitative PCR or digital PCR. It is most preferred to use the primers and probes determined in the present application for digital PCR.

[0044] The digital PCR reaction premix in this application contains primers, probes, enzymes (hot-start Taq enzyme and / or UDG enzyme), dNTPs, glycerol, betaine, NH4 + Mg 2+ , bovine serum albumin, etc.

[0045] Compared with the digital PCR reaction premix, the digital PCR amplification mixture in the present application contains an amplification template (such as a sample template, a positive control or a negative control) and distilled water.

[0046] In some embodiments, the present application provides a digital PCR reaction premix for detecting a target sequence, wherein the PCR reaction premix comprises the above-mentioned primer pair, or a combination of the above-mentioned primer pair and any probe.

[0047] In a specific embodiment, the present application targets the Plasmodium 18S ribosomal RNA gene sequence, designs multiple digital primers and probes that can be used for detection and typing through multiple sequence comparison and analysis, and realizes simultaneous detection of 4-plex digital PCR in one well by different probe colors.

[0048] To improve the efficiency of detection and typing, in one embodiment, the present application provides a method for efficiently detecting and typing Plasmodium using a PCR method, wherein an example of a universal upstream primer for the 18S ribosomal RNA gene is shown in SEQ ID NO: 1 5'-GGGAGTGAAGACGATCAGATAC-3', and an example of a universal downstream primer is shown in SEQ ID NO: 2 5'-GTGCCCTTCCGTCAATTCT-3'.

[0049] In one embodiment, the present application provides a probe that can be used to differentiate Plasmodium falciparum, an example of which is shown in SEQ ID NO: 3: 5'-ACACTTTCATCCAACACCTAGTCGGC-3'; a probe that can be used to differentiate Plasmodium vivax, an example of which is shown in SEQ ID NO: 4: 5'-TGCCGACTAGGCTTTGGATGAAAGA-3'; a probe that can be used to differentiate Plasmodium ovale, an example of which is shown in SEQ ID NO: 5: 5'-CCGTCGTAATCTTAACCATAAACTATGCCG-3'; a probe that can be used to differentiate Plasmodium malariae, an example of which is shown in SEQ ID NO: 6: 5'-CAACACCTAGTCGGCATAGT-3'.

[0050] In some embodiments, the present application provides a combination of primers and probes that can simultaneously detect and type four species of Plasmodium, wherein the combination of primers and probes includes any one or more of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0051] In some embodiments, the 5' end of the probe sequence used in the present application is labeled with a fluorescent group, and the 3' end of the probe sequence used in the present application is labeled with a fluorescent quenching group. When the above probes are located in the same reaction system, the spectral ranges of the fluorescent groups for screening different types of malarial parasites are different. The fluorescent group of the probe sequence used in the present application can be any fluorescent group commonly used in the art, including but not limited to FAM, VIC, ROX, Cy5, Cy5.5 and / or HEX; the fluorescent quenching group of the probe sequence used in the present application can be any fluorescent quenching group commonly used in the art, including but not limited to BHQ1, BHQ2, BHQ3, and / or Eclipse.

[0052] In some embodiments, each probe used in the present application and a fluorescent reporter group labeled at the 5' end and a fluorescent quencher group labeled at the 3' end are as follows: the probe for typing Plasmodium falciparum is shown in SEQ ID NO: 3, and its 5' end is fluorescently labeled with ROX, and its 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-ACACTTTCATCCAACACCTAGTCGGC-BHQ2-3'; the probe for typing Plasmodium vivax is shown in SEQ ID NO: 4, and its 5' end is fluorescently labeled with Cy5, and its 3' end is fluorescently labeled with BHQ2, i.e., 5'-Cy5-TGCCGACTAGGCTTTGGATGAAAGA-BHQ2-3'; the probe for typing Plasmodium ovale is shown in SEQ ID NO: 5, and its 5' end is fluorescently labeled with HEX, and its 3' end is fluorescently labeled with BHQ1, i.e., 5'-HEX-CCGTCGTAATCTTAACCATAAACTATGCCG-BHQ1-3'; the probe for typing Plasmodium malariae is shown in SEQ ID NO: As shown in NO:6, its 5' end is fluorescently labeled with FAM, and its 3' end is fluorescently labeled with BHQ1, that is, 5'-FAM-CAACACCTAGTCGGCATAGT-BHQ1-3'.

[0053] On the other hand, the present application provides a digital PCR reaction premix for specific detection of Plasmodium species, which, in addition to the above primer pairs, or a combination of any of the above primer pairs and probes, may also include any one, two, three, four, five, six, seven or eight components selected from the following: hot start Taq enzyme, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, NH4 + and / or Mg 2+ ion.

[0054] On the other hand, the present application provides a digital PCR microfluidic cartridge, which includes the above-mentioned digital PCR reaction premix for specifically detecting Plasmodium species of the present application.

[0055] On the other hand, the present application provides a kit for detecting and typing Plasmodium species, which includes the primer pairs mentioned above, or a combination of any primer pair and probe mentioned above, or a combination of the primer pair and any probe mentioned above. The kit of the present application may also include any one, two, three, four, five, six, seven or eight components selected from the following: hot start Taq enzyme, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, NH4 + , and Mg 2+ ion.

[0056] The kit of the present application may also include a positive control and / or a negative control.

[0057] In some embodiments, the present application provides a method for detecting Plasmodium species for non-disease diagnosis purposes,

[0058] The method comprises the following steps:

[0059] (1) Processing the sample to be tested;

[0060] The whole blood sample to be tested is 100-1000μl, preferably 100-200μl, 600-1000μl, 700-1000μl, 800-1000μl, 900-1000μl, and most preferably 200μl to improve the detection rate. It is recommended to use a commercial kit to extract DNA from the blood sample to be tested. After DNA extraction, it is recommended to test immediately, otherwise it is best to store it below -20°C for no more than 3 months.

[0061] (2) A digital PCR amplification mixture is prepared according to Table 1, which contains the sample to be detected provided in step (1) and the following digital PCR reaction premix; the final concentrations of the upstream and downstream primers in the digital PCR reaction premix are 50-900 nM, preferably 50-500 nM, 200-500 nM, and most preferably 450 nM; the final concentrations of each probe are 50-800 nM, preferably 50-500 nM, 100-400 nM, and most preferably 250 nM; Mg 2+The final concentration of ions is 0.5-5 mM, preferably 1-4.5 mM, 1.5-4 mM, 2-3.5 mM, and most preferably 3 mM; the final concentration of bovine serum albumin is 0.01-1 wt%, preferably 0.02-0.5 wt%, 0.03-0.1 wt%, 0.04-0.08 wt%, and most preferably 0.05%; the final concentration of hot-start Taq enzyme (which can be antibody-modified hot-start Taq enzyme, chemically modified hot-start Taq enzyme or aptamer-modified hot-start Taq enzyme) is 0.5-10 U / reaction, preferably 1-5 U / reaction, 1.5-4 U / reaction, 2-3.5U / reaction, and optimally 2.5 U / reaction; the final concentration of dNTP is 20-300µM, preferably 50-250µM, 100-230µM, 150-220µM, and most preferably 200µM. The digital PCR reaction premix may also contain UNG enzyme, with a final concentration of 0.1-10 U / reaction, 0.5-5 U / reaction, 1-2.5 U / reaction, 1-2 U / reaction, and most preferably 1.5 U / reaction. The pH value of the digital PCR reaction premix is ​​7-9, preferably pH 7.4-8.5, preferably pH 7.8-8.2, and most preferably pH 8.0.

[0062] Those skilled in the art will appreciate that the substances and concentrations listed in the above PCR amplification mixture are illustrative only, and those skilled in the art can make selections based on the actual system.

[0063] Table 1 Digital PCR amplification mixture

[0064]

[0065] (3) preparing droplets and performing PCR amplification reaction;

[0066] Step 1: Prepare microdroplets. First, add 50-100 μl of droplet generation oil into the square filling hole on the top of the microfluidic cartridge, preferably 70-100 μl, and most preferably 75 μl.

[0067] Step 2: Add 13-25 μl of digital PCR amplification mixture (containing amplified nucleic acid template, primer probe, etc.) into the circular sample well on the top of the microfluidic cartridge, preferably 15-25 μl, most preferably 20 μl;

[0068] Step 3: Cover the sample loading hole and oil filling hole at the top of the microfluidic cartridge and the waste liquid hole at the bottom with two sealing covers respectively; place the microfluidic cartridge containing the PCR amplification mixture on a PCR amplification instrument, preferably an SG-2000 PCR amplification instrument. The amplification reaction can be performed according to the amplification program recommended in Table 2:

[0069] Table 2 PCR amplification program

[0070]

[0071] (4) Signal Collection

[0072] Select DQ mode on the biochip reader (e.g., Dscanner4-1000, registration number: Su Xie Zhu Zhun 20202220818), set FAM, HEX, ROX and Cy5 in the fluorescence channels, take out the high-throughput digital PCR cartridge (e.g., microfluidic cartridge) from the PCR amplifier, preferably the SG-2000 PCR amplifier (registration number: Su Xie Zhu Zhun 20202220887), and transfer it smoothly to the biochip reader for signal collection.

[0073] (5) Results analysis

[0074] Click "Analyze" on the biochip reader to analyze the experimental data. First check that "Accepted Droplets" ≥ 10,000 before proceeding with subsequent analysis. Click "2D Amplitude" to view the 2D scatter plots of channel TAM, channel HEX, channel ROX, and channel Cy5. According to the positive control, click on the 2D graph to divide the positive droplets in each area. In the 2D graph or 1D graph, the darkest black dots represent droplets without amplified templates, blue dots represent droplets positive for Plasmodium malariae, green dots represent droplets positive for Plasmodium ovale, orange dots represent droplets positive for Plasmodium falciparum, and purple represents droplets positive for Plasmodium vivax ( Figure 1 ).

[0075] The primers, probes, digital PCR reaction premixes containing primer probes, kits, and microfluidic cartridges provided in this application can be used to prepare quantitative detection kits for detecting and typing four types of malarial parasites, and can also be used alone or together with other reagents for the detection of four types of malarial parasites in whole blood, wherein the processing steps for whole blood samples are: take 100-1000 μl of the whole blood sample to be tested, preferably 100-1000 μl, 600-1000 μl, 700-1000 μl, 800-1000 μl, 900-1000 μl, and most preferably 200 μl to improve the detection rate. It is recommended to use a commercial kit to extract DNA from whole blood samples. After the nucleic acid extraction is completed, it is recommended to test immediately, otherwise it is best to store it below -20°C for no more than 3 months.

[0076] In some embodiments, the sample types involved in the detection also include serum, plasma, throat swab, sputum, urine, feces, cerebrospinal fluid, body fluid or ascites.

[0077] A whole blood test involves drawing peripheral blood from a patient and examining the various blood cells and plasma components in the whole blood as a whole. In addition, the plasma or serum portion of the blood can also be tested as a sample as needed.

[0078] Throat swab testing is a medical testing method that uses a medical cotton swab to take a small amount of secretions from the human throat, inoculates it in a special culture dish, and then places it in a temperature-controlled device for culture. The throat swab sample in this application can be a sample directly obtained by a technician in this field from the clinic, or it can be a throat swab sample that has been cultured for a period of time, depending on the items that need to be tested.

[0079] Sputum is the secretion of alveoli, bronchi and trachea. Healthy people have very little sputum. Under normal circumstances, bronchial mucosal glands and goblet cells can secrete a small amount of mucus to keep the respiratory mucosa moist. Under pathological conditions, the respiratory mucosa and alveoli are stimulated and the mucosa becomes congested and edematous, serous fluid exudates, and mucus secretion increases. Various cells (red blood cells, white blood cells, macrophages, etc.), exudates such as fibrin and mucus, inhaled dust, certain tissue necrosis, etc. are mixed to form sputum. The sputum samples used in this application are samples that can be usually obtained by those skilled in the art.

[0080] Urine and feces samples are also samples that can be obtained by those skilled in the art through routine means.

[0081] Cerebrospinal fluid (CSF) is a colorless, transparent liquid that fills each ventricle, the subarachnoid space, and the central canal of the spinal cord. Cerebrospinal fluid is produced by the choroid plexus in the ventricles, and has properties similar to plasma and lymph, and is slightly viscous. Cerebrospinal fluid belongs to extracellular fluid. The cerebrospinal fluid sample used in this application can be a sample that is generally available to those skilled in the art.

[0082] In the present application, body fluid refers to various body fluids derived from blood, urine, feces, serous cavity fluid, cerebrospinal fluid, ascites, etc.

[0083] The ascites involved in this application refers to the fluid in the abdominal cavity of the human body, which can be obtained by conventional means.

[0084] For convenience, the sequences of primers and probes used in this application are summarized in the following table:

[0085] Table 3 Primer and probe sequences for the 18S ribosomal RMA gene of four Plasmodium species in this application

[0086]

[0087] Table 4 An example of fluorescent labeling of probes used in this application

[0088]

[0089] Example

[0090] Example 1 Four Plasmodium detection and typing kits

[0091] This application develops a digital PCR quantitative detection kit for detecting and typing four species of malarial parasites. The kit contains a primer and probe mixture, a digital PCR premix (digital PCR buffer, dNTPs, MgCl2, taq hot start enzyme mixture, glycerol, betaine), a negative quality control and a positive quality control.

[0092] Each sample needs to undergo three sets of tests. The primer-probe mixture and digital PCR premix in the kit are used to generate digital PCR droplets, reverse transcribe, and amplify the sample. The volume of the primer-probe mixture is 2 μL, the volume of the digital PCR premix is ​​10 μL, the DNA loading is 1 μL, the enzyme-free water is 7 μL, and the total reaction volume is 20 μL. The reaction system for digital PCR amplification is shown in Table 5:

[0093] Table 5 Multiplex digital PCR reaction system

[0094]

[0095] The components of the kit of the present application are shown in Table 6.

[0096] Table 6 Composition of the kit

[0097]

[0098] The kit of this application is provided with negative quality control products and positive quality control products, which are used to monitor the digital PCR amplification and chip reading of the sample and assist in the interpretation of the final results. The specific steps are as follows:

[0099] 1) Release nucleic acid from the sample to be tested;

[0100] 2) In one well of the microfluidic chip, using the nucleic acid obtained in step 1) as a template, adding a mixture of primers and fluorescently labeled probes and a digital PCR buffer;

[0101] 3) Perform digital PCR droplet generation and PCR amplification in a digital PCR amplifier; add 75 μl of oil-phase encapsulated reagent to the oil wells in the high-throughput digital PCR chip, add 20 μl of amplification mixture to the reagent wells, cover with a sealing cover, and place on a PCR amplifier for droplet generation and PCR amplification.

[0102] Perform reverse transcription and PCR amplification reactions according to the recommended amplification procedures in Table 2:

[0103] 4) Signal Collection

[0104] After PCR amplification, the chip is used as the detection object, and the fluorescence is detected by a biochip reader. The results are interpreted based on the presence and intensity of fluorescence in each droplet.

[0105] Result interpretation:

[0106] The results are shown in Table 7 and Figure 1 As shown, different fluorescent channels detect different types of Plasmodium.

[0107] Table 7 Different malarial parasites corresponding to different fluorescence channels

[0108] Fluorescence channels name Positive judgment criteria FAM Plasmodium malariae ≥3 positive droplets HEX Plasmodium ovale ≥3 positive droplets ROX Plasmodium falciparum ≥3 positive droplets Cy5 Plasmodium vivax ≥3 positive droplets

[0109] Example 2: Clinical blood sample detection by the detection kit

[0110] Referring to the method of Example 1, 39 whole blood samples from Huashan Hospital that had been confirmed by the thin and thick blood smear method were tested. Figure 2 The 1D image shows the test results of some samples. The black droplets are negative droplets, and the orange droplets are positive for Plasmodium falciparum ( Figure 2 A), red droplets are positive for Plasmodium vivax ( Figure 2 B), blue droplets are positive for Plasmodium malariae ( Figure 2 C), the green droplets are positive for Plasmodium ovale ( Figure 2 D). Figure 3 These are the results of digital PCR testing of clinical samples with mixed malaria infection. Figure 3 AB in the figure are the results of samples 31 and 33, indicating that sample 31 is infected with both Plasmodium falciparum and Plasmodium ovale. Figure 3 The CD in the figure is the result of sample 17, which shows that sample 17 is dual-infected with Plasmodium falciparum and Plasmodium malariae.

[0111] Example 3 Specificity Detection

[0112] The present application selects purple bacillus, intermedius streptococcus, bronchiseptica Bordetella, marcescens serratia, sobrinus streptococcus, listeria ei, cepacia Burkholderia, anginosus streptococcus, anaerobic peptostreptococcus, sanguis streptococcus, constellation streptococcus constellation subspecies, Veillonella parvula, agalactiae streptococcus, harmless listeria, banded bacillus, sobria aeromonas, salivarius streptococcus, caviae aeromonas, terrestrial raoulella (terrestrial klebsiella), capitate staphylococcus capitate subspecies, dysgalactiae streptococcus, micrococcus luteus, sonnei shigella, squirrel staphylococcus, listeria monocytogenes, aquatic raenella, salmonella typhimurium, lactobacillus acidophilus, hydrophilic aeromonas Bacteria, Bacillus agglutinans, Legionella pneumophila subspecies, Staphylococcus stutzeri subspecies, Pseudomonas stutzeri, Enterobacter carcinogenes, Sphingomonas paucimobilis, Neisseria lactis, Staphylococcus carnosus, Japanese Gouweiduo bacteria, Streptococcus canis, Enterococcus lead yellow, Proteus mirabilis, Pseudomonas rice, Bacteroides vulgaris, Acinetobacter pitei, Ralstonia pitei, Streptococcus bovis, Eikenella corrodens, Streptococcus pyogenes, Neisseria meningitidis, Enterococcus durable, Staphylococcus xylosus, Moraxella morrhagica, Morganella morganii, Pseudomonas mendocinosae, Enterococcus caecum, Streptococcus equi zooepidemicus subspecies, Rhodococcus equi, Basiliella marneffei, Gemini cocci of measles, Bacteroides ovale, Staphylococcus lugdunensis, Acinetobacter lwoffii, Haemophilus influenzae, Neisseria gonorrhoeae, Shigella dysenteriae, Oleomonas shigellae, Bacillus cereus, Bacillus subtilis, Streptococcus oralis, Acinetobacter radioresistance, Enterobacter cobe, Kingella, Streptococcus gallolyticus, Staphylococcus pseudointermedia, Enterobacter hallii, Streptococcus mitis, Enterococcus hirae, Listeria monocytogenes, Streptococcus gordonii, Neisseria dry, Streptococcus parasanguinis, Clostridium difficile, Bordetella parapertussis, Staphylococcus saprophyticus, Shigella flexneri, Yersinia flexneri, Hafnia alvei, Photobacterium non-symbioticum, Staphylococcus auris, Pseudomonas putida, Bacteroides thetaiotaomicron, Burkholderia polyphaga Specific reactions were performed on 115 strains, including Desmodiolus, Pasteurella multocida, Parabacteroides distanzi, Bacillus licheniformis, Bacteroides monothecae, Listeria monocytogenes, Propionibacterium acnes, Vibrio vulnificus, Pantoea agglomerans, Salmonella enteritidis, Yersinia enterocolitica subsp., Klebsiella oxytoca, Pseudomonas alcaligenes, Streptococcus mutans, Mobinacillus bayleyi, Acinetobacter berrezi, Shigella boydii, Actinobacillus actinomycetemcomitans, Cronobacter sakazakii, Bordetella pertussis, Enterococcus gallinarum, Clostridium perfringens, Staphylococcus warrenii and Haemophilus parainfluenzae. These strains are common pathogens that are easy to cause clinical symptoms in clinical practice. All of the above strains were confirmed to be undetectable. The detection steps are as follows:

[0113] (1) releasing nucleic acid of the strain to be tested;

[0114] (2) mixing the nucleic acids extracted from the above strains;

[0115] (3) Digital PCR droplet formation and amplification;

[0116] Digital PCR droplet formation and digital PCR amplification were performed according to the reaction system and reaction procedure described in Example 1;

[0117] (4) Signal collection:

[0118] The digital PCR chip is taken out from the digital PCR amplification instrument, and is smoothly transferred to the biochip reader for signal collection;

[0119] (5) Result interpretation and analysis:

[0120] Draw a threshold line according to the middle position between the negative droplets and the positive droplets of each strain positive quality control. If the number of positive droplets is greater than the threshold, the strain detection result corresponding to the positive droplet is determined to be positive. The software gives the copy number corresponding to the positive droplet. Draw a linear regression graph with the dilution as the horizontal axis and the measured value log of each dilution as the vertical axis.

[0121] The results of the cross-reaction of the 115 strains are shown in Figure 4. No cross-reaction of the 115 strains with the four Plasmodium primer probes in the kit was detected. Figure 4 only contains negative droplet clusters, and no positive droplet clusters were detected for the cross-reaction of the 115 strains.

[0122] The above are only optional embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A PCR primer and probe combination for detecting and typing a malarial parasite species, wherein the malarial parasite comprises at least one of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, wherein the primer and probe combination comprises: Universal PCR primers targeting the 18S ribosomal RNA gene of Plasmodium, the primers comprising an upstream primer as shown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2; a PCR probe specific for Plasmodium falciparum, the probe comprising the probe as shown in SEQ ID NO:3; a PCR probe specific for Plasmodium vivax, the probe comprising the probe as shown in SEQ ID NO:4; a PCR probe specific for Plasmodium ovale, the probe comprising the probe as shown in SEQ ID NO:5; a PCR probe specific for Plasmodium malariae, the probe comprising the probe as shown in SEQ ID NO:

6.

2. The PCR primer and probe combination according to claim 1, wherein the 5' end of the probe sequence is labeled with a fluorescent reporter group, and the 3' end of the probe sequence is labeled with a fluorescent quencher group; when the probes are in the same reaction system, the spectral range of the fluorescent reporter group for screening different Plasmodium species is different.

3. The PCR primer and probe combination according to claim 2, wherein the fluorescent reporter group is selected from FAM, VIC, ROX, Cy5, Cy5.5, and / or HEX; and the fluorescent quencher group is selected from BHQ1, BHQ2, BHQ3, and / or Eclipse.

4. The PCR primer and probe combination according to claim 3, wherein each probe used and its 5' end labeled fluorescent reporter group and 3' end labeled fluorescent quencher group are as follows: The probe for detecting Plasmodium falciparum is shown in SEQ ID NO:3, and its 5' end is fluorescently labeled with ROX and its 3' end is fluorescently labeled with BHQ2; the probe for detecting Plasmodium vivax is shown in SEQ ID NO:4, and its 5' end is fluorescently labeled with Cy5 and its 3' end is fluorescently labeled with BHQ2; the probe for detecting Plasmodium ovale is shown in SEQ ID NO:5, and its 5' end is fluorescently labeled with HEX and its 3' end is fluorescently labeled with BHQ1; the probe for detecting Plasmodium malariae is shown in SEQ ID NO:6, and its 5' end is fluorescently labeled with FAM and its 3' end is fluorescently labeled with BHQ1.

5. The combination according to any one of claims 1-4, which is a combination used for digital PCR (dPCR) reaction.

6. A digital PCR reaction premix for specifically detecting Plasmodium species, wherein the digital PCR reaction premix comprises the combination according to any one of claims 1-5.

7. The digital PCR reaction premix according to claim 6, wherein the digital PCR reaction premix further comprises one, two, three, four, five, six, seven or eight components selected from the following: hot start Taq enzyme, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, NH4 + , and Mg 2+ ion.

8. A digital PCR microfluidic cartridge, wherein the digital PCR microfluidic cartridge comprises the digital PCR reaction premix according to claim 6 or claim 7.

9. The microfluidic cartridge of claim 8, wherein the microfluidic cartridge comprises at least two wells.

10. A kit for detecting and typing Plasmodium species, wherein the kit comprises the digital PCR reaction premix for specifically detecting Plasmodium species according to claim 6 or claim 7.

11. The kit according to claim 10, wherein the kit further comprises a positive control, a negative control, and / or droplet generation oil.

12. The kit according to claim 11, wherein the kit further comprises a digital PCR microfluidic cartridge according to claim 8.

13. A method for detecting Plasmodium species for purposes other than disease diagnosis, the method comprising the following steps: (1) Release nucleic acid from the sample to be tested; (2) preparing a digital PCR amplification mixture, which contains the nucleic acid of the sample to be detected provided in step (1), the combination according to any one of claims 1 to 5, and / or a digital PCR reaction premix for specific detection of Plasmodium species according to claim 6 or claim 7; (3) preparing droplets in a digital PCR microfluidic cartridge and performing a PCR amplification reaction; (4) Use a digital PCR microfluidic card reader to read the fluorescence signal after the droplet PCR amplification reaction.

14. The method for detecting Plasmodium species for non-disease diagnosis purposes according to claim 13, wherein the sample to be tested is selected from whole blood, serum, plasma, throat swab, sputum, urine, feces, cerebrospinal fluid, body fluid or ascites.

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