A multiple primer set and kit for detecting lung abscess parasites

Abstract

The present invention discloses a multiple primer set and a kit for detecting lung abscess parasites. The kit includes amplification primers and fluorescent probe combinations of 10 parasites, including Toxoplasma gondii, Angiostrongylus cantonensis, Balamuthia baboons, Paragonimus westermani, Taenia solium, Leishmania donovani, Plasmodium falciparum, Leishmania chaga, Angiostrongylus malaysianus, and Trichinella spiralis. The kit of the present application contains UDG enzyme for anti-pollution of PCR amplification products to ensure the accuracy of the test results. The kit of the present application contains an internal reference control, which can show the occurrence of false negatives, and is used to monitor the collection, transportation, extraction and amplification process of the sample to be tested. The kit of the present application has a specificity of 100%, a detection sensitivity of 10 copies / mL, good specificity and high detection efficiency, simple operation steps, and a short detection cycle.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a multiple primer set and a kit thereof for detecting lung abscess parasites. Background Art

[0002] Lung abscess is a serious lung infection. Typical symptoms include persistent cough, coughing up large amounts of purulent and smelly sputum, fever, night sweats, weight loss, etc. If not treated promptly or thoroughly, lung abscess may become chronic, leading to symptoms such as chronic cough, coughing up purulent sputum, and repeated hemoptysis. In addition, lung abscess may also rupture into the chest cavity to form pneumothorax or bronchopleural fistula. Lung abscess not only has a serious impact on the patient's physical health, but may also reduce the patient's quality of life, as symptoms such as persistent cough and repeated infections can affect daily activities.

[0003] Lung abscesses are usually caused by pathogens such as bacteria, fungi or parasites. Accurate pathogen detection is essential for the diagnosis and treatment of lung abscesses. However, traditional detection methods, such as culture and microscopy, have many limitations, such as time-consuming, low sensitivity, and inability to detect multiple pathogens simultaneously. In recent years, molecular biology techniques, especially multiplex PCR technology, have become an important means of pathogen detection due to its high sensitivity, high specificity and ability to detect multiple pathogens simultaneously. Multiplex PCR technology can simultaneously amplify multiple target sequences in a single reaction by designing multiple primers for different pathogen-specific DNA sequences, thereby achieving simultaneous detection of multiple pathogens. Although patented technologies for primer sets and kits for multiplex PCR detection of blood parasites have been available, the prior art has not yet provided a rapid, accurate and easy-to-operate multiplex PCR detection method for the detection of parasites in lung abscesses. In addition, existing multiplex PCR kits may have limitations in primer design when detecting lung abscess parasites, resulting in a limited detection range and failure to cover all clinically relevant parasite species.

[0004] In order to overcome these limitations, the present invention proposes a new multiple primer set and kit specifically for detecting parasites in lung abscesses. This technology improves the sensitivity and specificity of detection by optimizing primer design, and can simultaneously detect multiple lung abscess-related parasites, providing a more effective diagnostic tool for clinical use. Summary of the invention

[0005] The present invention aims to solve the problems of difficulty in comprehensive screening and high missed detection rate in existing parasite detection technologies related to lung abscess, and provides a multiple primer set and a kit for detecting lung abscess parasites. The parasites include Toxoplasma gondii (TG), Angiostrongylus cantonensis (AC), Balamuthia mandrillaris (BM), Paragonimus westermani (PW), Taenia solium (TS), Leishmania donovani (LD), Plasmodium falciparum (PF), Leishmania chagasi (LC), Angiostrongylus malaysiansis (AM), and Trichinella spiralis (TSP).

[0006] Specifically, the technical solution provided by this application is as follows: In a first aspect, the present invention provides a multiplex primer set for detecting 10 types of lung abscess parasites, comprising: The forward primer for detecting Toxoplasma gondii is shown in SEQ ID NO. 1, the reverse primer is shown in SEQ ID NO. 2, and the fluorescent probe is shown in SEQ ID NO. 3; The forward primer for detecting Angiostrongylus cantonensis is shown in SEQ ID NO. 4, the reverse primer is shown in SEQ ID NO. 5, and the fluorescent probe is shown in SEQ ID NO. 6; The forward primer for detecting Balamuthiae baboon is shown in SEQ ID NO.7, the reverse primer is shown in SEQ ID NO.8, and the fluorescent probe is shown in SEQ ID NO.9; The forward primer for detecting Paragonimus westermani is shown in SEQ ID NO. 10, the reverse primer is shown in SEQ ID NO. 11, and the fluorescent probe is shown in SEQ ID NO. 12; The forward primer for detecting Taenia fasciatus is shown in SEQ ID NO. 13, the reverse primer is shown in SEQ ID NO. 14, and the fluorescent probe is shown in SEQ ID NO. 15; The forward primer for detecting Leishmania donovani is shown in SEQ ID NO. 16, the reverse primer is shown in SEQ ID NO. 17, and the fluorescent probe is shown in SEQ ID NO. 18; The forward primer for detecting Plasmodium falciparum is shown in SEQ ID NO. 19, the reverse primer is shown in SEQ ID NO. 20, and the fluorescent probe is shown in SEQ ID NO. 21; The forward primer for detecting Leishmania chagasii is shown in SEQ ID NO. 22, the reverse primer is shown in SEQ ID NO. 23, and the fluorescent probe is shown in SEQ ID NO. 24; The forward primer for detecting Angiostrongylus malaysianus is shown in SEQ ID NO. 25, the reverse primer is shown in SEQ ID NO. 26, and the fluorescent probe is shown in SEQ ID NO. 27; The forward primer for detecting Trichinella spiralis is shown in SEQ ID NO. 28, the reverse primer is shown in SEQ ID NO. 29, and the fluorescent probe is shown in SEQ ID NO. 30.

[0007] Based on the above technical solution, the 5' end of the fluorescent probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0008] Based on the above technical solution, the fluorescent group is selected from any one of Atto 425, FAM, ROX, CY5, CY5.5 and VIC, and the quenching group is any one of BHQ1, BHQ2 and BHQ3.

[0009] In a second aspect, based on the same invention, the present invention also provides a kit for detecting lung abscess parasites, wherein the kit comprises a multiple primer set for detecting 10 types of lung abscess parasites as described in the first aspect.

[0010] Based on the above technical solution, the kit comprises reaction solution A, reaction solution B, enzyme mixture, positive control substance and negative control substance.

[0011] Further, the reaction solution A includes a reaction premix, a Toxoplasma gondii forward primer, a Toxoplasma gondii reverse primer, a Toxoplasma gondii fluorescent probe, a Guangzhou roundworm forward primer, a Guangzhou roundworm reverse primer, a Guangzhou roundworm fluorescent probe, a baboon Balamuthia amoeba forward primer, a baboon Balamuthia amoeba reverse primer, a baboon Balamuthia amoeba fluorescent probe, a westermani forward primer, a westermani reverse primer, a westermani fluorescent probe, a chain tapeworm forward primer, a chain tapeworm reverse primer, a chain tapeworm fluorescent probe, a human ribonuclease forward primer, a human ribonuclease reverse primer, and a human ribonuclease fluorescent probe.

[0012] Further, the reaction solution B includes a reaction premix, a forward primer for Leishmania donovani, a reverse primer for Leishmania donovani, a fluorescent probe for Leishmania donovani, a forward primer for Plasmodium falciparum, a reverse primer for Plasmodium falciparum, a fluorescent probe for Plasmodium falciparum, a forward primer for Leishmania chagasii, a reverse primer for Leishmania chagasii, a fluorescent probe for Leishmania chagasii, a forward primer for Angiostrongylus malaysianus, a reverse primer for Angiostrongylus malaysianus, a fluorescent probe for Angiostrongylus malaysianus, a forward primer for Trichinella spiralis, a reverse primer for Trichinella spiralis, a fluorescent probe for Trichinella spiralis, a forward primer for human ribonuclease, a reverse primer for human ribonuclease, and a fluorescent probe for human ribonuclease.

[0013] Furthermore, the forward primer of the human ribonuclease (RP) is shown in SEQ ID NO. 31, the reverse primer is shown in SEQ ID NO. 32, and the fluorescent probe is shown in SEQ ID NO. 33; Furthermore, the reaction premix includes 1×PCR reaction buffer and RNase free H2O.

[0014] Furthermore, the enzyme mixture includes 1U / μL Taq DNA polymerase and 0.2U / μL UDG enzyme.

[0015] Furthermore, the positive control contains 10 parasites and RP gene sequences; and the negative control is TEBuffer.

[0016] Furthermore, in reaction solution A, the fluorescent groups of the six fluorescent probes, namely, Toxoplasma gondii (TG), Angiostrongylus cantonensis (AC), Balamuthiae baboon (BM), Paragonimus westermani (PW), Taenia solium (TS) and human ribonuclease RNase fragment, are different; in reaction solution B, the fluorescent groups of the six fluorescent probes, namely, Leishmania donovani (LD), Plasmodium falciparum (PF), Leishmania chagasii (LC), Angiostrongylus malaysianus (AM), Trichinella spiralis (TSP) and human ribonuclease RNase fragment, are different; but the fluorescent groups in reaction solution A and reaction solution B can be the same.

[0017] Further preferably, in reaction solution A, the 5' end of the fluorescent probe of TG is labeled with Atto 425 fluorescent group, and the 3' end is labeled with quencher group BHQ1; the 5' end of the fluorescent probe of AC is labeled with FAM fluorescent group, and the 3' end is labeled with quencher group BHQ1; the 5' end of the fluorescent probe of BM is labeled with ROX fluorescent group, and the 3' end is labeled with quencher group BHQ2; the 5' end of the fluorescent probe of PW is labeled with CY5 fluorescent group, and the 3' end is labeled with quencher group BHQ3; the 5' end of the fluorescent probe of TS is labeled with CY5.5 fluorescent group, and the 3' end is labeled with quencher group BHQ3; the 5' end of the fluorescent probe of RP is labeled with VIC fluorescent group, and the 3' end is labeled with quencher group BHQ1; Further preferably, in reaction solution B, the 5' end of the fluorescent probe of LD is labeled with Atto 425 fluorescent group, and the 3' end is labeled with quencher group BHQ1; the 5' end of the fluorescent probe of PF is labeled with FAM fluorescent group, and the 3' end is labeled with quencher group BHQ1; the 5' end of the fluorescent probe of LC is labeled with ROX fluorescent group, and the 3' end is labeled with quencher group BHQ2; the 5' end of the fluorescent probe of AM is labeled with CY5 fluorescent group, and the 3' end is labeled with quencher group BHQ3; the 5' end of the fluorescent probe of TSP is labeled with CY5.5 fluorescent group, and the 3' end is labeled with quencher group BHQ3; the 5' end of the fluorescent probe of RP is labeled with VIC fluorescent group, and the 3' end is labeled with quencher group BHQ1.

[0018] The reaction principle of the kit is as follows: if during the qPCR reaction, if the sample to be tested contains TG, the labeled Atto 425 probe in tube A containing reaction solution A generates a fluorescent signal; if the sample to be tested contains AC, the labeled FAM probe in tube A containing reaction solution A generates a fluorescent signal; if the sample to be tested contains BM, the labeled ROX probe in tube A containing reaction solution A generates a fluorescent signal; if the sample to be tested contains PW, the labeled CY5 probe in tube A containing reaction solution A generates a fluorescent signal; if the sample to be tested contains TS, the labeled CY5.5 probe in tube A containing reaction solution A generates a fluorescent signal; if the sample to be tested contains LD, the labeled Atto 425 probe generates a fluorescent signal; if the sample to be tested contains PF, the labeled FAM probe in tube B containing reaction solution B generates a fluorescent signal; if the sample to be tested contains LC, the labeled ROX probe in tube B containing reaction solution B generates a fluorescent signal; if the sample to be tested contains AM, the labeled CY5 probe in tube B containing reaction solution B generates a fluorescent signal; if the sample to be tested contains TSP, the labeled CY5.5 probe in tube B containing reaction solution B generates a fluorescent signal. In addition, if both tubes A and B contain internal reference genes, the labeled VIC probe should generate a fluorescent signal.

[0019] Furthermore, the specific primers and fluorescent probes of the present invention are shown in Table 1: Table 1: Primer and fluorescent probe sequence list

[0020] The present invention has the following advantages / benefits: The multiple primer set and kit for detecting lung abscess parasites of the present invention can simultaneously and specifically and sensitively detect 10 kinds of parasites. The multiple PCR kit for lung abscess parasites includes amplification primers and fluorescent probe combinations for 10 kinds of parasites. Among them, the amplification reaction of 10 kinds of parasites contains multiple fluorescent probes, primers, enzyme mixtures, PCR reaction buffers, etc. of different pathogen targets, and can detect the presence of Toxoplasma gondii (TG), Angiostrongylus cantonensis (AC), Balamuthiae baboon (BM), Paragonimus westermani (PW), Taenia solium (TS), Leishmania donovani (LD), Plasmodium falciparum (PF), Leishmania chaga (LC), Angiostrongylus malaysianus (AM), Trichinella spiralis (TSP) in the same reaction system; the kit also includes positive controls and negative controls, so as to monitor the accuracy of PCR detection, which is of great significance for the prevention and control of epidemics and public health safety.

[0021] The kit of this application contains UDG enzyme, which is used to prevent contamination of PCR amplification products and ensure the accuracy of the test results. The kit of this application contains an internal reference control, which can show the occurrence of false negatives and is used to monitor the collection, transportation, extraction and amplification process of the sample to be tested. The kit of this application has a specificity of 100%, a detection sensitivity of 10 copies / mL, good specificity and high detection efficiency.

[0022] The sample to be tested may include but is not limited to at least one of alveolar lavage fluid, deep sputum and pharyngeal swab. The multiple primer set and kit for lung abscess parasites of the present invention maintain high specificity while significantly improving the sensitivity of detection, are suitable for rapid detection of parasites, have simple operation steps and short detection cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0024] Figure 1 : TG test result diagram of the present invention; Figure 2 : AC test result diagram of the present invention; Figure 3 : BM detection result diagram of the present invention; Figure 4 : PW detection result diagram of the present invention; Figure 5 : TS detection result diagram of the present invention; Figure 6 : LD detection result diagram of the present invention; Figure 7 : PF test result diagram of the present invention; Figure 8 : LC detection result diagram of the present invention; Fig. 9 : AM detection result diagram of the present invention; Fig.10 : TSP test result diagram of the present invention; Fig.11 : The detection results of the five targets (TG, AC, BM, PW, TS) and RP of the positive control of the present invention; Fig.12 : The detection results of the five targets (LD, PF, LC, AM, TSP) and RP of the positive control of the present invention; Fig.13 : 10 targets and RP detection results of the negative control of the present invention. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0026] Embodiment 1: The kit for detecting 10 types of lung abscess parasites at the same time includes: reaction solution A, reaction solution B, enzyme mixture, positive control and negative control: Reaction solution A includes reaction premix, TG forward primer, TG reverse primer, TG fluorescent probe, AC forward primer, AC reverse primer, AC fluorescent probe, BM forward primer, BM reverse primer, BM fluorescent probe, PW forward primer, PW reverse primer, PW fluorescent probe, TS forward primer, TS reverse primer, and TS fluorescent probe; reaction solution B includes reaction premix, LD forward primer, LD reverse primer, LD fluorescent probe, PF forward primer, PF reverse primer, PF fluorescent probe, LC forward primer, LC reverse primer, LC fluorescent probe, AM forward primer, AM reverse primer, AM fluorescent probe, TSP forward primer, TSP reverse primer, and TSP fluorescent probe; reaction solution A and reaction solution B also include RP forward primer, RP reverse primer, and RP fluorescent probe, respectively.

[0027] The reaction premix includes 1×PCR reaction buffer and RNase free H2O, and the concentration of each primer pair and fluorescent probe is 100-200nmol / L; the primer pair concentration can be set to 200 nmol / L, and the fluorescent probe concentration can be set to 100 nmol / L. The enzyme mixture includes 1U / μL Taq DNA polymerase and 0.2U / μL UDG enzyme; the positive control contains 10 parasites and RNase P gene sequences; the negative control is TE Buffer.

[0028] In the same reaction system, the presence of Toxoplasma gondii (TG), Angiostrongylus cantonensis (AC), Balamuthiae baboon (BM), Paragonimus westermani (PW), Taenia solium (TS), Leishmania donovani (LD), Plasmodium falciparum (PF), Leishmania chaga (LC), Angiostrongylus malaysianus (AM), and Trichinella spiralis (TSP) were detected in two tubes A and B. Tube A was labeled with Atto 425, FAM, ROX, Cy5, and Cy5.5 for TG, AC, BM, PW, and TS, respectively, and tube B was labeled with Atto 425, FAM, ROX, Cy5, and Cy5.5 for LD, PF, LC, AM, and TSP, respectively. Reaction solution A was placed in A, and reaction solution B was placed in B. Both were equipped with internal reference controls, which were RNase P fragments and labeled with VIC. The internal reference control can show the occurrence of false negatives and is used to monitor the collection, transportation, extraction, and amplification processes of the samples to be tested.

[0029] The detection is carried out on a common qPCR detection platform. The steps are as follows: Step S1: Reagent preparation 1) First, take all reagents out of the refrigerator and equilibrate them to room temperature. The standard for PCR reaction solution is: take n×9μL of reaction solution A / B and n×1μL of enzyme mixture and add them to a 1.5 mL centrifuge tube. Vortex the centrifuge tube for a few seconds and centrifuge for a few seconds.

[0030] 2) Aliquoting of PCR reaction solution: Aliquot 10 μL of PCR reaction solution into thin-walled PCR reaction tubes.

[0031] Step S2, nucleic acid extraction: nucleic acid extraction is performed simultaneously on the sample to be tested (the sample to be tested is a plasmid simulation corresponding to each target, as shown in Table 4) and the positive control and negative control. Specifically, the nucleic acid extraction or purification reagent of Qingdao Han Tang Biotechnology Co., Ltd. is used. The operation refers to the instructions for nucleic acid extraction or purification reagent of Qingdao Han Tang Biotechnology Co., Ltd.; Step S3, adding samples: take out the PCR reaction tube containing the PCR reaction solution in step S1, add 40 μL of the nucleic acid extracted in step S2, the positive control substance and the negative control substance nucleic acid, cover the tube tightly, shake evenly and then centrifuge instantly at a speed of 3000 rpm / min for 20 s; Step S4, amplification detection: placing the PCR reaction tube obtained in step S3 into a fluorescent PCR amplification instrument for amplification detection, and the reaction program is set as shown in Table 2; Table 2 Fluorescence amplification instrument reaction program

[0032] Step S5, result determination: determine the sample test result according to the determination criteria in Table 3.

[0033] Table 3 Judgment criteria

[0034] Toxoplasma gondii (TG), Angiostrongylus cantonensis (AC), Balamuthiae baboon (BM), Paragonimus westermani (PW), Taenia solium (TS), Leishmania donovani (LD), Plasmodium falciparum (PF), Leishmania chaga (LC), Angiostrongylus malaysianus (AM), Trichinella spiralis (TSP) and positive and negative controls were tested and analyzed. The positive and negative controls met the requirements, indicating that the experiment was effective. TG, AC, BM, PW, TS, LD, PF, LC, AM and TSP were all detected on the corresponding targets, and no amplification was found in the negative controls, indicating that the detection kit and method have good specificity (the results are shown in Table 4).

[0035] Table 4 Sample test table

[0036] Example 2: Sensitivity detection The self-made standards of 10 targets were 10 6 Serial dilutions to 10 copies / mL 3 copies / mL, 10 2The templates were 10 copies / mL and 10 copies / mL. The negative samples were normal body fluids. The diluted templates and negative samples were amplified using the kit described in Example 1, respectively. The amplification conditions were the same as those in Example 1. The results were determined according to the result determination criteria in Example 1. The sensitivity results of the six target detections are shown in Table 5.

[0037] As can be seen from Table 5, when the target template is 10 copies / mL, the kit described in Example 1 can still use it as a template for amplification. Therefore, in the case of low-concentration templates, the kit described in the present invention can still perform detection and thus has a high sensitivity. 2 The results of pathogen detection in copies / mL are as follows Figures 1 to 9 When the kit of the present invention is used, there is no cross reaction between the primer pairs, and the sensitivity is as high as that of ordinary qPCR.

[0038] Table 5 Detection sensitivity results of 10 parasite nucleic acid sample kits

[0039] Example 3: Specific detection Other common parasites including Giardia lamblia, Acanthamoeba castellani, Entamoeba histolytica, Naegleria fowleri, Cryptosporidium parvum, Plasmodium vivax, Plasmodium malariae, Brugia malayi, and Bancrofti were selected as simulated interference samples, with a specific homemade concentration of 10 6 The specific detection was performed with the test process as shown in Example 1. The results showed that except for the positive control, the results of other samples were negative, proving that the present invention had no cross reaction with other pathogen nucleic acids in this example. The test results are shown in Table 6: Table 6: Sample test table

[0040] Example 4: Performance comparison of kits in different comparative examples This experiment provides kits of Comparative Example 1 and Comparative Example 2. The difference between these two kits and the kit of Example 1 is that different primer-probe compositions are used, as shown in Table 7. The fluorescent probes on each primer probe of each control group refer to Example 1 for comparative analysis, and the preparation method of the kit is the same as that of the aforementioned example.

[0041] The positive control substance in Example 1 was tested using the test kit prepared in Example 1 of the present application, and the test kits prepared in Control Group 1 and Control Group 2, respectively, to compare the experimental effects of each test kit.

[0042] Table 7 Primer probes for the control group

[0043] The experimental results show that compared with Example 1, the problems of Comparative Example 1 are: in Comparative Example 1, Atto 425 channel TG and LD cannot be detected at 10 copies / mL; FAM channel AC cannot be detected at 10 copies / mL, and PF has a negative false positive; ROX channel BM cannot be detected at 10 copies / mL, and LC has a negative false positive; CY5 channel PW and AM cannot be detected at 10 copies / mL; CY5.5 channel TS has a negative false positive, and TSP cannot be detected at 10 copies / mL.

[0044] Compared with Example 1, the problems of Comparative Example 2 are: in Comparative Example 1, Atto 425 channels TG and LD cannot detect 10 copies / mL; FAM channel AC has negative false positives, and PF cannot detect 10 copies / mL; ROX channels BM and LC cannot detect 10 copies / mL; CY5 channel PW cannot detect 10 copies / mL, and AM has negative false positives; CY5.5 channel TS cannot detect 10 copies / mL, and TSP has negative false positives.

[0045] In summary, the sensitivity and specificity of the multiple primer set and the detection kit thereof for detecting 10 types of lung abscess parasites provided by the present invention have significant effects.

[0046] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A multiplex primer set for detecting 10 types of lung abscess parasites, characterized in that: include: The forward primer for detecting Toxoplasma gondii is shown in SEQ ID NO. 1, the reverse primer is shown in SEQ ID NO. 2, and the fluorescent probe is shown in SEQ ID NO. 3; The forward primer for detecting Angiostrongylus cantonensis is shown in SEQ ID NO. 4, the reverse primer is shown in SEQ ID NO. 5, and the fluorescent probe is shown in SEQ ID NO. 6; The forward primer for detecting Balamuthiae baboon is shown in SEQ ID NO.7, the reverse primer is shown in SEQ ID NO.8, and the fluorescent probe is shown in SEQ ID NO.9; The forward primer for detecting Paragonimus westermani is shown in SEQ ID NO. 10, the reverse primer is shown in SEQ ID NO. 11, and the fluorescent probe is shown in SEQ ID NO. 12; The forward primer for detecting Taenia fasciatus is shown in SEQ ID NO. 13, the reverse primer is shown in SEQ ID NO. 14, and the fluorescent probe is shown in SEQ ID NO. 15; The forward primer for detecting Leishmania donovani is shown in SEQ ID NO. 16, the reverse primer is shown in SEQ ID NO. 17, and the fluorescent probe is shown in SEQ ID NO. 18; The forward primer for detecting Plasmodium falciparum is shown in SEQ ID NO. 19, the reverse primer is shown in SEQ ID NO. 20, and the fluorescent probe is shown in SEQ ID NO. 21; The forward primer for detecting Leishmania chagasii is shown in SEQ ID NO. 22, the reverse primer is shown in SEQ ID NO. 23, and the fluorescent probe is shown in SEQ ID NO. 24; The forward primer for detecting Angiostrongylus malaysianus is shown in SEQ ID NO. 25, the reverse primer is shown in SEQ ID NO. 26, and the fluorescent probe is shown in SEQ ID NO. 27; The forward primer for detecting Trichinella spiralis is shown in SEQ ID NO. 28, the reverse primer is shown in SEQ ID NO. 29, and the fluorescent probe is shown in SEQ ID NO.

30.

2. A multiplex primer set for detecting 10 types of lung abscess parasites according to claim 1, characterized in that: The 5' end of the fluorescent probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

3. A multiplex primer set for detecting 10 types of lung abscess parasites according to claim 2, characterized in that: The fluorescent group is selected from any one of Atto 425, FAM, ROX, CY5, CY5.5 and VIC, and the quenching group is any one of BHQ1, BHQ2 and BHQ3.

4. A kit for detecting lung abscess parasites, characterized in that: The kit comprises the multiple primer set for detecting 10 types of lung abscess parasites as described in claim 1.

5. A kit for detecting lung abscess parasites according to claim 4, characterized in that: The kit comprises a reaction solution A, a reaction solution B, an enzyme mixture, a positive control substance and a negative control substance.

6. A kit for detecting lung abscess parasites according to claim 5, characterized in that: The reaction solution A includes a reaction premix, a forward primer for Toxoplasma gondii, a reverse primer for Toxoplasma gondii, a fluorescent probe for Toxoplasma gondii, a forward primer for Angiostrongylus cantonensis, a reverse primer for Angiostrongylus cantonensis, a fluorescent probe for Angiostrongylus cantonensis, a forward primer for Balamuthiae baboon, a reverse primer for Balamuthiae baboon, a fluorescent probe for Balamuthiae baboon, a forward primer for Paragonimus westermani, a reverse primer for Paragonimus westermani, a fluorescent probe for Paragonimus westermani, a forward primer for Taenia chain, a reverse primer for Taenia chain, a fluorescent probe for Taenia chain, a forward primer for human ribonuclease, a reverse primer for human ribonuclease, and a fluorescent probe for human ribonuclease.

7. A kit for detecting lung abscess parasites according to claim 5, characterized in that: The reaction solution B includes a reaction premix, a forward primer for Leishmania donovani, a reverse primer for Leishmania donovani, a fluorescent probe for Leishmania donovani, a forward primer for Plasmodium falciparum, a reverse primer for Plasmodium falciparum, a fluorescent probe for Plasmodium falciparum, a forward primer for Leishmania chagasii, a reverse primer for Leishmania chagasii, a fluorescent probe for Leishmania chagasii, a forward primer for Angiostrongylus malaysianus, a reverse primer for Angiostrongylus malaysianus, a fluorescent probe for Angiostrongylus malaysianus, a forward primer for Trichinella spiralis, a reverse primer for Trichinella spiralis, a fluorescent probe for Trichinella spiralis, a forward primer for human ribonuclease, a reverse primer for human ribonuclease, and a fluorescent probe for human ribonuclease.

8. A kit for detecting lung abscess parasites according to any one of claims 6 and 7, characterized in that: The reaction premix includes 1×PCR reaction buffer and RNase free H2O.

9. A kit for detecting lung abscess parasites according to claim 5, characterized in that: The enzyme mixture includes 1U / μL Taq DNA polymerase and 0.2U / μL UDG enzyme.

10. A kit for detecting lung abscess parasites according to claim 5, characterized in that: The positive control substance contains 10 parasites and RP gene sequences; the negative control substance is TE Buffer.

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