Primer group, kit and method for detecting five food-borne pathogenic parasites
By designing specific primer sets and multiple PCR targeted second-generation sequencing methods, the problems of insufficient specificity, low sensitivity and long cycle of foodborne pathogenic parasite detection in the prior art are solved, and rapid and accurate detection of multiple parasites is achieved, with the detection time shortened to 12 hours and the sensitivity and specificity are improved.
Patent Information
- Application Number
- CN202510546444.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-08-19
AI Technical Summary
There is a lack of an efficient, specific and rapid detection method that can simultaneously, quickly and accurately detect foodborne pathogenic parasites such as Guangzhou Tube Circumac, Cryptosporidium, Giardia lancea, Toxoplasma gondii and Pork Taenia simultaneously, quickly and accurately, and there are problems such as insufficient detection specificity, low sensitivity, long detection cycle and high cost.
A primer set was designed, including 10 pairs of primers used to specifically detect the above-mentioned parasites. Each parasite corresponds to 2 pairs of specific primers. The second-generation sequencing method is targeted by multiple PCR, combining specific primer design and optimizing primer combinations to avoid inter-primer interference, and high-throughput detection is performed in combination with second-generation sequencing technology.
The rapid and accurate detection of five foodborne pathogenic parasites was achieved, and the detection time was shortened to 12 hours, and the sensitivity and specificity were improved, which avoided the problem of primer dimers in multiple PCR reactions, and the accuracy of the detection results reached 100%.
Smart Images
Figure CN120505429A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbial detection, and particularly relates to a primer set, a kit and a method for detecting five foodborne pathogenic parasites. Background Art
[0002] Foodborne parasitic diseases are one of the world's most pressing public health issues. They are diseases contracted through ingestion of food or water contaminated with parasites, their eggs, or larvae. In recent years, the infection rate of foodborne parasitic diseases in my country has been rising, and the number of infected people and regions has also expanded. In particular, the number of infected residents in urban areas of economically developed coastal regions has been on the rise. Furthermore, foodborne parasitic diseases in my country are widely distributed and pose serious risks to humans and animals.
[0003] Foodborne parasites mainly include protozoa, nematodes, trematodes, tapeworms, and sporozoans. Traditional detection techniques such as staining smear examination, floating egg collection method, sedimentation egg collection method, McFadden count method, and imaging are complex to operate, often face multiple tests, are time-consuming, and have low positive rates. They cannot meet the needs of rapid food safety testing and are not conducive to the handling of events such as foodborne parasitic infections. In recent years, a series of pathogen nucleic acid molecular detection technologies have been developed, including PCR, metagenomic sequencing, and multiplex PCR targeted second-generation sequencing. PCR has good specificity and sensitivity, but it can only detect one pathogen at a time and has the disadvantage of low throughput. Although metagenomic sequencing technology can detect a wide range of pathogens, it has a long cycle (24 hours), high cost, and is subject to host DNA interference. Multiplex PCR (polymerase chain reaction) targeted second-generation sequencing combines ultra-multiplex PCR targeted amplification technology with high-throughput sequencing technology to achieve accurate identification of targets. It has higher sensitivity and specificity, is low-priced, highly efficient (12 hours), and is not afraid of host DNA interference. It reduces sample usage, making it very suitable for pathogen detection in specific scenarios (such as the human respiratory tract and food safety).
[0004] Currently, infections caused by foodborne parasites such as Angiostrongylus cantonensis, Cryptosporidium, Giardia lamblia, Toxoplasma gondii, and Taenia solium are frequent. However, existing technologies lack efficient, specific, and rapid detection methods that can simultaneously detect all five of these foodborne parasites. Multiplex PCR-based targeted next-generation sequencing detection methods could provide improved technical support for infection diagnosis and treatment, as well as food safety testing. Summary of the Invention
[0005] The purpose of the present invention is to provide a primer set for detecting five foodborne pathogenic parasites, so as to solve the technical problems of insufficient specificity, low sensitivity, long detection cycle and high cost of the existing methods for detecting foodborne pathogenic parasites. The primer set can simultaneously, quickly and accurately detect five foodborne pathogenic parasites, namely, Angiostrongylus cantonensis, Cryptosporidium, Giardia lamblia, Toxoplasma gondii and Taenia solium.
[0006] The technical solution adopted by the present invention to solve its technical problem is:
[0007] A primer set for detecting five foodborne pathogenic parasites, comprising 10 pairs of primers for specifically detecting Angiostrongylus cantonensis, Cryptosporidium, Giardia lamblia, Toxoplasma gondii, and Taenia solium, with two pairs of specific primers corresponding to each pathogenic parasite; wherein,
[0008] The primers used for specific detection of Angiostrongylus cantonensis are the nucleotide sequences shown in SEQ ID Nos. 1 to 4, the primers used for specific detection of Cryptosporidium are the nucleotide sequences shown in SEQ ID Nos. 5 to 8, the primers used for specific detection of Giardia lamblia are the nucleotide sequences shown in SEQ ID Nos. 9 to 12, the primers used for specific detection of Toxoplasma gondii are the nucleotide sequences shown in SEQ ID Nos. 13 to 16, and the primers used for specific detection of Taenia solium are the nucleotide sequences shown in SEQ ID Nos. 17 to 20.
[0009] The primer sets were designed using the following methods: (1) constructing a database containing the genome sequences of five foodborne parasites; (2) using the Primer3 and blast methods to predict and screen the initial primer sequences with the highest inclusiveness and best species specificity based on the constructed database; (3) using MFEprimer to predict the possibility of dimer formation between primers, and optimizing the primer combinations through dynamic screening to ensure that each primer pair does not form more than three pairs of dimers; and (4) selecting two optimal primer pairs for each parasite. This design method ensures high primer specificity and stability and avoids the problem of primer interference in multiplex PCR reactions.
[0010] A kit for detecting five foodborne pathogenic parasites, comprising the primer set of the present invention, a PCR reaction mix, and library construction primers.
[0011] Preferably, the kit further comprises a genomic DNA extraction reagent, a nucleic acid amplification reagent, a fluorescent labeling reagent and / or a purification reagent for the sample to be tested.
[0012] A method for detecting five foodborne pathogenic parasites, comprising the following steps:
[0013] S1. Extract the total nucleic acid sequence of the sample to be tested;
[0014] S2. performing multiplex PCR amplification using a primer set specifically detecting Angiostrongylus cantonensis, Cryptosporidium parvum, Giardia lamblia, Toxoplasma gondii, and Taenia solium, and purifying the product; the primer set is the primer set for detecting five foodborne pathogenic parasites described in the present invention;
[0015] S3. Construct a library using primers containing sample splitting index sequences and sequencing adapter sequences; S4. Perform high-throughput sequencing on the constructed library using next-generation sequencing technology;
[0016] S5. Perform bioinformatics analysis on the sequencing data, including quality control, microbial annotation, and result determination, to detect the presence of the five foodborne pathogenic parasites in the samples.
[0017] Preferably, the multiplex PCR amplification reaction system comprises: nucleic acid extraction products from the sample to be tested, a pool of parasite detection primers, a PCR reaction mix, and nuclease-free water. This formulation ensures high efficiency and specificity of the multiplex PCR reaction, helping to improve detection sensitivity.
[0018] Preferably, the parasite detection primer pool is prepared by adding Rd1 SP: 5'-CCTACACGACGCTCTTCCGATCT-3' (SEQ ID No. 21) to the 5' end of the F primer and Rd2 SP: 5'-TTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID No. 22) to the 5' end of the R primer in the primer set for detecting five foodborne parasites described herein, and then synthesizing the resulting primer pool. More preferably, the synthesized primers are mixed at a concentration of 10 μM in a volume of 1 μL.
[0019] Preferably, the library construction reaction system includes: targeted enrichment reaction products, library construction primers, and amplification mix. This combination optimizes the library construction process and improves the quality and reliability of subsequent sequencing.
[0020] Preferably, the bioinformatics analysis comprises the following steps:
[0021] (1) Use fastp software to perform quality control on the raw sequencing data;
[0022] (2) Use blast software to align the quality control data with the targeted amplicon microbial library to obtain the microbial annotation results for each read;
[0023] (3) Microbial identification analysis: ① The alignment results were divided into species units, and the total number of annotated reads was counted; ② The data were normalized based on the sequencing data volume of 1Mb read to obtain RPM (Reads per million mapped reads). The specific calculation formula is: RPM = number of species annotated reads * 10^6 / total number of reads of the sample; ③ Only results with RPM ≥ 40 or 40 > RPM ≥ 10 but both primer pairs detected ≥ 3 reads of the species were retained;
[0024] (4) Sort the microbial annotation results from high to low according to the RPM number and interpret the results.
[0025] Preferably, the sample to be tested is blood, cerebrospinal fluid, alveolar lavage fluid, sputum, tissue fluid or food sample. This wide range of sample applicability enables the method to be applied in various scenarios such as clinical diagnosis and food safety testing.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] 1. The present invention successfully established a technical method for detecting five foodborne pathogenic parasites (including Angiostrongylus cantonensis, Cryptosporidium, Giardia lamblia, Toxoplasma gondii and Taenia solium) using a multiplex PCR targeted second-generation sequencing method. In the reaction system prepared by the present invention, the designed specific primers can specifically and sensitively bind to the genetic material (nucleic acid sequence) of the corresponding parasites, and the detection is stable.
[0028] 2. The present invention has established a targeted sequencing detection method with good specificity, high sensitivity, high timeliness (12 hours) and high accuracy (100%). Compared with the detection cycle of the metagenomic sequencing method (24 hours), the method of the present invention greatly shortens the detection time and has higher sensitivity.
[0029] 3. The present invention has designed highly specific primer sets for five important foodborne pathogenic parasites, with two pairs of primers for each parasite. Through a carefully optimized primer pool combination strategy, the primer dimer problem in the multiplex PCR reaction is effectively avoided, thereby improving the stability and reliability of the detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 The present invention provides a process flow chart for the method for detecting five foodborne pathogenic parasites. DETAILED DESCRIPTION
[0031] The technical solution of the present invention is further described in detail below through specific examples. It should be understood that the implementation of the present invention is not limited to the following examples, and any form of modification and / or change made to the present invention will fall within the scope of protection of the present invention.
[0032] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified.
[0033] Unless otherwise specified, the reagents used in the following examples can be purchased from conventional biochemical reagent stores.
[0034] Example 1: Primer Set and Kit for Detecting Five Foodborne Pathogenic Parasites
[0035] 1. Primer set design
[0036] The five foodborne parasites include Angiostrongylus cantonensis, Cryptosporidium, Giardia lamblia, Toxoplasma gondii, and Taenia solium. The specific steps are as follows:
[0037] (1) Genome database construction: The reference genome sequences contained in this database are used to construct specific primers for five foodborne pathogenic parasites. They were mainly obtained from multiple authoritative databases such as the NCBI NT library (ftp: / / ftp.ncbi.nlm.nih.gov / blast / db / FASTA / nt.gz), the GenBank library (ftp: / / ftp.ncbi.nlm.nih.gov / genomes), and the Parasite Reference Sequence Library (https: / / eupathdb.org / eupathdb). After removing the genome sequences with low credibility, the genome library (unit) was obtained, and the strains recorded as representative genome in the refseq_category column of the GenBank library were selected as the representative genome library (item) of each species.
[0038] (2) Specific primer design: a. Input the item library; b. Use primer3 v2.6.1 software, set the parameters: primer length: 18-25 nt (optimal 22 nt), primer TM value: 58℃-62℃ (optimal 60℃), primer GC content: 40%-60% (optimal 50%), primer length: 200-400 bp, and unbiasedly predict 1000 pairs of initial primer sets that meet the requirements; c. Use search v11 to align the initial primer set to the unit library, calculate the primer inclusiveness (parameters: -maxdiffs 6 -minamp50 -maxamp 1200), and retain primers with an inclusiveness higher than 60%; d. Use blast v2.16.0 to align the primer set to the NCBI NT library, analyze the nonspecific amplification of each primer pair, calculate the specificity, and retain primer sets with specificity greater than or equal to 95%; e. Finally, output the top five primer pairs with the highest inclusiveness and best specificity from the remaining primer sets; f. Obtain the initial primer pool, a total of 25 primer pairs;
[0039] (3) Primer pool combination optimization: ① Use MFEprimer v3.2.6 to predict the possibility of dimer formation for all primer pools, and only retain dimers with a dimer score (Score) greater than or equal to 10; ② Calculate the network relationship of all primer-dimer pairings and sort them from high to low according to the number of dimers formed; ③ Remove the primer with the highest number of dimers from the primer pool, and recalculate the network relationship until each primer pair does not form more than 3 pairs of dimers, completing the dynamic screening; ④ Finally, only 2 primer pairs are retained for each species, and the primer combinations correspond to the numbers 1 to 10 in Table 1.
[0040] Table 1. Example 1 - Primer information
[0041]
[0042]
[0043] 2. Preparation of reaction system
[0044] 1. The 5' end of the F primer in the screened parasite-specific primer set (Table 1) was added with Rd1 SP (Read1 Sequencing Primer, sequencing primer binding sequence): 5'-CCTACACGACGCTCTTCCGATCT-3' (SEQ ID No. 21), and the 5' end of the R primer was added with Rd2 SP (Read2 Sequencing Primer): 5'-TTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID No. 22), and then synthesized and mixed according to the concentrations and volumes shown in Table 1 to obtain a parasite detection primer pool.
[0045] 2. Prepare the reaction system according to Table 2 and Table 3.
[0046] Preparation of targeted enrichment reaction system: Mix the parasite detection primer pool, PCR reaction mix, and the nucleic acid of the sample to be tested, and add nuclease-free water to make up the total volume to 20 μL to prepare a multiplex parasite targeted amplification reaction system.
[0047] Preparation of library construction system: The library enrichment PCR reaction mix (VAHTS HiFi Amplification Mix, Novozymes), library construction primers (containing index sequences for sample splitting and sequencing adapter sequences for sequencing, UDB PCR Primer Mix, MGI), and target enrichment reaction products were mixed to prepare the library construction reaction system.
[0048] Table 2. Example 1 - Target region enrichment reaction system
[0049]
[0050] Table 3. Example 1 - Library construction reaction system
[0051]
[0052]
[0053] 3. Testing
[0054] 1. Sample experimental processing:
[0055] Nucleic acid extraction was performed using conventional methods in the art. The sample could be blood, cerebrospinal fluid, alveolar lavage fluid, sputum, tissue fluid, etc. This example uses the VAMNE Magnetic Pathogen DNA Kit (Novozymes), taking alveolar lavage fluid as an example:
[0056] (1) Instruments and materials: UltraClean Pathogen Multiplex PCR Mix for DNA & RNA (Novagen); VAHTS DNA Clean Beads (Novagen); VAHTS HiFi Amplification Mix (Novagen); PCR instrument; BGIseq E25 sequencer (MGI).
[0057] (2) DNA extraction from samples: DNA was extracted from bronchoalveolar lavage fluid samples according to the instructions of the VAMNE Magnetic Pathogen DNA Kit, and the extracted DNA was frozen in a -20°C medical refrigerator.
[0058] (3) Add the extracted DNA product to the targeted enrichment reaction system and perform the reaction on a PCR instrument. Purify the reaction system using VAHTS DNA Clean Beads to obtain the targeted enrichment reaction product.
[0059] (4) Add the target enrichment reaction products to the library construction reaction system and perform the reaction on a PCR instrument. Purify the reaction products using VAHTS DNA Clean Beads to obtain a DNA library.
[0060] (5) The DNA library was sequenced using the SE50 mode of BGIseq E25.
[0061] 2. Bioinformatics analysis and result interpretation
[0062] (1) Data quality control: Fastp v0.23.4 sequencing raw data were used for quality control with the parameters set to -q 25 -u15 -l 17 -n 3. Reads containing more than 3 N bases were filtered, short fragment sequences with a quality value lower than Q15 were removed, sequences with a length lower than 17 bp were filtered, and low-complexity sequences and adapter sequences were filtered. After all the above filtering steps were passed, sequences with a length lower than 15 bp were filtered;
[0063] (2) Microbial alignment analysis: blast v2.16.0 was used to align the above data to the targeted amplicon microbial library. The results were sorted based on the score obtained by identity*alignment_cov, and the results with the highest scores were retained to obtain the microbial annotation results of each read.
[0064] (3) Microbial identification analysis: ① The above alignment results were counted based on species as units, and the total number of annotated reads was calculated; ② The data was normalized according to the sequencing data volume of 1Mb read to obtain RPM (Reads per million mapped reads). The specific calculation formula is: RPM = number of species annotated reads * 10^6 / total number of reads of the sample; ③ Only results with RPM ≥ 40 or 40 > RPM ≥ 10 but both pairs of primers for the species detected ≥ 3 reads were retained; finally, the microbial annotation results were sorted from high to low by RPM number for interpretation by medical personnel.
[0065] Example 2: Timeliness test of the method
[0066] 1. Experimental Data
[0067] To test the computational time of Example 1 of the present invention, cerebrospinal fluid specimens from seven patients suspected of parasitic infection were collected. The specimens were split into two halves: one was tested using the present invention (using the processing method of Example 1), with each sample measuring 0.5 Mreads; the other was subjected to metagenomic sequencing, with each sample measuring 20 Mb reads. Metagenomic computational analysis was performed using 12 CPUs. Finally, the differences in detection cycle between the present invention and metagenomic sequencing gene detection methods were compared.
[0068] 2. Experimental Results
[0069] Table 4. Example 2 - Comparison of the timeliness of the present invention and the metagenomic sequencing gene detection method
[0070]
[0071] As shown in Table 4, the entire metagenomic sequencing gene detection process takes 24 hours, while the method of the present invention only takes 11.5 hours, a time reduction of 100%. Furthermore, the data analysis and report interpretation of the present invention are simpler than those of metagenomic sequencing gene detection methods, thus lowering the threshold for application and enabling more rapid detection of potential pathogenic parasites in test samples.
[0072] Example 3: Accuracy testing of the method
[0073] 1. Experimental Data
[0074] Cerebrospinal fluid specimens (Samples 1-7) were collected from seven individuals whose clinical diagnosis and PCR results indicated infection with foodborne parasites. A saliva sample (Sample 8) from a healthy individual was also collected as a negative control. All specimens were divided into two halves: one was tested using the present invention (using the treatment method described in Example 1); the other was subjected to metagenomic sequencing genetic analysis to detect pathogens. The differences in the detection results between the present invention and the metagenomic sequencing genetic analysis method were compared.
[0075] 2. Experimental Results
[0076] Table 5. Example 3 - Comparison of pathogen detection between the present invention and the metagenomic sequencing gene detection method
[0077] sample Clinical diagnosis + PCR results RPM of the present invention The present invention Metagenomic sequencing Sample 1 Angiostrongylus cantonensis 5365(2) + + Sample 2 Toxoplasma gondii 9564(2) + - Sample 3 Toxoplasma gondii 10456(2) + + Sample 4 Angiostrongylus cantonensis 6873(2) + + Sample 5 pork tapeworm 1530(2) + + Sample 6 Giardia lamblia 346(1) + - Sample 7 Cryptosporidium 5691(2) + + Sample 8 - 0 - -
[0078] Table 5 Description: The numbers in the brackets of the RPM column of the present invention represent the number of primers with detection signals; + indicates detection (positive); - indicates no detection / discovery (negative).
[0079] As can be seen from Table 5, the detection sensitivity and specificity of the present invention are both 100%, while the sensitivity of metagenomic sequencing is 71.5% (5 / 7) and the specificity is 100%. The species-specific primers designed by the present invention are more sensitive and are particularly suitable for low pathogen loads.
[0080] Example 4: Detection of food samples using a kit
[0081] 1. Experimental Data
[0082] One specimen was collected from each of four vegetables (usually susceptible to parasitic infection), namely cauliflower, shepherd's purse, lotus root and water celery, and tested according to the processing method of Example 1.
[0083] 2. Experimental Results
[0084] Table 6. Example 4 - Pathogen detection results of food samples
[0085]
[0086]
[0087] As shown in Table 6, sample 1 was found to be contaminated with Angiostrongylus cantonensis, while sample 3 was found to be contaminated with Giardia lamblia.
[0088] In summary, the detection method of the present invention has the advantages of good specificity, high sensitivity, high timeliness (12 hours) and high accuracy (100%). Compared with the detection cycle of the metagenomic sequencing method (24 hours), the method of the present invention greatly shortens the detection time and has higher sensitivity.
Claims
1. A primer set for detecting five foodborne pathogenic parasites, characterized in that: The primer set includes 10 pairs of primers for specifically detecting Angiostrongylus cantonensis, Cryptosporidium, Giardia lamblia, Toxoplasma gondii and Taenia solium, with each pathogenic parasite corresponding to 2 pairs of specific primers; wherein, The primers used for specific detection of Angiostrongylus cantonensis are the nucleotide sequences shown in SEQ ID No. 1 to 4, The primers used for specific detection of Cryptosporidium are the nucleotide sequences shown in SEQ ID No. 5 to 8, The primers used for specific detection of Giardia lamblia are the nucleotide sequences shown in SEQ ID No. 9 to 12, The primers used for specific detection of Toxoplasma gondii are the nucleotide sequences shown in SEQ ID No. 13 to 16, The primers used for specific detection of pork tapeworm are the nucleotide sequences shown in SEQ ID No. 17 to 20.
2. A kit for detecting five foodborne pathogenic parasites, characterized in that: The kit comprises the primer set according to claim 1, PCR reaction mix and library construction primers.
3. The kit according to claim 2, wherein The kit also includes a genomic DNA extraction reagent for the sample to be tested, a nucleic acid amplification reagent, a fluorescent labeling reagent and / or a purification reagent.
4. A method for detecting five foodborne pathogenic parasites, characterized in that: The method comprises the following steps: S1. Extract the total nucleic acid sequence of the sample to be tested; S2. performing multiplex PCR amplification using a primer set specifically detecting Angiostrongylus cantonensis, Cryptosporidium parvum, Giardia lamblia, Toxoplasma gondii, and Taenia solium, and purifying the product; the primer set is the primer set for detecting five foodborne pathogenic parasites according to claim 1; S3. Perform library construction using library construction primers containing sample split index sequences and sequencing adapter sequences; S4. Perform high-throughput sequencing of the constructed library using next-generation sequencing technology; S5. Perform bioinformatics analysis on the sequencing data, including quality control, microbial annotation, and result determination, to detect the presence of the five foodborne pathogenic parasites in the samples.
5. The method according to claim 4, characterized in that The multiplex PCR amplification reaction system includes: nucleic acid extraction products of the sample to be tested, a parasite detection primer pool, PCR reaction mix and nuclease-free water.
6. The method according to claim 4, characterized in that Preparation method of the parasite detection primer pool: In the primer set for detecting five foodborne pathogenic parasites described in claim 1, Rd1SP: 5'-CCTACACGACGCTCTTCCGATCT-3' (SEQ ID No. 21) is added to the 5' end of the F primer, and Rd2 SP: 5'-TTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID No. 22) is added to the 5' end of the R primer, and then synthesized to obtain a parasite detection primer pool.
7. The method according to claim 6, characterized in that The synthesized primers were mixed at a concentration of 10 μM and a volume of 1 μL.
8. The method according to claim 4, characterized in that The reaction system for library construction includes: targeted enrichment reaction products, library construction primers and amplification mix.
9. The method according to claim 4, characterized in that The bioinformatics analysis includes the following steps: (1) Use fastp software to perform quality control on the raw sequencing data; (2) Use blast software to align the quality control data with the targeted amplicon microbial library to obtain the microbial annotation results for each read; (3) Microbial identification analysis: ① The alignment results were divided into species units, and the total number of annotated reads was counted; ② The data were normalized based on the sequencing data volume of 1Mb read to obtain RPM (Reads per million mapped reads). The specific calculation formula is: RPM = number of species annotated reads * 10^6 / total number of reads of the sample; ③ Only results with RPM ≥ 40 or 40 > RPM ≥ 10 but both primer pairs detected ≥ 3 reads of the species were retained; (4) Sort the microbial annotation results from high to low according to the RPM number and interpret the results.
10. The method according to claim 4, characterized in that: The sample to be tested is blood, cerebrospinal fluid, alveolar lavage fluid, sputum, tissue fluid or food sample.