EP4010499A1 - Method and kit for detecting nematodes, tapeworms and flukes using the multiplex pcr technique, and oligonucleotides - Google Patents
Method and kit for detecting nematodes, tapeworms and flukes using the multiplex pcr technique, and oligonucleotidesInfo
- Publication number
- EP4010499A1 EP4010499A1 EP20732476.5A EP20732476A EP4010499A1 EP 4010499 A1 EP4010499 A1 EP 4010499A1 EP 20732476 A EP20732476 A EP 20732476A EP 4010499 A1 EP4010499 A1 EP 4010499A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- dna
- seq
- primers
- multiplex pcr
- taenia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Definitions
- the present invention relates to a method and kit for detecting nematodes, tapeworms, and flukes using the multiplex PCR technique via the parasite DNA detection.
- the resultant method and kit of the present invention which are based on the PCR reaction are intended to detect the presence of genetic material (DNA) of the parasites in a biological material, such as food, samples taken from the environmental, human and animal feces, etc.
- DNA genetic material
- Parasite DNA detection in the test samples may indicate the presence of live and invasive forms of these parasites.
- the great parasite egg survival rate poses a serious sanitation problem.
- Health hazards caused by the parasites are digestive, respiratory and neurological disorders, allergies, and in case of toxocariasis, vision disorders. Parasites are also associated with the growing number of liver and pancreatic cancer cases.
- PCR polymerase chain reaction
- Multiplex PCR is a technique used in molecular biology for amplification multiple matrices during one PCR reaction. Thanks to the simultaneous use of different sets of primers, this method provides a great saving of time and allows reducing the cost of testing while maintaining high quality of results.
- the presence of many primers can lead to their cross-hybridization and increases the risk of errors when binding to the target sequence. Therefore, a proper design of primers plays a very important role, being a key step affecting the efficiency of the multiplex PCR. It is recommended that the primers used in one test have similar reaction kinetics. They should have the right length (18-30 bp) and comparable melting point, which depends on the percentage of guanine and cytosine in the chain. Due to the competition occurring between the primers in one reaction mixture, the key feature of the primers is their specificity to the amplified target sequence.
- the object of the present invention was to develop PCR primers for the amplification of the mitochondrial cox1 fragment and 18S rDNA of some helminths, which in a fairly wide spectrum of PCR reaction parameters, amplify the relevant parasite DNA fragments without entering into any reaction with plant DNA or DNA obtained from the feces.
- the present invention provides a method for detecting in the biological material nematodes, tapeworms, and flukes, selected from the group including Toxocara cati, Toxocara canis, Taenia solium, Taenia saginata, Echinococcus multilocularis, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, Dipylidium caninum, Taenia taeniaeformis, Fasciola hepatica, Ascaris suum, Toxascaris leonina, Parascaris equorum, and Echinococcus granulosus, comprising the following steps: a) Providing a sample of the biological material; b) Isolation of DNA from the sample; c) Carrying out a multiplex PCR reaction with a selected set of primers; d) Size analysis of the multiplex PCR reaction products; wherein for the identification of one of the above species in the multiplex PCR reaction a species-specific
- the method comprises an additional step of sequencing the multiplex PCR reaction product.
- the present invention provides also a kit for detecting in the biological material nematodes, tapeworms, and flukes, selected from the group including Toxocara cati, Toxocara canis, Taenia solium, Taenia saginata, Echinococcus multilocularis, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, Dipylidium caninum, Taenia taeniaeformis, Fasciola hepatica, Ascaris suum, Toxascaris leonina, Parascaris equorum, and Echinococcus granulosus, using the multiplex PCR reaction, characterized in that it comprises at least one set of primers among such as either SEQ ID NO. 1 -3 or SEQ ID NO. 4-9 or SEQ ID NO. 10-12 or SEQ ID NO. 13-21.
- the kit comprises a species-specific set of primers selected from the list:
- the invention relates also to an oligonucleotide having sequence selected from SEQ ID NO. 1 -21.
- Figure 3 Alignment of the 18S rDNA sequence fragment with marked sites for nematode (Ascaris lumbricoides, Ascaris suum, Toxascaris leonina, Parascaris equorum, Toxocara cati, Toxocara canis, and Enterobius vermicularis) PCR primer binding.
- nematode Ascaris lumbricoides, Ascaris suum, Toxascaris leonina, Parascaris equorum, Toxocara cati, Toxocara canis, and Enterobius vermicularis
- Figure 9 Alignment of the 18S rDNA sequence fragment obtained by sequencing the PCR reaction products with a pair of primers Nc1 F (SEQ ID NO. 10) and Nc452R (SEQ ID NO. 12).
- Figure 10. S11 and S12 sequences matching Toxocara canis sequences.
- Tortoise pinworm sequence (S151) is identical, in the analyzed section, with the horse and goat pinworm sequences.
- Figure 13 Alignment of the 18S rDNA sequence fragment with marked sites for tapeworm (Dipylidium caninum, Taenia solium, Taenia saginata, Hydatigera (Taenia) taeniaeformis, Echinococcus multilocularis, Echinococcus granulosus); nematode (Ascaris lumbricoides, Ascaris suum, Toxascaris leonina, Parascaris equorum, Toxocara cati, Toxocara canis, Enterobius vermicularis), and fluke Fasciola hepatica PCR primer binding.
- tapeworm Dipylidium caninum, Taenia solium, Taenia saginata, Hydatigera (Taenia) taeniaeformis, Echinococcus multilocularis, Echinococcus granulosus
- nematode Ascaris lumbricoides, Ascaris suum
- FIG. 14 Photo of the gel with multiplex PCR reaction products employing 9 primers (NA950F (SEQ ID NO. 13), C950F (SEQ ID NO. 15), NTr950F (SEQ ID NO. 14), Fh950F (SEQ ID NO. 16), Em950F (SEQ ID NO. 17), Tt950F (SEQ ID NO. 18), Nc1200R (SEQ ID NO. 19), C1200R (SEQ ID NO. 20), and Fh1200R (SEQ ID NO. 21)).
- 9 primers NA950F (SEQ ID NO. 13), C950F (SEQ ID NO. 15), NTr950F (SEQ ID NO. 14), Fh950F (SEQ ID NO. 16), Em950F (SEQ ID NO. 17), Tt950F (SEQ ID NO. 18), Nc1200R (SEQ ID NO. 19), C1200R (SEQ ID NO. 20), and Fh1200R (SEQ ID NO. 21)
- Figure 18 Did sequence matching Hydatigera spp. and Taenia taeniaeformis sequences.
- the method and kit of the present invention are based on the multiplex PCR reaction by which the presence of nematode (Toxocara canis, Toxocara cati, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis), tapeworm (Echinococcus multilocularis, Dipylidium caninum, Hydatigera taeniaeformis — formerly Taenia taeniaeformis), and fluke (Fasciola hepatica) DNA could be detected.
- nematode Toxocara canis, Toxocara cati, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis
- tapeworm Echinococcus multilocularis, Dipylidium caninum, Hydatigera taeniaeformis — formerly Taenia taeniaeformis
- fluke Fesciola hepatica
- a set of multiplex PCR primers was designed for the assay, which amplify a specific DNA fragment of the aforementioned parasites, while not entering into any reaction with neither plant DNA nor DNA obtained from the feces.
- the developed assay can be used as a tool to control the biological purity of food for the presence of parasites potentially dangerous to humans (nematodes, tapeworms, and flukes).
- the main source of parasites occurring in food could be the natural fertilizers used for plant cultivation, wild animals being intermediate hosts of the parasites and the lack of hygiene maintenance by the producer during harvesting, processing and packaging.
- Example 1 Isolation of the parasite eggs from the test material
- Isolation of the parasite eggs is carried out from soil, feces or plant material samples by flotation method consisting in a thorough mixing of 2 g of the test sample with 10% NaCI solution with the addition of sucrose (65 g per 100 mL). Plant material should be ground beforehand. Test samples should be homogeneous. The 50 mL test tube containing the sample covered with the flotation solution to the rim should be covered with a glass plate and left to stand for 2 hours at room temperature. Eggs accumulated on the glass plate should be flushed with 1 mL of distilled water into a 1.5 mL test tube and centrifuged (7000xg, 1 minute, room temperature) and then the water from above the eggs should be pipetted off.
- the centrifuged eggs should be covered with 100 pL distilled water, stirred and counted in a hemocytometer using a light microscope, choosing the egg sample volume and the degree of its dilution with distilled water depending on the hemocytometer type and the egg number.
- kits for DNA isolation comprising glass beads or other "abrasive" material with similar properties, can be used. Dried and thoroughly ground plant material should be soaked in water in a sterile glass container (e.g. a watch glass) for 5 minutes, with addition of 0.15 g sterile distilled water to 0.05 g dry plant material. Further steps should follow the instructions of the commercial DNA isolation kit manufacturer.
- Example 4 Detection of Toxocara cati and Toxocara canis based on cox1 sequence
- the PCR assay primers for Toxocara cati and Toxocara canis DNA detection were developed based on the sequence analysis of genes encoding the cytochrome c oxidase subunit 1 (cox1).
- NCBI reference sequences of the complete Toxocara cati (NC_010773.1) and Toxocara canis (NC_010690.1) mitochondrial genome were used.
- COIF280TS (SEQ ID NO. 1) - 5’-3’TTGATGTTGGGGGCTCCTGA
- COIR497CN (SEQ ID NO. 2) - 5’-3’CCAAGAATAGAACTAACTCCCGCAC
- COIR634CT (SEQ ID NO. 3) - 5 -3’
- Figure 1 shows a photograph of an agarose gel with products of Toxocara cati and Toxocara canis cox1 DNA fragment amplification obtained by PCR, where
- T. cati and T. canis DNA isolated from purified eggs.
- DNA isolation a commercial kit for isolation from tissues with the addition of quartz sand;
- DNA isolation a commercial kit for isolation from tissues with the addition of quartz sand;
- DNA isolation a commercial kit for isolation from tissues
- DNA isolation a commercial kit for isolation from stool
- DNA isolation a commercial kit for isolation from tissues
- Amplified DNA fragment sizes Toxocara canis - 220 base pairs, Toxocara cati - 360 base pairs.
- Example 5 Detection of Taenia solium, T. saginata, and Echinococcus multilocularis based on cox1 sequence
- the PCR assay primers for Taenia solium, T. saginata, and Echinococcus multilocularis DNA detection were developed based on sequence analysis of genes encoding the cytochrome c oxidase subunit 1 (cox1).
- sequence analysis and design of reaction primers Taenia solium mitochondrial DNA, complete genome GenBank: AB086256.1, Taenia saginata mitochondrion, complete genome GenBank: AY684274, and Echinococcus multilocularis mitochondrial cox1 gene for cytochrome c oxidase subunit 1 GenBank: AB461413.1 sequences were used.
- Em656F (SEQ ID NO. 4) 5-3': TTTTGATCCGTTAGGTGGGGGT
- Em912R (SEQ ID NO. 5) 5'-3': AAAAAACCGCCGTCTTCACA
- Tsl663F (SEQ ID NO. 6) 5'-3’: CCGTTAGGAGGTGGTGATCC
- Tsl954R (SEQ ID NO. 7) 5'-3': TAATCCCCGTAGGCACTCCAA
- Tsg661 F (SEQ ID NO. 8) 5 -3': ATCCATTGGGTGGTGGTGATCCT
- Tsg1078R (SEQ ID NO. 9) 5'-3': GACAACACAATACCAGTCACACCA
- Biological material prepared according to the methods of Examples 1 and 2 was subjected to PCR reaction using the following conditions:
- Echinococcus multilocularis tapeworm DNA was obtained from the digestion of the tapeworm uterine proglottid (which contains 200 to 600 eggs). DNA isolated from a single uterine proglottid was suspended in 200 pL of water. 1 pL of DNA was used for the PCR reaction, which corresponds to the amount of DNA obtained from 1 to 3 eggs.
- PCR reactions were carried out with DNA isolated from the two tapeworm species commonly found in cats and dogs: Dipylidium caninum, and Taenia taeniaeformis. The polymerase activity is strongly inhibited by the DNA isolated from historical specimens of Taenia solium and Taenia saginata preserved with formalin (fresh specimens of these tapeworms are currently not available in Poland).
- Figure 2 shows a photograph of an agarose gel with products of Echinococcus multilocularis, Dipylidium caninum and Taenia taeniaeformis cox1 DNA fragment amplification obtained by PCR, where
- Echinococcus multilocularis DNA fragment size - 220 base pairs.
- a 'ladder' of amplified DNA fragments is formed.
- Toxocara canis Toxocara canis, Toxocara cati, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, tapeworms: Echinococcus multilocularis, Dipylidium caninum, Taenia taeniaeformis, and flukes Fasciola hepatica PCR primers were designed and tested allowing to amplify fragments of 18S rDNA genes encoding a small subunit (SSU) of ribosomal RNA.
- material parasites
- DNA from Toxocara cati (S142) and Toxocara canis (S12) adult specimens was isolated with a standard tissue DNA isolation kit according to the manufacturer's protocol (Qiagen).
- Toxocara cati obtained from cat feces after deworming
- the isolated DNA was additionally purified (to remove the PCR inhibitors) by additional precipitation in ethyl alcohol.
- Pinworm eggs S15 - 5 eggs, S151 - 30 eggs
- Hermann's tortoise Testudo hermanni
- a stool DNA isolation kit Qiagen
- Toxocara canis (S11) egg DNA was isolated directly from the feces of an infected dog.
- NcF1 (SEQ ID NO. 10) 5'-3’: GGTTATATGCTTATCTCAAAGGCTAAG
- Nc291 R (SEQ ID NO. 11) 5 -3': GGCAGACACTTGATAGACACGTCG
- Nc452R (SEQ ID NO. 12) 5'-3': CGAGAGTGGGTAATTTGCGCGCCT
- Figure 3 shows the alignment of the 18S rDNA sequence fragment with marked sites for nematode (Ascaris lumbricoides, Ascaris suum, Toxascaris leonina, Parascaris equorum, Toxocara cati, Toxocara canis, and Enterobius vermicularis) PCR primer binding.
- nematode Ascaris lumbricoides, Ascaris suum, Toxascaris leonina, Parascaris equorum, Toxocara cati, Toxocara canis, and Enterobius vermicularis
- DNA isolates from dandelion were used in the PCR reaction. DNA from dried plants was isolated with a stool DNA isolation kit (Qiagen).
- the reaction was carried out in a volume of 25 pL: 2.5 pL 10x PCR buffer (pH 8.6; 15 mM MgClz), 0.25 pL of each primer (20 mM), 0.5 pL dNTPs mix (20 mM), 0.3 pL Taq DNA polymerase (1 U/pL), and 2.5 pL DNA of the analyzed sample. 2.5 pL sterile water was used In the control sample.
- the reaction was carried out in TM100 Thermal Cycler (Bio-Rad).
- the prepared biological material was subjected to PCR reaction using the following conditions:
- Figure 4 shows the result of PCR reaction employing NcF1 and Nc452R primers, where
- the PCR reaction products (NcF1 - Nc452R) for the isolates: S151 , S142, and S12 were purified with a commercial PCR product purification kit (QIAquick - PCR Purification Kit, QIAGEN). The purified DNA was sequenced (Genomed, Warsaw). PCR primers (NcF1 - Nc452R) were used as sequencing primers.
- the 18S rDNA sequences obtained were analyzed and compiled using ContigExpress software, which is a part of the Vector NTI Advance 11 molecular analysis software package (Invitrogen, USA). The compiled sequences were checked against GenBank database using Blast software.
- Figure 5 shows the visualization of sequencing result of the 18S tortoise pinworm sequence fragment (S151 ).
- Figure 6 shows the visualization of sequencing result of the 18S Toxocara cati sequence fragment (S142).
- Figure 7 shows the visualization of sequencing result of the 18S Toxocara canis sequence fragment (S12).
- Figure 8 shows the visualization of sequencing result of the 18S Toxocara canis sequence fragment (S11).
- Figure 9 shows the alignment of the 18S rDNA sequence fragment obtained by sequencing the PCR reaction products with a pair of primers Nc1 F, and Nc452R.
- Toxocara spp. (S11, S12, and S142) a sequence of 371 base pairs was obtained, for S151 sample - a sequence of 369 base pairs. S11 and S12 sequences were found to be identical.
- sequences obtained were compared with the sequences in GenBank using the Blast software.
- FIG 10 shows that the S11 and S12 sequences are matching the Toxocara canis sequences.
- Figure 11 shows that the S142 sequence matches the Toxocara cati sequences.
- Figure 12 shows that the tortoise pinworm sequence (S151) is identical, in the analyzed section, with the horse and goat pinworm sequences.
- a multiplex PCR assay was designed in which a short (250 base pairs) fragment of nematode, tapeworm, and fluke 18S rDNA is amplified.
- Nine PCR primers were designed for this assay:
- NA950F (SEQ ID NO. 13) 5'-3': ACGGGGGCATTCGTATCGCTGC
- NTr950F (SEQ ID NO. 14) 5 -3': ACGGGGACATTCGTATTGCTGCG
- Fh950F (SEQ ID NO. 16) 5'- 3': ACGGGGGCATTTGTATGGCGG
- Em950F (SEQ ID NO. 17) 5'-3': GCGGGGGCGTTTGTATGGCTG
- Tt950F (SEQ ID NO. 18) 5 -3': GCGGGGACGTTTGTATGGTTGCG
- Nc1200R (SEQ ID NO. 19) 5’ -3': CAACCATACTTCCCCCGGAACCGAAA
- C1200R (SEQ ID NO. 20) 5 -3': CAACCATACTTCCCCCGGAACCGAAA Fh1200R (SEQ ID NO. 21) 5’-3': AACCATACTTCCCCCGGAGCCCAAA
- Figure 13 shows the alignment of the 18S rDNA sequence fragment with marked sites for tapeworm (Dipylidium caninum, Taenia solium, Taenia saginata, Hydatigera (Taenia) taeniaeformis, Echinococcus multilocularis, Echinococcus granulosus); nematode (Ascaris lumbricoides, Ascaris suum, Toxascaris leonina, Parascaris equorum, Toxocara cati, Toxocara canis, Enterobius vermicularis), and fluke Fasciola hepatica PCR primer binding.
- tapeworm Dipylidium caninum, Taenia solium, Taenia saginata, Hydatigera (Taenia) taeniaeformis, Echinococcus multilocularis, Echinococcus granulosus
- nematode Ascaris lumbricoides, Ascaris suum
- Figure 14 shows the multiplex PCR reaction products with 9 primers (NA950F, C950F, NTr950F, Fh950F, Em950F, Tt950F, Nc1200R, C1200R, and Fh1200R).
- Figure 15 shows the visualization of sequencing result of the 18S sequence fragment of the Did sample.
- Figure 16 shows the visualization of sequencing result of the 18S sequence fragment of the Em2 sample.
- sequences obtained were compared to the sequences in GenBank using the Blast software.
- Figure 17 shows that the Em2 sequence is matching the Echinococcus spp. sequences.
- Figure 18 shows that the Did sequence matches Hydatigera spp. and Taenia taeniaeformis sequences.
- the reaction was carried out in a volume of 35 pL: 3.5 pL 10x PCR buffer (pH 8.6, 2.2 mM MgClz), 0.4 pL of each primer (20 mM), 0.5 pL dNTPs mix (20 mM), 1 pL Taq DNA polymerase (1 U/pL), 24.5 pL sterile water, and 3.5 pL DNA of the analyzed sample. In the control sample 3.5 pL sterile water was added instead of DNA.
- the reaction was carried out in TM100 Thermal Cycler (Bio-Rad) according to the program: 95°C - 1 minute
- An assay was developed to detect nematode, tapeworm, and fluke DNA by determining experimentally the appropriate concentration of magnesium chloride (MgClz) and Taq DNA polymerase as well as the parameters of the PCR reaction program (time, temperatures, and number of cycles).
- MgClz magnesium chloride
- Taq DNA polymerase the parameters of the PCR reaction program (time, temperatures, and number of cycles).
- PCR primers have been developed to amplify the mitochondrial cox1 fragment and 18S rDNA of some helminths, which in a fairly wide spectrum of PCR reaction parameters amplify the relevant parasite DNA fragments without entering into any reaction with plant DNA or DNA obtained from feces.
- a universal multiplex PCR assay has been developed for many parasites that pose a serious threat to human health (nematodes - Toxocara canis, Toxocara cati, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis; tapeworms - Echinococcus multilocularis, Dipylidium caninum, Taenia taeniaeformis, and a fluke - Fasciola hepatica).
- the helminth 18S rDNA fragments obtained during assays allow confirmation of the parasite species after sequencing.
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PCT/PL2020/050036 WO2022093051A1 (en) | 2020-05-09 | 2020-05-09 | Method and kit for detecting nematodes, tapeworms and flukes using the multiplex pcr technique, and oligonucleotides |
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EP20732476.5A Withdrawn EP4010499A1 (en) | 2020-05-09 | 2020-05-09 | Method and kit for detecting nematodes, tapeworms and flukes using the multiplex pcr technique, and oligonucleotides |
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CN110592233B (en) * | 2019-09-26 | 2022-11-08 | 吉林大学 | Gene chip for detecting common food-borne parasites and application thereof |
CN110656187B (en) * | 2019-10-28 | 2023-06-02 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Kit for detecting pathological tissues or echinococcus in canine feces by multiple RAA and multiple PCR and detection method |
CN110714087B (en) * | 2019-11-14 | 2021-10-26 | 中国农业科学院兰州兽医研究所 | Primer, kit and identification method for simultaneously and rapidly identifying 4 taenia solium larvae |
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