Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum
INTRODUCTION: Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis.
OBJECTIVE: To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus.
MATERIALS AND METHODS: Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control.
RESULTS: The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%).
CONCLUSIONS: A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2018 |
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| Erschienen: |
2018 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:38 |
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| Enthalten in: |
Biomedica : revista del Instituto Nacional de Salud - 38(2018), 1 vom: 15. März, Seite 111-119 |
| Sprache: |
Spanisch |
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| Beteiligte Personen: |
Varela-M, Rubén E [VerfasserIn] |
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| Links: |
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| Themen: |
Angiostrongylus |
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| Anmerkungen: |
Date Completed 16.07.2018 Date Revised 23.10.2018 published: Electronic Citation Status MEDLINE |
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| doi: |
10.7705/biomedica.v38i0.3407 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM283163429 |
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| 500 | |a Date Revised 23.10.2018 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a INTRODUCTION: Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis | ||
| 520 | |a OBJECTIVE: To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus | ||
| 520 | |a MATERIALS AND METHODS: Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control | ||
| 520 | |a RESULTS: The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%) | ||
| 520 | |a CONCLUSIONS: A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Angiostrongylus | |
| 650 | 4 | |a Angiostrongylus cantonensis | |
| 650 | 4 | |a multiplex polymerase chain reaction | |
| 650 | 4 | |a real-time polymerase chain reaction | |
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| 700 | 1 | |a Arias, Jinney Stefany |e verfasserin |4 aut | |
| 700 | 1 | |a Velásquez, Luz Elena |e verfasserin |4 aut | |
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