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 meni...
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| Format: | Article |
| Language: | English |
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Instituto Nacional de Salud
2018-03-01
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| Series: | Biomédica: revista del Instituto Nacional de Salud |
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| Online Access: | https://www.revistabiomedica.org/index.php/biomedica/article/view/3407 |
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| author | Rubén E. Varela-M Jinney Stefany Arias Luz Elena Velásquez |
| author_facet | Rubén E. Varela-M Jinney Stefany Arias Luz Elena Velásquez |
| author_sort | Rubén E. Varela-M |
| collection | DOAJ |
| description | 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. |
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| issn | 0120-4157 0120-4157 |
| language | English |
| last_indexed | 2024-12-10T21:07:04Z |
| publishDate | 2018-03-01 |
| publisher | Instituto Nacional de Salud |
| record_format | Article |
| series | Biomédica: revista del Instituto Nacional de Salud |
| spelling | doaj.art-86039adb4c544a36aee6f6cfb7f567f92022-12-22T01:33:35ZengInstituto Nacional de SaludBiomédica: revista del Instituto Nacional de Salud0120-41570120-41572018-03-0138111111910.7705/biomedica.v38i0.34072066Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorumRubén E. Varela-M0Jinney Stefany Arias1Luz Elena Velásquez2Unidad de Biología Molecular y Computacional, Programa de Estudio y Control de Enfermedades Tropicales (PECET), Universidad de Antioquia, Medellín, Colombia Facultad de Ciencias Básicas, Universidad Santiago de Cali, Cali, ColombiaGrupo de Microbiología Ambiental, Universidad de Antioquia, Medellín, ColombiaGrupo de Microbiología Ambiental, Universidad de Antioquia, Medellín, Colombia Unidad de Malacología Médica y Trematodos, Programa de Estudio y Control de Enfermedades Tropicales (PECET), Universidad de Antioquia, Medellín, ColombiaIntroduction: 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.https://www.revistabiomedica.org/index.php/biomedica/article/view/3407AngiostrongylusAngiostrongylus cantonensisreal-time polymerase chain reactionmultiplex polymerase chain reaction |
| spellingShingle | Rubén E. Varela-M Jinney Stefany Arias Luz Elena Velásquez Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum Biomédica: revista del Instituto Nacional de Salud Angiostrongylus Angiostrongylus cantonensis real-time polymerase chain reaction multiplex polymerase chain reaction |
| title | Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum |
| title_full | Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum |
| title_fullStr | Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum |
| title_full_unstemmed | Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum |
| title_short | Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum |
| title_sort | standardization of a multiplex real time pcr test for the identification of angiostrongylus cantonensis a costaricensis and a vasorum |
| topic | Angiostrongylus Angiostrongylus cantonensis real-time polymerase chain reaction multiplex polymerase chain reaction |
| url | https://www.revistabiomedica.org/index.php/biomedica/article/view/3407 |
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