TY - JOUR
T1 - Real-time PCR for sexing Schistosoma mansoni cercariae
AU - Chevalier, Frédéric D.
AU - Le Clec'h, Winka
AU - Alves De Mattos, Ana Carolina
AU - Loverde, Philip T.
AU - Anderson, Timothy JC
N1 - Funding Information:
This study was supported by NIH grants [ R01-AI097576 (T.J.C.A.) , 1R01AI115691 and P50 AI098507 (P.T.L.) ], World Health Organization [ HQNTD1206356 (P.T.L.) ], and the UTHSCSA Presidents Collaborative Research Fund (P.T.L.) . Biomphalaria glabrata snails were provided by the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, MD) through NIH-NIAID Contract HHSN272201000005I for distribution through BEI Resources. The molecular work at TBRI was conducted in facilities constructed with support from Research Facilities Improvement Program Grant ( C06 RR013556 ) from the National Center for Research Resources (NIH). W.L was supported by a Cowles fellowship from Texas Biomedical Research Institute. A.C.A.M received support from CAPES (17766/12-5). We thank the Theodor Bilharz Research Institute (Egypt) for providing the SmEG parasites.
Publisher Copyright:
© 2016 Elsevier B.V. All rights reserved.
PY - 2016/1/1
Y1 - 2016/1/1
N2 - The gender of cercarial larvae can only be determined using molecular methods. End point PCR methods that amplify repetitive markers on the W chromosome of the female (ZW) parasites have been developed, but sometimes results are ambiguous or incorrect. To more effectively distinguish sexes, and to determine why end point PCR can be incorrect, we quantified the W6 repeat sequence and a specific Z chromosome gene using real-time PCR. The ratio between copy number of W6 and a Z chromosome marker unambiguously identifies gender: females have higher ratios (421-4371) than males (0-21). However, some males have low numbers of W6 elements in their genome, and qPCR demonstrated significantly higher W6/Z marker ratios for male genotypes giving ambiguous end point PCR results compared with males giving clear end point results. The quantitative PCR sexing method developed will be particularly useful where reliable sexing of cercariae is critical, for example when staging genetic crosses.
AB - The gender of cercarial larvae can only be determined using molecular methods. End point PCR methods that amplify repetitive markers on the W chromosome of the female (ZW) parasites have been developed, but sometimes results are ambiguous or incorrect. To more effectively distinguish sexes, and to determine why end point PCR can be incorrect, we quantified the W6 repeat sequence and a specific Z chromosome gene using real-time PCR. The ratio between copy number of W6 and a Z chromosome marker unambiguously identifies gender: females have higher ratios (421-4371) than males (0-21). However, some males have low numbers of W6 elements in their genome, and qPCR demonstrated significantly higher W6/Z marker ratios for male genotypes giving ambiguous end point PCR results compared with males giving clear end point results. The quantitative PCR sexing method developed will be particularly useful where reliable sexing of cercariae is critical, for example when staging genetic crosses.
KW - Cercariae
KW - Schistosome
KW - Sexing methods
KW - qPCR
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U2 - 10.1016/j.molbiopara.2016.03.010
DO - 10.1016/j.molbiopara.2016.03.010
M3 - Article
C2 - 27021570
AN - SCOPUS:84962724280
SN - 0166-6851
VL - 205
SP - 35
EP - 38
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1-2
ER -