Development of a multiplex PCR assay to detect gastroenteric pathogens in the feces of Mexican children.
Acute gastroenteritis (AGE) is a major cause of childhood morbidity and mortality worldwide; the etiology of AGE includes viruses, bacteria, and parasites. A multiplex PCR assay to simultaneously identify human Astrovirus (HAstV), Calicivirus (HuCVs), Entamoeba                         histolytica (E. histolytica), and enteroinvasive Escherichia coli (EIEC) in stool samples is described. A total of 103 samples were individually analyzed by ELISA (enzyme-linked immunosorbent assays) and RT-PCR/PCR. HAstV and HuCVs were detected in four out of 103 samples (3.8 %) by RT-PCR, but ELISAs found only one sample as positive for HuCVs (2.5 %). E. histolytica was identified in two out of 19 samples (10.5 %) and EIEC in 13 out of 20 samples (70 %) by PCR, and all PCR products were sequenced to verify their identities. Our multiplex PCR results demonstrate the simultaneous amplification of different pathogens such as HAstV, EIEC, and E. histolytica in the same reaction, though the HuCVs signal was weak in every replicate. Regardless, this multiplex PCR protocol represents a novel tool for the identification of distinct pathogens and may provide support for the diagnosis of AGE in children.
Programa Institucional de Biomedicina Molecular, Sección de Estudios de Posgrado e Investigación, Escuela Nacional de Medicina y Homeopatía-IPN, Instituto Politécnico Nacional Guillermo Massieu Helguera No. 239, Col. Fracc La Escalera-Ticomán, 07320 México, DF, México.
R
R
Tolentino-Ruiz
D
D
Montoya-Varela
M
M
García-Espitia
M
M
Salas-Benito
A
A
Gutiérrez-Escolano
C
C
Gómez-García
P
P
Figueroa-Arredondo
J
J
Salas-Benito
M
M
De Nova-Ocampo
D002139:Caliciviridae / Q000302:isolation & purification*; D002648:Child; D004748:Entamoeba histolytica / Q000302:isolation & purification*; D004926:Escherichia coli / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology / Q000469:parasitology / Q000821:virology; D005759:Gastroenteritis / Q000175:diagnosis* / Q000382:microbiology / Q000469:parasitology / Q000821:virology; D006801:Humans; D001257:Mamastrovirus / Q000302:isolation & purification*; D008800:Mexico; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*