Differential Diagnosis ofEntamoebaspp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

Abstract

Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T m) was 73°C and 70°C, respectively. For E. hartmanni, the T m was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

Frequency and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba hartmanni in the context of water scarcity in northeastern Brazil

“…dispar/E. moshkovskii complex and E. hartmanni parasites actually harbour low-pathogenicity species such as E. dispar , E. moshkovskii , or even E. hartmanni ( Gomes et al 2014 , Nair & Variyam 2014 , Efunshile et al 2015 , , Nath et al 2015) . The proportions of these subjects are variable, but can be quite high.…”

“…Nested-PCR was performed with primers Eh-L (5-ACATTTTGAAGACTTTATGTAAGTA-3) and Eh-R (5-CAGATCTAGAAACAATGCTTCTCT-3), which are specific for E. histolytica and amplify a 427 bp fragment, Ed-L (5-GTTAGTTATCTAATTTCGATTAGAA-3) and Ed-R (5-ACACCACTTACTATCCCTACC-3), which are specific for E. dispar and amplify a 195 bp product, and Mos 1 (5-GAAACCAAGAGTTTCACAAC-3) and Mos 2 (5-CAATATAAGGCTTGGATGAT-3), which are specific for E. moshkovskii and yield a 553 bp product ( Paglia & Visca 2004 , Lau et al 2013) . Molecular characterisation of E. hartmanni was performed essentially as described by Gomes et al (2014) , but with minor modifications. Briefly, primers EhartR1 mod (5-ATTGTCTTCACTATTCCATGCC-3) and EhartF mod (5-CCAGCTTTCCAAACATGATG-3) were used to amplify a 186 bp product.…”

Differentiation of Entamoeba histolytica from Entamoeba dispar by nested multiplex polymerase chain reaction

“…Traditionally, the laboratory diagnosis of Entamoeba spp. is based on their morphology on microscopic examination of stool [7] . However, morphological identification fails to differentiate E. histolytica from the identical nonpathogenic E. dispar [8] .…”

Development of a multiplex qPCR in real time for quantification and differential diagnosis ofSalmonellaGallinarum andSalmonellaPullorum

“…The use of the SYBr Green system in multiplex PCR in real time has been reported in different studies to identify pathogens, allergens, microbiological violations in contaminated food and antibiotic-resistant genes ( Note: Primers pSG were designed based on ROD43 and primers pSP were designed based on ROD24 (Batista, 2013). Varga & James, 2005;Liu et al, 2006;Fan et al, 2007;Pafundo et al, 2010;Rajtak et al, 2011;Kagkli et al, 2012;Cheng et al, 2013;Gomes et al, 2014;Singh & Mustapha, 2014). However, this fluorescent dye, differently from probe systems, binds indistinctly to doublestranded DNA, emitting interfering levels of fluorescence in case of nonspecific amplification.…”