Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples

Foo, Phiaw Chong; Yean, Chan Yean; Too, Wei Cun See; Tan, Zi Ning; Kin, Wong Weng; Lalitha, Pattabhiraman; Lim, Boon Huat

doi:10.1099/jmm.0.044552-0

Cited by 15 publications

(14 citation statements)

References 21 publications

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“…The detection limit of thermostabilized multiplex PCR for H. influenzae and K. pneumoniae at bacteria level was at 1.0 × 10 3 CFU/ml, whereas the sensitivity of both multiplex PCR assays was at 1.0 × 10 4 CFU/ml. The sensitivity level achieved in this study was found to be comparable to the ones obtained in prior studies [3, 11, 12]. …”

Section: Discussionsupporting

confidence: 88%

“…Two parameters (enzyme stabilizer and Taq DNA polymerase) were optimized during this process. Trehalose was added as an enzyme stabilizer to protect and prevent any conformation changes of Taq DNA polymerase enzyme during and after dehydration [11, 12]. It stabilizes the PCR reagents, especially Taq DNA polymerase against stresses brought by vacuum drying and freeze thawing [13].…”

Section: Discussionmentioning

confidence: 99%

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A Thermostabilized, One-Step PCR Assay for Simultaneous Detection ofKlebsiella pneumoniaeandHaemophilus influenzae

Khazani

Zuraina

Yean

et al. 2017

Journal of Tropical Medicine

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Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp). In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using the Q10 method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

A Thermostabilized, One-Step PCR Assay for Simultaneous Detection ofKlebsiella pneumoniaeandHaemophilus influenzae

Khazani

Zuraina

Yean

et al. 2017

Journal of Tropical Medicine

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“…DNA extracts were subsequently tested using a polymerase chain reaction (PCR) assay adapted from Foo et al . (2012). First, a segment (~748 bp) of the Entamoeba spp.…”

Section: Methodsmentioning

confidence: 99%

“…All primers used are from Foo et al . (2012). PCR amplification cycles were performed in an Eppendorf Mastercycler pro (Hamburg, Germany) thermal cycler.…”

Section: Methodsmentioning

confidence: 99%

Entamoeba histolyticainfection in humans, chimpanzees and baboons in the Greater Gombe Ecosystem, Tanzania

et al. 2018

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Entamoeba histolytica is an enteric parasite that infects approximately 50 million people worldwide. Although E. histolytica is a zoonotic parasite that has the potential to infect nonhuman primates, such transmission is poorly understood. Consequently, this study examined whether E. histolytica is present among humans, chimpanzees and baboons living in the Greater Gombe Ecosystem (GGE), Tanzania. The primary aims were to determine patterns of E. histolytica infection in a system with human-nonhuman primate overlap and to test associations between infection status and potential risk factors of disease. Entamoeba spp. occurred in 60.3% of human, 65.6% of chimpanzee and 88.6% of baboon samples. Entamoeba histolytica occurred in 12.1% of human, 34.1% of chimpanzee and 10.9% of baboon samples. Human E. histolytica infection was associated with gastrointestinal symptoms. This was the first study to confirm the presence of E. histolytica in the GGE. The high sample prevalence of E. histolytica in three sympatric primates suggests that zoonotic transmission is possible and stresses the need for further phylogenetic studies. Interventions targeting better sanitation and hygiene practices for humans living in the GGE can help prevent E. histolytica infection in humans, while also protecting the endangered chimpanzees and other primates in this region.

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“…Additionally, PCR has been used to evaluate the effectiveness of routine methods, such as formalin-ether sedimentation and trichrome staining, for the detection of E. histolytica (240). Additionally, there have been rigorous assessments of various types of nucleic acid amplification assays in an effort to determine the best approach for the detection of this pathogen (235,(240)(241)(242)(243)(244). In addition to a variety of PCR-based methods, other methods of nucleic acid amplification, such as loop-mediated isothermal amplification (LAMP), have been used to detect E. histolytica (245).…”

Section: Laboratory-developed Tests For Gastrointestinal Parasites Mmentioning

confidence: 99%

Practical Guidance for Clinical Microbiology Laboratories: Laboratory Diagnosis of Parasites from the Gastrointestinal Tract

Garcia¹,

et al. 2018

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This Practical Guidance for Clinical Microbiology document on the laboratory diagnosis of parasites from the gastrointestinal tract provides practical information for the recovery and identification of relevant human parasites. The document is based on a comprehensive literature review and expert consensus on relevant diagnostic methods. However, it does not include didactic information on human parasite life cycles, organism morphology, clinical disease, pathogenesis, treatment, or epidemiology and prevention. As greater emphasis is placed on neglected tropical diseases, it becomes highly probable that patients with gastrointestinal parasitic infections will become more widely recognized in areas where parasites are endemic and not endemic. Generally, these methods are nonautomated and require extensive bench experience for accurate performance and interpretation.

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Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples

Cited by 15 publications

References 21 publications

A Thermostabilized, One-Step PCR Assay for Simultaneous Detection ofKlebsiella pneumoniaeandHaemophilus influenzae

A Thermostabilized, One-Step PCR Assay for Simultaneous Detection ofKlebsiella pneumoniaeandHaemophilus influenzae

Entamoeba histolyticainfection in humans, chimpanzees and baboons in the Greater Gombe Ecosystem, Tanzania

Practical Guidance for Clinical Microbiology Laboratories: Laboratory Diagnosis of Parasites from the Gastrointestinal Tract

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