Multiplex Real-Time PCR Assay Targeting Eight Parasites Customized to the Korean Population: Potential Use for Detection in Diarrheal Stool Samples from Gastroenteritis Patients

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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Evaluation of a New Multiplex Real-Time PCR Assay for Detecting Gastroenteritis-Causing Viruses in Stool Samples

Annals of Laboratory Medicine ◽

10.3343/alm.2018.38.3.220 ◽

2018 ◽

Vol 38 (3) ◽

pp. 220-225 ◽

Cited By ~ 9

Author(s):

Jungwon Hyun ◽

Dae-Hyun Ko ◽

Su-Kyung Lee ◽

Han-Sung Kim ◽

Jae-Seok Kim ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Assay ◽

Stool Samples

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Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis , Cryptosporidium parvum / Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review

Clinical Microbiology and Infection ◽

10.1016/j.cmi.2015.10.019 ◽

2016 ◽

Vol 22 (2) ◽

pp. 190.e1-190.e8 ◽

Cited By ~ 28

Author(s):

A. Laude ◽

S. Valot ◽

G. Desoubeaux ◽

N. Argy ◽

C. Nourrisson ◽

...

Keyword(s):

Literature Review ◽

Multiplex Pcr ◽

Real Time ◽

Cryptosporidium Parvum ◽

Real Time Pcr ◽

Giardia Intestinalis ◽

Pcr Assay ◽

Cryptosporidium Hominis ◽

Multiplex Pcr Assay ◽

Stool Samples

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Detection and species identification of microsporidial infections using SYBR Green real-time PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.026781-0 ◽

2011 ◽

Vol 60 (4) ◽

pp. 459-466 ◽

Cited By ~ 29

Author(s):

Spencer D. Polley ◽

Samuel Boadi ◽

Julie Watson ◽

Alan Curry ◽

Peter L. Chiodini

Keyword(s):

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Enterocytozoon Bieneusi ◽

Sybr Green ◽

Pcr Assay ◽

Real Time Analysis ◽

Highly Sensitive ◽

Stool Samples

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)−1, whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.

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Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

Parasitology International ◽

10.1016/j.parint.2011.06.010 ◽

2012 ◽

Vol 61 (1) ◽

pp. 183-186 ◽

Cited By ~ 23

Author(s):

Xian-Quan Cai ◽

Hai-Qiong Yu ◽

Jian-Shan Bai ◽

Jian-Dong Tang ◽

Xu-Chu Hu ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clonorchis Sinensis ◽

Pcr Assay ◽

Stool Samples ◽

Human Stool

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Improving the specific diagnosis of trematode, cestode and nematode infections by a multiplex single-tube real-time PCR assay

Journal of Clinical Pathology ◽

10.1136/jclinpath-2018-205590 ◽

2019 ◽

Vol 72 (7) ◽

pp. 487-492 ◽

Cited By ~ 1

Author(s):

Samson S Y Wong ◽

Rosana W S Poon ◽

Kelvin K W To ◽

Jasper F W Chan ◽

Gang Lu ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Molecular Diagnostic ◽

Rrna Gene ◽

28S Rrna ◽

Pcr Assay ◽

Single Tube ◽

Histological Sections ◽

Pcr Products ◽

Stool Samples

AimsHelminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay.MethodsWe designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested.ResultsThe PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples.ConclusionsThe highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.

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Isolation and detection of Shiga toxin-producing Escherichia coli in clinical stool samples using conventional and molecular methods

Journal of Medical Microbiology ◽

10.1099/jmm.0.007732-0 ◽

2009 ◽

Vol 58 (7) ◽

pp. 905-911 ◽

Cited By ~ 27

Author(s):

Matthew W. Gilmour ◽

Linda Chui ◽

Theodore Chiu ◽

Dobryan M. Tracz ◽

Kathryn Hagedorn ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Molecular Methods ◽

Genetic Profile ◽

Pcr Assay ◽

Suspension Array ◽

Stool Samples ◽

Pcr Targeting

The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.

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