Real-time PCR using SYBR Green for the detection of Shigella spp. in food and 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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Differential Diagnosis ofEntamoebaspp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

The Scientific World JOURNAL ◽

10.1155/2014/645084 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Thiago dos Santos Gomes ◽

Mariana Coimbra Garcia ◽

Flavia de Souza Cunha ◽

Heloisa Werneck de Macedo ◽

José Mauro Peralta ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Laboratory Diagnosis ◽

Sybr Green ◽

Similar Species ◽

Green Is ◽

Stool Samples ◽

Polymerase Chain ◽

Negative Controls

Amoebiasis, a disease caused byEntamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of theE. histolytica/E. disparcomplex. Furthermore, morphologically similar species such asEntamoeba hartmannicontribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system forE. histolyticaandE. disparand a single real-time PCR forE. hartmanni. The multiplex protocol detected up to 0.0143 pg ofE. histolyticaDNA and 0.5156 pg ofE. disparDNA, and the average melting temperature (Tm) was 73°C and 70°C, respectively. ForE. hartmanni, theTmwas 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. disparDNA was detected in 37; none exhibitedE. histolyticaDNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however,E. hartmanniDNA 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.

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Molecular diagnosis of Shigella, Salmonella and Campylobacter by multiplex Real-time PCR in stool culture samples in Ouagadougou (Burkina Faso)

Sudan Journal of Medical Sciences ◽

10.18502/sjms.v12i3.931 ◽

2017 ◽

Vol 12 (3) ◽

pp. 163

Author(s):

Salfo Sawadogo ◽

Birama Diarra ◽

Cyrille BIsseye ◽

Tegwindé Rebeca Compaore ◽

Florencia W. Djigma ◽

...

Keyword(s):

Burkina Faso ◽

Real Time ◽

Real Time Pcr ◽

Bacterial Culture ◽

Stool Culture ◽

Salmonella Spp ◽

E Coli ◽

Shigella Spp ◽

Stool Samples ◽

Campylobacter Spp

ABSTRACT:Background: Bacteriological diagnosis of Campylobacter spp, Salmonella spp and Shigella spp could be necessary in the case of infectious gastroenteritis syndrome.The objective of this study was to diagnose concomitantly the three enteropathogenic bacteria by multiplex Real-Time PCR in stool culture samples in Ouagadougou (Burkina Faso).Materials and Methods: The study was conducted from February 5th to March 9th, 2013. Two hundred stool samples were received during the study period. The bacteria were identified by bacterial culture following by multiplex Real-Time PCR.Results: Shigella spp and Campylobacter spp were sought by culture in all 200 samples. Enteropathogenic E. coli was sought only in 37 samples from all children under 2 years old. The bacterial culture was positive in 12 stool samples. Shigella spp and Salmonella spp. were isolated respectively in 5 (2.5%) and 3 samples (1.5%). Enteropathogenic E. coli was isolated in 10.8% (4/37) of the samples tested.The multiplex real-time PCR identified bacteria in 20 patients, including 17 cases of Shigella spp., 1 case of Salmonella spp. and 2 cases of Campylobacter spp.Conclusions: This study has highlighted the low frequency of 3 sought bacterial genera in stool samples. It has also demonstrated a significant difference between the culture and the multiplex Real-Time PCR method in the diagnosis of Shigella.

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Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC)

Microorganisms ◽

10.3390/microorganisms8111801 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1801

Author(s):

Michael Bording-Jorgensen ◽

Brendon D. Parsons ◽

Gillian A.M. Tarr ◽

Binal Shah-Gandhi ◽

Colin Lloyd ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Gene Detection ◽

Culture Positivity ◽

Hands On ◽

Culture Independent ◽

Chromogenic Agar ◽

Stool Samples

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes’ amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3–5 days until a sample is negative by culture.

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Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

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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Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples

Pathogens ◽

10.3390/pathogens10060656 ◽

2021 ◽

Vol 10 (6) ◽

pp. 656

Author(s):

Konstantin Tanida ◽

Andreas Hahn ◽

Kirsten Alexandra Eberhardt ◽

Egbert Tannich ◽

Olfert Landt ◽

...

Keyword(s):

Real Time ◽

Indigenous People ◽

Real Time Pcr ◽

Latent Class ◽

Enterocytozoon Bieneusi ◽

Enteric Disease ◽

Immunosuppressed Patients ◽

Study Population ◽

Stool Samples ◽

Pcr Assays

Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class analysis-based test comparison without a reference standard with perfect accuracy. Thereby, two one-tube real-time PCR assays and two two-tube real-time PCR assays targeting Enterocytozoon bieneusi and Encephalocytozoon spp. were included in the assessment with reference stool material (20), stool samples from Ghanaian HIV-positive patients (903), and from travelers, migrants and Colombian indigenous people (416). Sensitivity of the assays ranged from 60.4% to 97.4% and specificity from 99.1% to 100% with substantial agreement according to Cohen’s kappa of 79.6%. Microsporidia DNA was detected in the reference material and the stool of the HIV patients but not in the stool of the travelers, migrants, and the Colombian indigenous people. Accuracy-adjusted prevalence was 5.8% (n = 78) for the study population as a whole. In conclusion, reliable detection of enteric disease-associated microsporidia in stool samples by real-time PCR could be demonstrated, but sensitivity between the compared microsporidia-specific real-time PCR assays varied.

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High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

Journal of Fish Diseases ◽

10.1111/jfd.13053 ◽

2019 ◽

Vol 42 (10) ◽

pp. 1359-1368 ◽

Cited By ~ 4

Author(s):

Yolanda Torres‐Corral ◽

Clara Fernández‐Álvarez ◽

Ysabel Santos

Keyword(s):

Real Time ◽

High Throughput ◽

Real Time Pcr ◽

Melting Curve ◽

Curve Analysis ◽

Sybr Green ◽

Sybr Green I ◽

Melting Curve Analysis ◽

Identification And Quantification

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Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

Microorganisms ◽

10.3390/microorganisms9010054 ◽

2020 ◽

Vol 9 (1) ◽

pp. 54

Author(s):

Salem Belkessa ◽

Daniel Thomas-Lopez ◽

Karim Houali ◽

Farida Ghalmi ◽

Christen Rune Stensvold

Keyword(s):

Real Time ◽

Molecular Characterization ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Giardia Duodenalis ◽

Specific Pcr ◽

Stool Samples ◽

Pcr Targeting ◽

Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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