Real-time PCR detection of Toxoplasma gondii in surface water samples in São Paulo, Brazil

In this study, a quantitative species-specific polymerase chain reaction (PCR) method to rapidly detect E. histolytica in water is developed. First, the specificity of E. histolytica PCR detection was verified by using species-specific primers of 16S-like rRNA genes to clearly differentiate it from the closely related amoebae species E. dispar and E. moshkovskii. The sensitivity of this method was subsequently determined using purified E. histolytica genomic DNA and culture cells as PCR reaction templates. Results indicated that conventional PCR visualized on 1% agarose gel was able to detect as low as 0.02 pg genomic DNA and 5 cells, while real-time PCR could detect 0.01 pg genomic DNA and 2 cells of E. histolytica. The protocols for E. histolytica PCR detection in real water samples were then optimized by spiking E. histolytica cells into tap water and reservoir raw water samples. A two-round centrifugation treatment to concentrate amoeba cells directly as a PCR template was the most effective way to detect E. histolytica in spiked tap water samples, while DNA extraction after concentrating amoeba cells was required for spiked reservoir raw water samples. The detection limit of 50 E. histolytica cells in 100 ml tap water was achieved in 2 h from sample collection to real-time PCR data readout. With these established protocols, 78 tap water samples, 11 reservoir raw water samples and 4 feed water samples from Singapore water supply systems were analyzed by both conventional PCR and real-time PCR methods. No E. histolytica cell was detected in tested samples.

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Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis

Water Science & Technology ◽

10.2166/wst.2011.838 ◽

2011 ◽

Vol 64 (12) ◽

pp. 2453-2459 ◽

Cited By ~ 2

Author(s):

G. N. van Blerk ◽

L. Leibach ◽

A. Mabunda ◽

A. Chapman ◽

D. Louw

Keyword(s):

Surface Water ◽

High Resolution ◽

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Curve Analysis ◽

Pcr Assay ◽

High Resolution Melt ◽

Gene Target ◽

Enrichment Step

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16–18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

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Serological survey of Rickettsia in equids from Vale do Paraíba, São Paulo, Brazil, and their tick identification and molecular investigation of Rickettsia

Brazilian Journal of Veterinary Research and Animal Science ◽

10.11606/issn.1678-4456.bjvras.2019.158159 ◽

2019 ◽

Vol 56 (4) ◽

pp. e158159

Author(s):

Claudia Iorio Budweg ◽

Amanda Oliveira de Sousa ◽

Tânia Regina Vieira de Carvalho ◽

Zahi Êni Santos Souza ◽

Maria Carolina de Azevedo Serpa ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Spotted Fever ◽

Sao Paulo ◽

Serological Response ◽

São Paulo ◽

Serological Survey ◽

Blood Samples ◽

Rickettsia Rickettsii ◽

Brazilian Spotted Fever

Brazilian spotted fever is a serious and lethal illness for humans and is caused by the Rickettsia rickettsii bacteria. In the state of São Paulo/SP (Brazil), the etiological agent of this disease is transmitted by the Amblyomma sculptum tick. It was already shown that horses infected with this bacteria produce a strong immune response and could be important sentinels for the detection of the disease in a proper region. The present investigation performed a serological survey in horses from five farms of Vale do Paraíba, São Paulo state, Brazil, searching for antibodies against, Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommatis, Rickettsia rhipicephali, and Rickettsia bellii. In each farm, ticks were also collected that were taxonomically identified and examined by real-time PCR for Rickettsia spp DNA. Blood samples were collected from 206 horses, and 334 ticks were picked up from these animals from January to December 2017. Eighty ticks wereA. sculptum and 254 Dermacentor nitens. Of the blood samples, 7.3% seroconverted to Rickettsia spp. Of these, 0.97% had a positive serological response to R. bellii. None of the 80 A. sculptum ticks were positive through real-time PCR for Rickettsia spp. Although there was no detection of ticks infected by Rickettsia spp in five farms of Paraíba Valley, the horses presented serological positive reactions against this agent. Thus, further large studies should be conducted in the area targeting hosts and vectors to generate data for control measures of the transmission of Brazilian spotted fever.

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Elevated mRNA Expression of FGF2 and TGFB1 Genes in Myelofibrosis and Essential Thrombocythemia Patients

Blood ◽

10.1182/blood.v124.21.5179.5179 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5179-5179

Author(s):

Daniela Prudente Teixeira Nunes ◽

Luciene Terezina Lima ◽

Guilherme Wataru Gomes ◽

Maria de Lourdes L. F. Chauffaille ◽

Maria Regina Regis Silva ◽

...

Keyword(s):

Mrna Expression ◽

Real Time ◽

Essential Thrombocythemia ◽

Real Time Pcr ◽

Sao Paulo ◽

World Health ◽

Mrna Levels ◽

São Paulo ◽

Mutation Status ◽

Allele Burden

Abstract Background: Myelofibrosis (MF) and essential thrombocythemia (ET) are clonal hematopoietic stem cell malignancies classified as myeloproliferative neoplasms (MPNs). FGF2 and TGF-β were associated with pathophysiology and progression of these diseases. Although FGF2 and TGF-β roles in angiogenesis and fibrosis are known, their mRNA expression in leukocytes of MPNs patients was not yet evaluated. Furthermore, the detailed mechanism of TGF-β signaling in the pathophysiology of these diseases remains unclear. In regular signaling process, Smads are a group of proteins critical for transmitting signals from TGF-β superfamily of the cell surface to the nucleus. Thus, Smads signaling plays an important role in the regulation of hematopoiesis and it could modulate TGF-β action in MPNs. Aims: The aim of this study was to investigate if there are differences in mRNA expression of FGF2, TGFB1 and SMADS 1 to 7 in MPNs patients and healthy controls and if it is related to JAK2 mutation status and allele burden. Methods: Fifteen primary myelofibrosis (PMF), 15 myelofibrosis post essential thrombocytemia (MPET), and 22 essential trombocythemia (ET) patients diagnosed according to the World Health Organization criteria (2008) were selected from the Hematology Department of the Federal University of de Sao Paulo and from the Hematology Division of the Catholic University of Sao Paulo. Other group with healthy subjects was also included. The control group was matched by age and gender with MPNs patients: 30 for PMF, 17 for MPET, and 34 for ET. Expressions of FGF2, TGFB1 and SMADs 1 to 7 mRNA were evaluated in total leukocytes by Real Time PCR, using Taqman assays. JAK2V617F mutation status and allele burden and MPL W515K/L analysis were performed in total leukocytes DNA by Real Time PCR, using Taqman MGB probes. Results: No difference was found between frequencies of JAK2V617F mutated subjects (P=0.520) and in allele burden percentage (P=0.415) in MPNs groups. Allele burden was also not associated with mRNA levels of studied genes in each group of MPNs, and none of the patients presented MPL W515K/L mutations. MF and ET patients had higher TGFB1 and FGF2 mRNA levels than its controls (P<0.05), while MPET patients had higher FGF2 levels than controls (P< 0.001). Both MPET and ET patients had higher SMAD6 expression than its controls (P<0.05), whereas ET patients also presented higher SMAD1 levels than controls (P=0.01). The SMADs expressions were similar in MF and their controls. Conclusions: Our findings suggest that FGF mRNA is overexpressed in leukocytes of MF, MPET and ET patients. It seems that higher TGFB1 mRNA levels do not affect the molecular signaling by SMADs in MF patients, although SMAD6 and SMAD1 mRNA levels in ET and MPET patients might be upregulated. Financing: FAPESP 2012/ 12957-5 Disclosures No relevant conflicts of interest to declare.

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Detection of Giardia intestinalis in water samples collected from natural water reservoirs and wells in northern and north-eastern Poland using LAMP, real-time PCR and nested PCR

Journal of Water and Health ◽

10.2166/wh.2017.039 ◽

2017 ◽

Vol 15 (5) ◽

pp. 775-787 ◽

Cited By ~ 8

Author(s):

Anna Lass ◽

Beata Szostakowska ◽

Krzysztof Korzeniewski ◽

Panagiotis Karanis

Keyword(s):

Surface Water ◽

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Nested Pcr ◽

Lamp Assay ◽

Giardia Intestinalis ◽

Detection Methods ◽

Rrna Gene ◽

Water Reservoirs

Giardia intestinalis is a protozoan parasite, transmitted to humans and animals by the faecal–oral route, mainly through contaminated water and food. Knowledge about the distribution of this parasite in surface water in Poland is fragmentary and incomplete. Accordingly, 36 environmental water samples taken from surface water reservoirs and wells were collected in Pomerania and Warmia-Masuria provinces, Poland. The 50 L samples were filtered and subsequently analysed with three molecular detection methods: loop-mediated isothermal amplification (LAMP), real-time polymerase chain reaction (real-time PCR) and nested PCR. Of the samples examined, Giardia DNA was found in 15 (42%) samples with the use of LAMP; in 12 (33%) of these samples, Giardia DNA from this parasite was also detected using real-time PCR; and in 9 (25%) using nested PCR. Sequencing of selected positive samples confirmed that the PCR products were fragments of the Giardia intestinalis small subunit rRNA gene. Genotyping using multiplex real-time PCR indicated the presence of assemblages A and B, with the latter predominating. The results indicate that surface water in Poland, as well as water taken from surface wells, may be a source of Giardia strains which are potentially pathogenic for humans. It was also demonstrated that LAMP assay is more sensitive than the other two molecular assays.

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