Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples

[EN] Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the ß-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower C t values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier C t values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits. © 2011 Springer-Verlag.This work was supported by the Spanish Ministerio de Ciencia e Innovacion grants AGL2005-07776-C03-03 and AGL2008-05275-C03-03/ALI). Part of this work was also funded by the Fondo Europeo de Desarrollo Regional (Feder) grant POICV 2000-2006. We thank staff at the wastewater treatment plants and the Entidad de Saneamiento de Aguas for assistance in sample collection.Alonso Molina, JL.; Amoros, I.; Cañigral Cárcel, I. (2011). Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples. Applied Microbiology and Biotechnology. 89(4):1203-1211. https://doi.org/10.1007/s00253-010-2984-6S1203121189

A novel real-time PCR assay for the detection of Helicobacter pullorum-like organisms in chicken products

A novel real-time PCR assay was developed for the direct detection in food of Helicobacter pullorum-like bacteria, which are occasionally associated with human enteric disease. Experiments using control strains showed that the realtime PCR assay was specific and reproducible, with a detection level of 1 colony-forming unit (CFU)/g. The assay was then applied to determine contamination rates in 30 samples of three types of chicken-meat products obtained from five retail outlets in Spain (Valencia); all of the samples were initially considered to be culture-negative for Helicobacter even after an enrichment period. H.pullorum-like DNA was detected in seven out of ten chicken carcasses and in one chicken-burger sample (without enrichment), as well as in one liver sample (after enrichment). Sequencing of three randomly selected PCR products confirmed concordance (99% homology) with the H. pullorum 16S rDNA gene. The advantages of real-time PCR over conventional PCR assays are the improved detection level, speed of testing, and validation of specificity by melting-point analysis. The fact that bacteria are frequently present in chicken carcasses sold in retail stores highlights the importance of more widely monitoring contamination rates. The novel assay described herein allows better assessment of potential human health risks posed by H. pullorum

Desarrollo de métodos moleculares para la detección y caracterización de bacterias patógenas emergentes del género Vibrio en aguas y alimentos

Vibrio parahaemolyticus y Vibrio vulnificus son microorganismos patógenos pertenecientes a la familia Vibrionaceae y al género Vibrio. Son microorganismos con morfología ligeramente curvada, gram negativos y oxidasa positivos. En cuanto a sus aspectos ecológicos son halófilos, por tanto suelen estar presentes en aguas marinas costeras y en el interior de moluscos, crustáceos y peces, y tienden a encontrarse en aguas cálidas. La patología que producen está asociada al consumo de mariscos, moluscos y pescado crudo o poco cocinado y a la exposición a aguas contaminadas. Ambas especies son consideradas patógenos emergentes debido al aumento en su incidencia y a la mayor distribución geográfica de los casos de intoxicación alimentaria, sobre todo en países asiáticos. La detección e identificación de ambas especies en alimentos de origen marino y agua, presenta gran dificultad, ya que los procedimientos convencionales son largos y tediosos, y pueden proporcionar falsos negativos. Los métodos de detección molecular pueden suponer una alternativa más rápida, sensible y fiable. Objetivos: Debido a esto, en este trabajo se han desarrollado métodos de detección y cuantificación de Vibrio spp., Vibrio parahaemolyticus y Vibrio vulnificus mediante PCR tradicional, PCR a tiempo real e hibridación in situ con sondas fluorescentes (FISH) para su aplicación en matrices alimentarias y en agua. En este estudio se han aplicado los distintos métodos a matrices ambientales (agua de playa y agua residual) y a alimentos de origen marino. Resultados: Los resultados muestran que la PCR a tiempo real es el método más rápido y sensible para la detección de las diferentes especies estudiadas, además de permitir la cuantificación del microorganismo en las muestras de forma fiable. En este trabajo hemos demostrado la presencia de bacterias del género Vibrio en ambos tipos de matrices y el potencial de aguas y alimentos de origen marino para actuar como vehículos de transmisión de V. parahaemolyticus y V. vulnificusCañigral Cárcel, I. (2011). Desarrollo de métodos moleculares para la detección y caracterización de bacterias patógenas emergentes del género Vibrio en aguas y alimentos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11799Palanci

Single-Nucleotide Polymorphism Array-Based Karyotyping of Acute Promyelocytic Leukemia

<div><p>Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), but additional chromosomal abnormalities (ACA) and other rearrangements can contribute in the development of the whole leukemic phenotype. We hypothesized that some ACA not detected by conventional techniques may be informative of the onset of APL. We performed the high-resolution SNP array (SNP-A) 6.0 (Affymetrix) in 48 patients diagnosed with APL on matched diagnosis and remission sample. Forty-six abnormalities were found as an acquired event in 23 patients (48%): 22 duplications, 23 deletions and 1 Copy-Neutral Loss of Heterozygocity (CN-LOH), being a duplication of 8(q24) (23%) and a deletion of 7(q33-qter) (6%) the most frequent copy-number abnormalities (CNA). Four patients (8%) showed CNAs adjacent to the breakpoints of the translocation. We compared our results with other APL series and found that, except for dup(8q24) and del(7q33-qter), ACA were infrequent (≤3%) but most of them recurrent (70%). Interestingly, having CNA or <i>FLT3</i> mutation were mutually exclusive events. Neither the number of CNA, nor any specific CNA was associated significantly with prognosis. This study has delineated recurrent abnormalities in addition to t(15;17) that may act as secondary events and could explain leukemogenesis in up to 40% of APL cases with no ACA by conventional cytogenetics.</p></div

Comparison of type, size and number of SNP-A abnormalities in reported APL series.

<p>*one Uniparental Tetrasomy has been included both in duplication and CN-LOH group.</p><p>CNA: Copy-Number Abnormality. CN-LOH: Copy-Neutral Loss of Heterozygocity.</p

Schematic representation of CNA adjacent to the translocation breakpoints found in our series.

<p>On the left panel, the Smooth Signal of chromosome 15 and 17 from case APL_32 are represented. Results from diagnosis sample are shown in blue and from complete remission sample, in green. On the right, chromosomes 15 and 17 are depicted with G-banding and arrows pointing the location of the <i>PML</i> and <i>RARA</i> genes. Areas shaded in blue show regions duplicated and in red, deleted. Panel (A) corresponds to isochromosome der(17)t(15;17); Panel (B) shows a small duplication of the <i>PML</i> gene, and in panel (C) both the <i>PML</i> and the <i>RARA</i> genes are duplicated. These two cases had a cryptic t(15;17) by CC, that was revealed by FISH. In panel (D) two small deletions are found distally to the translocation breakpoints.</p

Detailed list of abnormalities found in our series (n = 48).

<p>Detailed list of abnormalities found in our series (n = 48).</p

Karyogram of APL according to SNP-A analysis in our series.

<p>Coloured bars depict the extension of abnormalities. Gains appear in blue at the right of each chromosome; losses in red and CN-LOH in green, at the left side of it.</p

Heatmap and pie chart depicting abnormalities found in our series in addition to the t(15;17).

<p>Each column represents one patient. Abnormalities are listed in the Y axis and coloured in the corresponding row of the heatmap. Abnormalities detected by Conventional Cytogenetics (CC) (red) and <i>FLT3</i> mutations (green) are frequent events; however, SNP-A revealed an important proportion of patients with potentially leukemogeneic abnormalities (blue). Dup: duplication: del: deletion; CNA: copy-number abnormality.</p

Main characteristics of our series.

<p>*PML-RARA was diagnosed by FISH or RT-PCR.</p