New Triplex Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Feces▿

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqManmgb and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 102 CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method

Toward an international standard for PCR-based detection of Escherichia coli O157 - Part 1. Assay development and multi-center validation

ilustracionesEscherichia coli del serogrupo O157 pertenece al patotipo de las enterohemorrágicas, la más virulenta de E. coli, porque además de producir endotoxinas, libera dos exotoxinas conocidas como toxinas Shiga, Stx1 y Stx2 (Stx, Shiga toxin), causantes de graves patologías que pueden ser letales, como Colitis Hemorrágica (CH) y Síndrome Urémico Hemolítico (SUH), para personas con un sistema inmune deficiente o poco desarrollado (niños menores de cinco años y personas de la tercera edad). Esta STEC O157 (Shiga-Toxin producing E. coli) puede contaminar alimentos como productos cárnicos, derivados lácteos y vegetales, encontrándose también en aguas de consumo no tratadas. En la actualidad, la detección y la cuantificación de microrganismos patógenos en diferentes matrices alimenticias representan un gran reto para la industria. La técnica molecular qPCR-SYBR Green, entre otras, es sensible, rápida y específica, permitiendo detectar ADN de STEC del serogrupo O157, en un proceso automatizado que reduce ampliamente los tiempos de los procedimientos, con obtención de resultados en cuestión de horas, con un mínimo de contaminaciones y falsos positivos. En esta investigación se validaron cuatro protocolos de qPCR-SYBR Green para la detección específica de los genes rfbE (dos secuencias de oligonucleótidos), stx1 y sxt2 expresados por E. coli del serogrupo O157 (ATCC 43895), en muestras de carne molida bovina contaminada de forma artificial. Los parámetros de validación desarrollados fueron la selectividad, la sensibilidad y la robustez. Además, se hizo una comparación entre la qPCR-SYBR Green y otros métodos tradicionales como los gold standar, para determinar la confiabilidad de los protocolos. Las qPCR desarrolladas presentaron eficiencias entre 77 % y 97 % y una elevada linealidad (R2 0,99). Los límites de corte para cada secuencia de primers fueron: 3,1667 x 10-2 ng µL-1 para rfbE (primers rfbE y O157); 1,7228 x 10-3 ng µL-1 para stx1 y 3,5185 x 10-3 ng µL-1 para stx2. Tanto la inclusividad y la exclusividad fueron del 100 %, así como la precisión analítica, valor predictivo positivo y negativo. Además, fueron procesos bastante robustos. En la matriz contaminada se logró detectar hasta 4 UFC mL-1. Por los resultados obtenidos, los protocolos de qPCR-SYBR Green podrían implementarse para rastrear la presencia de E. coli O157 en el análisis rutinario de carne molida bovina, o como una prueba diagnóstica sencilla, rápida, altamente sensible y específica. (Tomado de la fuente)Escherichia coli from serogroup O157 belongs to the enterohemorrhagic pathotype, the most virulent of E. coli, because in addition to producing endotoxins, it releases two exotoxins known as Shiga toxins, Stx1 and Stx2 (Stx, Shiga toxin), causing serious pathologies that can be lethal, such as Hemorrhagic Colitis (CH) and Hemolytic Uremic Syndrome (HUS), for people with a poor or poorly developed immune system (children under five and the elderly). This STEC (Shiga-Toxin producing E. coli) O157 can contaminate foods such as meat products, dairy products, vegetables, and is also found in untreated drinking water. Currently, the detection and quantification of pathogenic microorganisms in different food matrices represents a great challenge for the industry; the qPCR-SYBR Green molecular technique, among others, is sensitive, fast and specific, allowing the detection of STEC DNA from serogroup O157, in an automated process that greatly reduces procedure times, obtaining results in a matter of hours, with a minimum of contaminations and false positives. In this investigation, four qPCR-SYBR Green protocols were validated for the specific detection of the rfbE (two oligonucleotide sequences), stx1 and sxt2 genes expressed by E. coli from serogroup O157 (ATCC 43895), in samples of contaminated ground beef artificially. The validation parameters developed were selectivity, sensitivity and robustness. Also, a comparison was made between the qPCR-SYBR Green and other traditional methods such as the gold standard, to determine the reliability of the protocols. The developed qPCRs presented efficiencies between 77% and 97% and high linearity (R2 0.99). The cutoff limits for each sequence of primers were: 3.1667 x 10-2 ng µL-1 for rfbE (primers rfbE and O157); 1.7228 x 10-3 ng µL-1 for stx1 and 3.5185 x 10-3 ng µL-1 for stx2. Both inclusivity and exclusivity were 100%, as well as analytical precision, positive and negative predictive value. They were also quite robust processes. Up to 4 CFU mL-1 were detected in the contaminated matrix. Based on the results obtained, the qPCR-SYBR Green protocols could be implemented to track the presence of E. coli O157 in the routine analysis of bovine ground beef, or as a simple, rapid, highly sensitive and specific diagnostic test. (Tomado de la fuente)MaestríaMagister en Ciencias - QuímicaBioquímica, Biología Molecula