Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

Liang, Zhanbei; Keeley, Ann

doi:10.1016/j.watres.2012.08.014

Cited by 41 publications

(28 citation statements)

References 53 publications

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“…On the other hand, it was also found that PMA treatment does not always lead to complete removal of the qPCR signal of dead bacteria. Fittipaldi et al (2010) summarized that incomplete suppression of the signal from dead will occur if (i) the amplicon size of the qPCR assay is short (Luo et al, 2010; Li and Chen, 2012; Schnetzinger et al, 2013); (ii) the target bacteria is at high concentration (Elizaquivel et al, 2012; Zhu et al, 2012; Li and Chen, 2013; Pacholewicz et al, 2013); (iii) the concentration of Mg 2 + in the PCR reaction is not adapted (Nocker et al, 2006); or (iv) the fat content of food sample is high (Yang et al, 2011), and may also vary according to the “killing” treatment (Nocker et al, 2007a; Kobayashi et al, 2010; Yang et al, 2011; Liang and Keeley, 2012). Additionally, the turbidity of food samples may hamper light penetration and samples dilution is required for a thorough light exposure.…”

Section: Limitations Of the Pma Approach And The Remedies To Address mentioning

confidence: 99%

Advances and Challenges in Viability Detection of Foodborne Pathogens

et al. 2016

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Foodborne outbreaks are a serious public health and food safety concern worldwide. There is a great demand for rapid, sensitive, specific, and accurate methods to detect microbial pathogens in foods. Conventional methods based on cultivation of pathogens have been the gold standard protocols; however, they take up to a week to complete. Molecular assays such as polymerase chain reaction (PCR), sequencing, microarray technologies have been widely used in detection of foodborne pathogens. Among molecular assays, PCR technology [conventional and real-time PCR (qPCR)] is most commonly used in the foodborne pathogen detection because of its high sensitivity and specificity. However, a major drawback of PCR is its inability to differentiate the DNA from dead and viable cells, and this is a critical factor for the food industry, regulatory agencies and the consumer. To remedy this shortcoming, researchers have used biological dyes such as ethidium monoazide and propidium monoazide (PMA) to pretreat samples before DNA extraction to intercalate the DNA of dead cells in food samples, and then proceed with regular DNA preparation and qPCR. By combining PMA treatment with qPCR (PMA-qPCR), scientists have applied this technology to detect viable cells of various bacterial pathogens in foods. The incorporation of PMA into PCR-based assays for viability detection of pathogens in foods has increased significantly in the last decade. On the other hand, some downsides with this approach have been noted, particularly to achieve complete suppression of signal of DNA from the dead cells present in some particular food matrix. Nowadays, there is a tendency of more and more researchers adapting this approach for viability detection; and a few commercial kits based on PMA are available in the market. As time goes on, more scientists apply this approach to a broader range of pathogen detections, this viability approach (PMA or other chemicals such as platinum compound) may eventually become a common methodology for the rapid, sensitive, and accurate detection of foodborne pathogens. In this review, we summarize the development in the field including progress and challenges and give our perspective in this area.

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Section: Limitations Of the Pma Approach And The Remedies To Address mentioning

confidence: 99%

Advances and Challenges in Viability Detection of Foodborne Pathogens

et al. 2016

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“…Reverse transcriptase real-time quantitative PCR (qRT-PCR) has aimed to replace vital dye staining as a measure of cyst and oocyst viability by detecting and quantifying changes in gene expression by measuring the level of heat-induced hsp70 mRNA, as well as other molecular viability indicators such as beta tubulin mRNA and 18S rRNA, in Cryptosporidium (Liang and Keeley, 2012) and Giardia (Baque et al, 2011). The labile nature of quantifying rRNA and especially mRNA, however, makes working with it more challenging.…”

Section: Molecular-based Detection Of Parasite Viability In Food Matrmentioning

confidence: 99%

“…Ethidium monoazide and propidium monoazide treatments coupled with traditional or real-time PCR or LAMP have been extensively used in bacteriology for the differentiation of viable and nonviable cells and, more recently, protozoa (Brescia et al, 2009). A good correlation between what is now called viability PCR and RT-qPCR for viability detection of fresh Cryptosporidium oocysts was reported following disinfection of oocysts in water samples (Liang and Keeley, 2012). A good correlation between what is now called viability PCR and RT-qPCR for viability detection of fresh Cryptosporidium oocysts was reported following disinfection of oocysts in water samples (Liang and Keeley, 2012).…”

Section: Molecular-based Detection Of Parasite Viability In Food Matrmentioning

confidence: 99%

Laboratory diagnostic methods

Traub

Cuttell

2015

Foodborne Parasites in the Food Supply Web

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“…A avaliação da infectividade de protozoários de veiculação hídrica com métodos que associam o cultivo celular e os corantes vitais com a reação em cadeia de polimerase (polymerase chain reaction -PCR) tem sido realizada e se mostra promissora (ALUM et al, 2012;LIANG & KEELEY, 2012).…”

Section: Introductionunclassified

Inativação de cistos de Giardia duodenalis por peroxidação e peroxidação assistida por radiação ultravioleta

Guimarães¹,

Santos

Franco³

et al. 2015

Eng. Sanit. Ambient.

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No presente trabalho foi avaliado um processo oxidativo avançado (POA), peróxido de hidrogênio assistido por radiação ultravioleta (H2O2/UV), e um processo oxidativo, peroxidação (H2O2), na inativação de cistos de Giardia duodenalis. Foram utilizadas concentrações de peróxido de hidrogênio de 15 mg.L-1 e 6x103 mg.L-1 e dose de UV de 44, 2.736 e 5.472 mW s.cm-2. A eficiência dos processos foi aferida com base na observação de danos à parede dos cistos por meio da reação de imunofluorescência direta (RID) e da microscopia eletrônica de varredura (MEV). Quando utilizada a menor dose de radiação e concentração de oxidante, o POA se mostrou mais eficiente que a peroxidação tanto em causar dano na parede de G. duodenalis quanto em reduzir o número de cistos. Com a aplicação do POA e da peroxidação houve uma redução média de 45,2 e 22,6% do número de cistos de G. duodenalis, sendo que, em média, 42 e 29,4% dos cistos restantes apresentaram danos, respectivamente.

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Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

Cited by 41 publications

References 53 publications

Advances and Challenges in Viability Detection of Foodborne Pathogens

Advances and Challenges in Viability Detection of Foodborne Pathogens

Laboratory diagnostic methods

Inativação de cistos de Giardia duodenalis por peroxidação e peroxidação assistida por radiação ultravioleta

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