A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples

Abstract A non-radio-labeled probe-based detection method was developed for rapid enumeration of Salmonella in seafood and water samples. A Salmonella-specific invA gene probe was developed using a digoxigenin-based non-radio labeling assay, which was evaluated with naturally contaminated seafood and water samples. The probe-based technique was further compared with the quantitative PCR assay. The method was specific for detection of different Salmonella serovars without any nonspecific hybridization with other Salmonella-related Enterobacteriaceae. The optimum labeling efficiency was determined for the labeled probe, and 10 pg/μL probe concentration was observed to be most efficient for detection of Salmonella colonies on nylon membrane. Quantification of Salmonella in naturally contaminated seafood and water samples (n = 21) was in the range 10–102 CFU/mL. The assay successfully quantified Salmonella in spiked seafood and water samples in the presence of background flora, and the entire assay was completed within 48 h. The probe-based assay was further evaluated with real-time PCR, and results showed that the assay was comparable to real-time PCR assay. Thus, this probe-based assay can be a rapid, useful, and alternative technique for quantitative detection of Salmonella in food, feed, and water samples.

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Multiplex Real-Time PCR Assay for Simultaneous Detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri

Journal of Clinical Microbiology ◽

10.1128/jcm.00875-06 ◽

2006 ◽

Vol 44 (10) ◽

pp. 3589-3595 ◽

Cited By ~ 230

Author(s):

Y. Qvarnstrom ◽

G. S. Visvesvara ◽

R. Sriram ◽

A. J. da Silva

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Naegleria Fowleri ◽

Pcr Assay ◽

Balamuthia Mandrillaris ◽

Nægleria Fowleri

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Molecular Detection of Adenovirus in Tap Water from Coastal Areas Of Karachi Pakistan:The Real-Time PCR Assay

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab191.299 ◽

2021 ◽

Vol 156 (Supplement_1) ◽

pp. S140-S140

Author(s):

A Kalam

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Low Income ◽

Water Samples ◽

Real Time Pcr ◽

Tap Water ◽

Watery Diarrhea ◽

Acid Extraction ◽

Pcr Assay ◽

Viral Contamination

Abstract Introduction/Objective Diarrhea is a major source of morbidity and mortality in low-income and middle-income countries. In underdeveloped countries, diseases caused by viruses identified in environmental samples cause major health problems. Little knowledge about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. Adenovirus which causes watery diarrhea, particular has been recognized as important causal pathogen. Adenovirus remains a global threat to public health and an indicator of inequity and lack of social development. Tap water samples from coastal sites in Karachi between 2019 and 2020 over a period of 11 months. The total of 40 tap water sample was examined for infectious Adenovirus by a real time polymerase chain reaction (PCR) amplification. Methods/Case Report This Pilot study is conducted on tap water samples from Karachi Pakistan, n=40 are processed. Extraction of nucleic acid from all filtered water samples collected with Sterivex filter units by using Qiagen DNeasy Power Water Sterivex Kit. As per the manufacturer’s instruction. Phocine herpesvirus(PhHV) is added as an external positive control to monitor the efficiency of nucleic acid extraction and amplification. TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) is being used in probe based real time PCR assay,the below 35 Ct value is considered as a positive sample. Results (if a Case Study enter NA) Results showed the total of 37.7% of the sources were positive for adenovirus.The level of viral contamination was moderate to high. Conclusion The results has been showed that no seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Further the Real time PCR assay increases the sensitivity and provides the high resolution of pathogen detection.

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