Recommendations for Validation of Real-Time PCR Methods for Molecular Diagnostic Identification of Botanicals

2019 ◽  
Vol 102 (6) ◽  
pp. 1767-1773 ◽  
Author(s):  
Steven G. Newmaster ◽  
Dhivya Shanmughanandhan ◽  
Prasad Kesanakurti ◽  
Hanan Shehata ◽  
Adam Faller ◽  
...  
Keyword(s):  
Real Time ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Target Material ◽  
Test Method ◽  
Alternative Methods ◽  
Molecular Diagnostic ◽  
Data Systems ◽  
Target Species ◽  
Commercial Laboratory

Background: PCR methods are the most commonly used DNA-based identity tool in the commercial food, beverage, and natural health product markets. These methods are routinely used to identify foodborne pathogens and allergens in food. Proper validation methods for some sectors have been established, while there are none in other markets, such as botanicals. Results: A survey of the literature indicates that some validation criteria are not addressed when developing PCR tests for botanicals. Objective: We provide recommendations for qualitative real-time PCR methods for validating identity tests for botanical ingredients. Methods: These include common criteria that underpin the development and validation of rigorous tests, including (1) the aim of the validation test, (2) the applicability of different matrix variants, (3) specificity in identifying the target species ingredient, (4) sensitivity in detecting the smallest amount of the target material, (5) repeatability of methods, (6) reproducibility in detecting the target species in both raw and processed materials, (7) practicability of the test in a commercial laboratory, and (8) comparison with alternative methods. In addition, we recommend additional criteria, according to which the practicability of the test method is evaluated by transferring the method to a second laboratory and by comparison with alternative methods. Conclusions and Highlights: We hope that these recommendations encourage further publication on the validation of PCR methods for many botanical ingredients. These properly validated PCR methods can be developed on small, real-time biotechnology that can be placed directly into the supply chain ledger in support of highly transparent data systems that support QC from the farm to the fork of the consumer.

Recommendations for Validation of Real-Time PCR Methods for Molecular Diagnostic Identification of Botanicals

2019 ◽  
Vol 102 (6) ◽  
pp. 1767-1773 ◽  
Author(s):  
Steven G Newmaster ◽  
Dhivya Shanmughanandhan ◽  
Prasad Kesanakurti ◽  
Hanan Shehata ◽  
Adam Faller ◽  
...  
Keyword(s):  
Real Time ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Target Material ◽  
Test Method ◽  
Alternative Methods ◽  
Molecular Diagnostic ◽  
Data Systems ◽  
Target Species ◽  
Commercial Laboratory

Abstract Background: PCR methods are the most commonly used DNA-based identity tool in the commercial food, beverage, and natural health product markets. These methods are routinely used to identify foodborne pathogens and allergens in food. Proper validation methods for some sectors have been established, while there are none in other markets, such as botanicals. Results: A survey of the literature indicates that some validation criteria are not addressed when developing PCR tests for botanicals. Objective: We provide recommendations for qualitative real-time PCR methods for validating identity tests for botanical ingredients. Methods: These include common criteria that underpin the development and validation of rigorous tests, including (1) the aim of the validation test, (2) the applicability of different matrix variants, (3) specificity in identifying the target species ingredient, (4) sensitivity in detecting the smallest amount of the target material, (5) repeatability of methods, (6) reproducibility in detecting the target species in both raw and processed materials, (7) practicability of the test in a commercial laboratory, and (8) comparison with alternative methods. In addition, we recommend additional criteria, according to which the practicability of the test method is evaluated by transferring the method to a second laboratory and by comparison with alternative methods. Conclusions and Highlights: We hope that these recommendations encourage further publication on the validation of PCR methods for many botanical ingredients. These properly validated PCR methods can be developed on small, real-time biotechnology that can be placed directly into the supply chain ledger in support of highly transparent data systems that support QC from the farm to the fork of the consumer.

Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

2005 ◽  
Vol 68 (6) ◽  
pp. 1217-1221 ◽  
Author(s):  
PAVEL KRCMAR ◽  
EVA RENCOVA
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Single Species ◽  
Absolute Quantification ◽  
Quantitative Detection ◽  
Vertebrate Species ◽  
Target Species ◽  
Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

scholarly journals A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e89395 ◽  
Author(s):  
Jiyoun Yeo ◽  
Erin L. Crawford ◽  
Thomas M. Blomquist ◽  
Lauren M. Stanoszek ◽  
Rachel E. Dannemiller ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Testing ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Testing ◽  
Ffpe Samples ◽  
Pcr Method

Developing a multiplex real-time PCR with a new pre-enrichment to simultaneously detect four foodborne bacteria in milk

2019 ◽  
Vol 14 (10) ◽  
pp. 885-898 ◽  
Author(s):  
Moezi Parichehr ◽  
Kargar Mohammad ◽  
Doosti Abbas ◽  
Khoshneviszadeh Mehdi
Keyword(s):  
Real Time ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Pcr Assay ◽  
Reliable Technique ◽  
E Coli ◽  
Foodborne Bacteria ◽  
Multiple Pathogens ◽  
Enrichment Step

Aim: The aim of this study is to formulate a new single nonselective pre-enrichment medium (ELSS) that can support the concurrent growth of four major foodborne pathogens containing E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica serovar Entertidis to develop a multiplex TaqMan Real-time PCR (mRT-PCR). Methods: The mRT-PCR with a new pre-enrichment was carried out for simultaneous detection and quantification of these foodborne bacteria. Results: By using mRT-PCR after 16 h pre-enrichment in ELSS, the detection limit of each pathogen was 1 CFU/25 ml contaminated milk, as well as inclusivity and exclusivity reached 100%. Conclusion: The mRT-PCR assay with pre-enrichment step is a fast and reliable technique for detecting single or multiple pathogens in food products.

Development of 23 individual TaqMan® real-time PCR assays for identifying common foodborne pathogens using a single set of amplification conditions

2014 ◽  
Vol 43 ◽  
pp. 35-40 ◽  
Author(s):  
Paola Cremonesi ◽  
Laura Francesca Pisani ◽  
Cristina Lecchi ◽  
Fabrizio Ceciliani ◽  
Pieranna Martino ◽  
...  
Keyword(s):  
Real Time ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Pcr Assays ◽  
Single Set

Parasitological versus molecular diagnosis of strongyloidiasis in serial stool samples: how many?

2017 ◽  
Vol 92 (1) ◽  
pp. 12-16 ◽  
Author(s):  
E. Dacal ◽  
J.M. Saugar ◽  
T. Soler ◽  
J.M. Azcárate ◽  
M.S. Jiménez ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Correct Diagnosis ◽  
Molecular Techniques ◽  
Maximum Sensitivity ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Techniques ◽  
Molecular Technique ◽  
Asymptomatic Patients ◽  
Stool Samples

AbstractStrongyloidiasis is usually an asymptomatic disease in immunocompetent patients, caused by Strongyloides stercoralis. However, in immunocompromised patients it can produce a severe clinical profile. Therefore, a correct diagnosis is necessary in these cases and in those chronic asymptomatic patients. The low sensitivity of classical parasitological techniques requires the analysis of multiple serial stool samples. Molecular diagnostic techniques represent an improvement in the detection of the parasite. The objective of this study was to evaluate the minimum number of samples necessary to achieve maximum sensitivity by real-time polymerase chain reaction (PCR). A total of 116 stool samples from 39 patients were analysed by direct microscopic observation, agar culture, Harada–Mori and real-time PCR, in one, two, three and four or more consecutive samples. After two serial samples, 6 out of 39 patients were positive by parasitological and molecular techniques, while 16 of them were real-time PCR positive, and all the patients detected by parasitology were also detected by the molecular technique, reaching 100.00% sensitivity versus 83.00% when analysing a single sample. These data also reflect apparently low specificity (51.52%) and positive predictive value (PPV) (27.27 %) values, due to the high number of cases detected by real-time PCR and not by parasitological techniques. These cases were confirmed as true positives when analysing three, four or more samples from the same patient. In conclusion, the application of molecular techniques decreases the number of serial stool samples necessary to give a diagnosis with the maximum sensitivity.

scholarly journals Unexpected cases in field diagnosis of African swine fever virus in Vietnam: The needs consideration when performing molecular diagnostic tests

2020 ◽  
Vol 10 (2) ◽  
Author(s):  
Anh Duc Truong ◽  
Duc Viet Ly ◽  
Thi Hao Vu ◽  
Van Tuan Hoang ◽  
Thi Chinh Nguyen ◽  
...  
Keyword(s):  
Binding Site ◽  
Real Time ◽  
Real Time Pcr ◽  
Virus Isolation ◽  
False Negative ◽  
African Swine Fever ◽  
Molecular Diagnostic ◽  
Conventional Pcr ◽  
Sequencing Method ◽  
Single Mismatch

Background: The first confirmed case of African swine fever (ASF) in Vietnam was reported officially in February 2019. To date, ASF virus (ASFV) have been detected in 63/63 provinces in Vietnam. Currently, real-time polymerase chain reaction (PCR) is considered to be a powerful tool for viral detection in field samples, including ASFV. However, some recent reports have suggested that mismatches in primer and probe binding regions may directly affect real-time PCR qualification, leading a false-negative result.Aim: This study aims to further examine a conflicting result obtained from two OIE recommended methods, conventional PCR and real-time PCR, for ASFV detection.Methods: Two ASF suspected pigs from different provinces in the north of Vietnam were selected for this study based on clinical signs and postmortem lesions. The different results obtained by OIE-recommended conventional PCR and real-time PCR were further analyzed by the Sanger sequencing method and virus isolation in combination with hemadsorption (HAD) test using porcine alveolar macrophages cells.Results: The results showed that when the primer sequence matched perfectly with the sequences of field isolates, a mutation in probe binding region was found, indicating that a single mismatch in the probe binding site may cause a false-negative result by real-time PCR in detecting ASFV in clinical samples in Vietnam. An agreement between conventional PCR, using PPA1/PPA2 primers and two golden standard methods, virus isolation in combination with HAD assay, and sequencing method was observed in this study.Conclusion: A single mismatch in the probe binding site caused a failse-negative result by realtime PCR method in field diagnosis of ASFV. The needs consideration when selecting the appropriate molecular diagnostic methods is based on the current databases of ASFV sequences,  particularly for epidemiological surveillance of ASF. Keywords: African swine fever, PCR, Pigs, Real-time PCR, Vietnam

scholarly journals Performance of Three Commercial Molecular Diagnostic Assays for the Simultaneous Detection of Mycoplasma genitalium and Macrolide Resistance

2021 ◽  
Author(s):  
Chloé Le Roy ◽  
Cécile Bébéar ◽  
Sabine Pereyre
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Molecular Diagnostic ◽  
Handling Characteristics ◽  
Significant Difference ◽  
Commercial Kit ◽  
Resistance Detection

The increasing frequency of macrolide resistance is an emerging issue in the treatment of Mycoplasma genitalium infection. Because evaluation of new commercial kit detecting M. genitalium and macrolide resistance is needed, we evaluated the performance and handling characteristics of the Allplex MG & AziR (Seegene), the Macrolide-R/MG ELITe MGB (ELITechGroup), and the ResistancePlus MG FleXible kits (SpeeDx-Cepheid) in comparison with an in-house real-time PCR and 23S rRNA gene sequencing used as reference. A total of 239 urogenital specimens (135 M. genitalium-positive and 104 M. genitalium-negative specimens) collected between April and December 2019 at the French National Reference Center for bacterial Sexually Transmitted Infections were assessed. The overall agreement for M. genitalium detection of the three commercial kits compared with the in-house real-time PCR was 94.6–97.6%, and there was no significant difference. A total of 97 specimens were found M. genitalium-positive with the three kits and were used to assess macrolide resistance detection. The clinical sensitivity for resistance detection was 74.5% (95% confidence interval 61.7–84.2%), 96.2% (87.2–99.0%), and 92.8% (82.7–97.1%) for the Allplex MG & AziR, Macrolide-R/MG ELITe MGB, and ResistancePlusMG FleXible kits, respectively. The sensitivity of the Macrolide-R/MG ELITe MGB kit was significantly higher than that of the Allplex MG & AziR kit. The clinical specificity for resistance detection of the three kits was 97.4–97.6%. The random-access possibility, input sample volume, and DNA extract availability for detecting resistance to other antibiotics may also influence the selection of a commercial kit by diagnostic laboratories.

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