Development of a real-time PCR assay with an internal amplification control for the detection of spoilage fungi in fruit preparations

Cronobacter sakazakii is a severe virulent strain that is frequently detected in powdered infant formula (PIF). Therefore, it is necessary to develop a fast and specific detection method. The specificity of our newly developed quantitative real-time PCR (qRT–PCR) was validated with DNA from 46 strains. Among them, 12 C. sakazakii strains were correctly amplified, whereas no positive florescent signal was observed from 34 nontarget controls. The detection limit of C. sakazakii was about 110 CFU/mL in broth and 1100 CFU/g in PIF. After enrichment in buffered peptone water for 6 h, our developed qRT–PCR assay could reliably detect C. sakazakii when the inoculation level was as low as 2 CFU/25 g (0.08 CFU/g) in PIF. The growth of C. sakazakii could be inhibited by the presence of Lactobacillus pentosus and Bacillus cereus, which used a longer enrichment period before the isolation was accomplished. However, at 5 and 50 CFU/25 g inoculation levels of C. sakazakii in the presence of 4 × 106 CFU L. pentosus/25 g or of 2 × 104 CFU B. cereus/25 g, the qRT–PCR assay could detect the presence of Cronobacter even though these artificially spiked samples were negative in culture. Therefore, our results indicated that the qRT–PCR assay could detect samples containing inhibitors and could avoid false negatives by using an internal amplification control.

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Development of an internal amplification control system for a real-time PCR assay for detection of Neisseria meningitidis in CSF and EDTA blood

Pathology ◽

10.1097/pat.0000000000000098 ◽

2014 ◽

Vol 46 (4) ◽

pp. 344-347 ◽

Cited By ~ 4

Author(s):

Christopher J. McIver ◽

Sydney M. Bell ◽

Noel Er

Keyword(s):

Control System ◽

Neisseria Meningitidis ◽

Real Time ◽

Real Time Pcr ◽

Internal Amplification Control ◽

Pcr Assay ◽

Edta Blood

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Development of a real-time PCR assay with an internal amplification control for the screening of Shiga toxin-producingEscherichia coliin foods

Letters in Applied Microbiology ◽

10.1111/j.1472-765x.2009.02561.x ◽

2009 ◽

Vol 48 (5) ◽

pp. 554-559 ◽

Cited By ~ 27

Author(s):

F. Auvray ◽

C. Lecureuil ◽

F. Dilasser ◽

J. Taché ◽

S. Derzelle

Keyword(s):

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Internal Amplification Control ◽

Pcr Assay

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Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

Applied and Environmental Microbiology ◽

10.1128/aem.00460-07 ◽

2007 ◽

Vol 73 (18) ◽

pp. 5840-5847 ◽

Cited By ~ 227

Author(s):

Jessica L. Nordstrom ◽

Michael C. L. Vickery ◽

George M. Blackstone ◽

Shelley L. Murray ◽

Angelo DePaola

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

The United States ◽

Internal Amplification Control ◽

Most Probable Number ◽

Specific Marker ◽

Pcr Assay ◽

Probable Number ◽

The Real

ABSTRACT Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh + and trh + strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

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An Optimised Quantitative Real-Time PCR Assay for Listeria Monocytogenes Including and Internal Amplification Control

10.1051/iufost:20060642 ◽

2006 ◽

Author(s):

D. Rodríguez-Lázaro ◽

M. Pla ◽

M. Scortti ◽

H.J. Monzó ◽

J.A. Vázquez-Boland

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Internal Amplification Control ◽

Quantitative Real Time Pcr ◽

Pcr Assay

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Real-time PCR assay with an endogenous internal amplification control for detection and quantification of Anaplasma marginale in bovine blood

Ticks and Tick-borne Diseases ◽

10.1016/j.ttbdis.2019.101334 ◽

2020 ◽

Vol 11 (2) ◽

pp. 101334

Author(s):

Svetlana N. Kovalchuk ◽

Anna V. Babii ◽

Anna L. Arkhipova

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Anaplasma Marginale ◽

Internal Amplification Control ◽

Bovine Blood ◽

Pcr Assay ◽

Detection And Quantification

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Development of a Multiplex Real-Time PCR Assay with Internal Amplification Control for the Detection of Shigella Species and Enteroinvasive Escherichia coli

Journal of Food Protection ◽

10.4315/0362-028x-73.9.1618 ◽

2010 ◽

Vol 73 (9) ◽

pp. 1618-1625 ◽

Cited By ~ 22

Author(s):

DEANNE M. DEER ◽

KEITH A. LAMPEL

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Real Time ◽

Real Time Pcr ◽

The United States ◽

Internal Amplification Control ◽

Control Strain ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Shigella Species

Shigella species, particularly S. sonnei and S. flexneri, remain some of the leading bacterial etiological agents of gastrointestinal diseases in the United States and globally. The isolation and detection of these foodborne pathogens are critical for preventing the spread of disease and facilitating epidemiological investigations aimed at determining the source of a Shigella infection outbreak. A multiplex real-time PCR-based assay was developed that targets all four species of Shigella plus enteroinvasive Escherichia coli. The assay incorporates primers directed to the ipaH genes located on both the virulence plasmid and chromosome, the plasmid-encoded virulence gene mxiC, a mutated mxiC gene (mxiC::kan) that differentiates wild-type strains from a laboratory control strain, and an internal amplification control. More than 50 isolates of all four Shigella species were tested for inclusivity and specificity of the multiplex PCR assay, and more than 30 non-Shigella isolates were tested for exclusivity of the assay. The sensitivity of the assay was 1 to 3 CFU and 5 to 50 fg of target (total) DNA for the ipaH, mxiC, and mxiC::kan gene targets. The assay performed equally well and with no measurable inhibition in the Shigella target reactions when rinsates of several high-risk produce commodities (parsley, cilantro, alfalfa sprouts, and lettuce) were added to the reactions. This multiplex PCR assay is sensitive and specific and has the added dimension of discriminating all Shigella species from the positive control strain so that in any sample analysis other strains can be excluded as a source of contamination.

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