Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks

ABSTRACTA specific real-time PCR quantification method combined with a propidium monoazide sample treatment step was developed to determine quantitatively the viable population of thePhotobacterium phosphoreumspecies group in raw modified-atmosphere-packed salmon. Primers were designed to amplify a 350-bp fragment of the gyrase subunit B gene (gyrB) ofP. phosphoreum. The specificity of the two primers was demonstrated by using purified DNA from 81 strains of 52 different bacterial species. When these primers were used for real-time PCR in pure culture, a good correlation (R2of 0.99) was obtained between this method and conventional enumeration on marine agar (MA). Quantification was linear over 5 log units as confirmed by using inoculated salmon samples. On naturally contaminated fresh salmon, the new real-time PCR method performed successfully with a quantification limit of 3 log CFU/g. A correlation coefficient (R2) of 0.963 was obtained between the PCR method and classic enumeration on MA, followed by identification of colonies (290 isolates identified by real-time PCR or by 16S rRNA gene sequencing). A good correlation with anR2of 0.940 was found between the new PCR method and an available specific conductance method forP. phosphoreum. This study presents a rapid tool for producing reliable quantitative data on viableP. phosphoreumbacteria in fresh salmon in 6 h. This new culture-independent method will be valuable for future fish inspection, the assessment of raw material quality in fish processing plants, and studies on the ecology of this important specific spoilage microorganism.

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Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Mouse Feces

Applied and Environmental Microbiology ◽

10.1128/aem.01906-15 ◽

2015 ◽

Vol 81 (19) ◽

pp. 6749-6756 ◽

Cited By ~ 62

Author(s):

Yun-Wen Yang ◽

Mang-Kun Chen ◽

Bing-Ya Yang ◽

Xian-Jie Huang ◽

Xue-Rui Zhang ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Bacterial Species ◽

Chronic Inflammatory Bowel Disease ◽

Trinitrobenzene Sulfonic Acid ◽

Rrna Gene ◽

Specific Primers ◽

Content Type

ABSTRACTMouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers forFirmicutes,Actinobacteria,Bacteroidetes,Deferribacteres, “CandidatusSaccharibacteria,”Verrucomicrobia,Tenericutes, andProteobacteriawere designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level.

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Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7430-7434.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7430-7434 ◽

Cited By ~ 77

Author(s):

Trevor G. Phister ◽

David A. Mills

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Primers ◽

Rrna Gene ◽

Dekkera Bruxellensis ◽

Pcr Assay ◽

Specific Pcr ◽

Spoilage Yeast ◽

Pcr Method ◽

26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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The ZKIR Assay, a Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

Applied and Environmental Microbiology ◽

10.1128/aem.02711-19 ◽

2020 ◽

Vol 86 (7) ◽

Cited By ~ 1

Author(s):

Elodie Barbier ◽

Carla Rodrigues ◽

Geraldine Depret ◽

Virginie Passet ◽

Laurent Gal ◽

...

Keyword(s):

Public Health ◽

Klebsiella Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Health Concern ◽

Complex Matrices ◽

Content Type ◽

Novel Method ◽

Pcr Method

ABSTRACT Klebsiella pneumoniae is of growing public health concern due to the emergence of strains that are multidrug resistant, virulent, or both. Taxonomically, the K. pneumoniae complex (“Kp”) includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here, we analyzed 1,001 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR (zur-khe intergenic region) assay, was developed and used to detect Kp in 96 environmental samples. The results were compared to a culture-based method using Simmons citrate agar with 1% inositol medium coupled to matrix-assisted laser desorption ionization–time of flight mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10−1 CFU g−1 after enrichment for 24 h in lysogeny broth supplemented with ampicillin, and it was 1.5 × 103 to 1.5 × 104 CFU g−1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 multilocus sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific, and sensitive novel method to detect the presence of Kp in complex matrices and indicates that Kp isolates from environmental samples differ from clinical isolates. IMPORTANCE The Klebsiella pneumoniae species complex Kp includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic-resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and we show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.

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Molecular Diagnosis of Invasive Aspergillosis and Detection of Azole Resistance by a Newly Commercialized PCR Kit

Journal of Clinical Microbiology ◽

10.1128/jcm.01032-17 ◽

2017 ◽

Vol 55 (11) ◽

pp. 3210-3218 ◽

Cited By ~ 33

Author(s):

Eric Dannaoui ◽

Frédéric Gabriel ◽

Manuel Gaboyard ◽

Gaëlle Lagardere ◽

Lucile Audebert ◽

...

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Clinical Samples ◽

Azole Resistance ◽

Rrna Gene ◽

28S Rrna ◽

Serum Samples ◽

Content Type ◽

28S Rrna Gene

ABSTRACTAspergillus fumigatusis the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance inA. fumigatusis worrisome. The aim of this study was to validate the new MycoGENIEA. fumigatusreal-time PCR kit and to evaluate its performance on clinical samples for the detection ofA. fumigatusand its azole resistance. This multiplex assay detects DNA from theA. fumigatusspecies complex by targeting the multicopy 28S rRNA gene and specific TR34and L98H mutations in the single-copy-numbercyp51Agene ofA. fumigatus. The specificity ofcyp51Amutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinicalA. fumigatusisolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and 13 patients without aspergillosis. The limit of detection was <1 copy for theAspergillus28S rRNA gene and 6 copies for thecyp51Agene harboring the TR34and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR34and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection ofA. fumigatusDNA and azole resistance due to TR34and L98H mutations in clinical samples.

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Analysis of community structures in anaerobic processes using a quantitative real-time PCR method

Water Science & Technology ◽

10.2166/wst.2005.0502 ◽

2005 ◽

Vol 52 (1-2) ◽

pp. 85-91 ◽

Cited By ~ 52

Author(s):

Y. Yu ◽

C. Lee ◽

S. Hwang

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rrna Gene ◽

Quantitative Real Time Pcr ◽

Community Structures ◽

Specific Primer ◽

Anaerobic Processes ◽

Promising Tool ◽

Pcr Method ◽

Group Specific Primer

The methanogenic community structures of four different anaerobic processes were characterized using a quantitative real-time PCR with group-specific primer and probe sets targeting the 16S rRNA gene (rDNA). The group specific primer and probe sets were developed and used to detect the orders Methanosarcinales, and the families Methanosarcinaceae and Methanosaetaceae. Two separate sets targeting the domains Archaea and Bacteria were also used. Each microbial population in different anaerobic processes was determined and the relative abundance in the system was compared with each other. Dominant methanogenic populations and the community structures in the processes were varied by hydraulic retention time and acetate concentration. This indicates that the real-time PCR method with the primer and probe sets is a promising tool to analyze community structures in anaerobic processes.

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Development and Validation of a Real-Time PCR Method To Quantify Rumen Protozoa and Examination of Variability between Entodinium Populations in Sheep Offered a Hay-Based Diet

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.200-206.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 200-206 ◽

Cited By ~ 53

Author(s):

Lucy C. Skillman ◽

Andrew F. Toovey ◽

Andrew J. Williams ◽

André-Denis G. Wright

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Primers ◽

18S Rrna Gene ◽

Rrna Gene ◽

Advantages And Disadvantages ◽

Pcr Efficiency ◽

Rumen Protozoa ◽

Pcr Method ◽

Serial Dilutions

ABSTRACT PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (ε) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R 2 = 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (100 and 106 entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.

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Detection of Plasmodium Infection by the illumigene Malaria Assay Compared to Reference Microscopy and Real-Time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.00806-17 ◽

2017 ◽

Vol 55 (10) ◽

pp. 3037-3045 ◽

Cited By ~ 22

Author(s):

Candace Rypien ◽

Barbara Chow ◽

Wilson W. Chan ◽

Deirdre L. Church ◽

Dylan R. Pillai

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Lamp Assay ◽

Content Type ◽

Pcr Method ◽

Blood Specimens ◽

Significant Cost Reduction ◽

Low Prevalence ◽

Peripheral Blood Smears ◽

The Tropics

ABSTRACT Malaria is one of the leading causes of infectious disease in travelers returning from the tropics. The diagnosis of malaria is typically performed by examining Giemsa-stained thick and thin peripheral blood smears, which is time consuming, labor intensive, and requires high levels of proficiency. Alternatively, loop-mediated isothermal amplification (LAMP) is a new molecular method, which is rapid, sensitive, and requires less capital equipment and technological training. We conducted a retrospective study comparing two formats of a commercial LAMP assay (Meridian illumi gene malaria [M] and malaria Plus [MP]) versus reference microscopy on archived blood specimens ( n = 140) obtained from unique returning travelers suspected of having malaria. Discrepant results were resolved by either repeat testing or a laboratory developed ultrasensitive real-time PCR method. On initial testing, the Meridian illumi gene M and MP kits had sensitivities of 97.3% (95% confidence interval [CI], 90.7 to 99.7%) and 100.0% (95.1 to 100.0%) and specificities of 93.8% (84.8 to 98.3%) and 91.5% (81.3 to 97.2%), respectively, versus reference microscopy. We project a significant cost reduction in low prevalence settings where malaria is not endemic with LAMP-based malaria screening given the excellent negative predictive value achieved with LAMP.

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Development of a Quantitative Real-Time PCR Method To Enumerate Total Bacterial Counts in Ready-to-Eat Fruits and Vegetables

Journal of Food Protection ◽

10.4315/0362-028x-69.10.2504 ◽

2006 ◽

Vol 69 (10) ◽

pp. 2504-2508 ◽

Cited By ~ 12

Author(s):

HAJIME TAKAHASHI ◽

HIROTAKA KONUMA ◽

YUKIKO HARA-KUDO

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fruits And Vegetables ◽

Bacterial Species ◽

Culture Method ◽

Plate Count ◽

Rrna Gene ◽

Pcr Assay ◽

Cycle Number ◽

Wide Range

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the β subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.

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New PCR-based assay for the identification of Pectobacterium carotovorum causing potato soft rot

Plant Disease ◽

10.1094/pdis-08-21-1676-re ◽

2021 ◽

Author(s):

M. Belén Suárez ◽

Marta Diego ◽

F. J. Feria ◽

M J Martín-Robles ◽

Sergio Moreno ◽

...

Keyword(s):

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Bacterial Species ◽

Intergenic Spacer ◽

Soft Rot ◽

Pcr Analysis ◽

Rrna Gene ◽

Accurate Identification ◽

Intergenic Spacer Region

Soft rot on potato tuber is a destructive disease caused by pathogenic bacterial species of the genera Pectobacterium and Dickeya. Accurate identification of the causal agent is necessary to ensure adequate disease management, since different species may have distinct levels of aggressiveness and host range. One of the most important potato pathogens is P. carotovorum, a highly heterogeneous species capable of infecting multiple hosts. The complexity of this species, until recently divided into several subspecies, has made it difficult to develop precise diagnostic tests. This study proposes a PCR assay based on the new pair of primers Pcar1F/R to facilitate the identification of potato isolates of P. carotovorum according to the most recent taxonomic description of this species. The new primers were designed on a variable segment of the 16S rRNA gene and the intergenic spacer region (ITS) of available DNA sequences from classical and recently established species in the genus Pectobacterium. The results of the PCR analysis of genomic DNA from 32 Pectobacterium and Dickeya strains confirmed that the Pcar1F/R primers have sufficient nucleotide differences to discriminate between P. carotovorum and other Pectobacterium species associated with damage to potato crops, with the exception of P. versatile, which improves the specificity of the currently available primers. The proposed assay was originally developed as a conventional PCR but was later adapted to the real-time PCR format for application in combination with the existing real-time PCR test for the potato-specific pathogen P. parmentieri. This should be useful for the routine diagnosis of potato soft rot.

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Development of a Peptide Nucleic Acid-Based Multiprobe Real-Time PCR Method Targeting thehsp65Gene for Differentiation among Mycobacterium abscessus Strains: TABLE 1

Journal of Clinical Microbiology ◽

10.1128/jcm.03424-14 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1403-1405 ◽

Cited By ~ 2

Author(s):

Kijeong Kim ◽

Byoung-Jun Kim ◽

Tae Sun Shim ◽

Seok-Hyun Hong ◽

Yoon-Hoh Kook ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Peptide Nucleic Acid ◽

Mycobacterium Abscessus ◽

Content Type ◽

Pcr Method

Recently, the need to distinguish between members of theMycobacterium abscessusgroup has gained increasing attention. Here, we introduced a novel peptide nucleic acid (PNA) real-time PCR method targeting thehsp65gene in order to distinguish between four subspecies within theM. abscessusgroup (M. abscessusand 3 types ofM. massiliense).

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