Duplex real-time PCR assay for rapid identification ofStaphylococcus aureusisolates from dairy cow milk

Staphylococcus aureusisolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification ofStaph. aureusisolates, targeting bothnucandSa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detectnucorSa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124Staph. aureusisolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent withnucandSa442were revealed, while 2 isolates showed only the peak consistent withSa442. Four isolates bacteriologically identified asStaph. aureus, were PCR-negative and were further identified asStaph. pseudintermediusby sequencing.Staph. pseudintermediusand coagulase-negative staphylococci did not carrynucorSa442. The results showed the correct identification of all isolates, comprehending also coagulase—or nuc-negativeStaph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, onStaph. aureus-positive or—negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypicalStaph. aureusisolates causing dairy cow mastitis. Also, it could be applied directly to milk samples, to detectStaph. aureusmammary infections avoiding bacteriological analysis.

Download Full-text

Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture

Journal of Dairy Research ◽

10.1017/s0022029915000084 ◽

2015 ◽

Vol 82 (2) ◽

pp. 200-208 ◽

Cited By ~ 20

Author(s):

Heidi Hiitiö ◽

Rauna Riva ◽

Tiina Autio ◽

Tarja Pohjanvirta ◽

Jani Holopainen ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bacterial Species ◽

Bovine Mastitis ◽

Antimicrobial Treatment ◽

Reference Laboratory ◽

Pcr Assay ◽

Coagulase Negative Staphylococci ◽

Staph Aureus ◽

Milk Samples

Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85·7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83·0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97·0 and 95·8% compared with BC. For Staphylococcus spp., the corresponding figures were 86·7 and 75·4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37·0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.

Download Full-text

Rapid Detection and Differentiation ofClonorchis sinensisandOpisthorchis viverriniUsing Real-Time PCR and High Resolution Melting Analysis

The Scientific World JOURNAL ◽

10.1155/2014/893981 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 7

Author(s):

Xian-Quan Cai ◽

Hai-Qiong Yu ◽

Rong Li ◽

Qiao-Yun Yue ◽

Guo-Hua Liu ◽

...

Keyword(s):

High Resolution ◽

Real Time ◽

Real Time Pcr ◽

Genomic Dna ◽

High Resolution Melting ◽

Clonorchis Sinensis ◽

Public Health Problem ◽

Rapid Identification ◽

Cox1 Gene ◽

Liver Flukes

Clonorchis sinensisandOpisthorchis viverriniare both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification ofC. sinensisandO. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria ofC. sinensis. Moreover,C. sinensisandO. viverriniwere able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection ofC. sinensisandO. viverrini.

Download Full-text

Evaluation of High Resolution Melting (HRM) Analysis for Meat Species Identification of Raw and Cooked Meat

Separations ◽

10.3390/separations8080116 ◽

2021 ◽

Vol 8 (8) ◽

pp. 116

Author(s):

Peyman Gholamnezhad ◽

Hamed Ahari ◽

Gholamreza Nikbakht Brujeni ◽

Seyed Amir Ali Anvar ◽

Abbasali Motallebi

Keyword(s):

High Resolution ◽

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

High Resolution Melting ◽

Meat Products ◽

Pcr Assay ◽

Hrm Analysis ◽

Mitochondrial Cytochrome B ◽

Meat Samples

The current study aimed to examine a real-time PCR assay with high-resolution melting (HRM) analysis for the species identification of minced meat samples. Meat samples from several animal species were purchased and minced separately or as a mixture of two species. DNA was extracted from all meat samples and subjected to real-time PCR assay by amplifying species-specific mitochondrial cytochrome b regions. Regarding the meat mixtures, two separate melting curves with specific melt peak temperatures (Tm) were detected. Additionally, DNA from each species was quantified, based on the calibration curves. The results showed that a real-time PCR assay with HRM analysis is suitable for the species identification of meat products, and could be used for the detection of meat frauds.

Download Full-text

Genotype-specific real-time PCR combined with high-resolution melting analysis for rapid identification of red-spotted grouper nervous necrosis virus

Archives of Virology ◽

10.1007/s00705-017-3375-4 ◽

2017 ◽

Vol 162 (8) ◽

pp. 2315-2328 ◽

Cited By ~ 5

Author(s):

Dimitra K. Toubanaki ◽

Evdokia Karagouni

Keyword(s):

High Resolution ◽

Real Time ◽

Real Time Pcr ◽

High Resolution Melting ◽

Rapid Identification ◽

High Resolution Melting Analysis ◽

Nervous Necrosis Virus ◽

Necrosis Virus ◽

Melting Analysis

Download Full-text

PP-040 Real-time PCR assay for identification of Brucella abortus in bulk milk samples in Iran

International Journal of Infectious Diseases ◽

10.1016/s1201-9712(10)60108-7 ◽

2010 ◽

Vol 14 ◽

pp. S36-S37 ◽

Cited By ~ 1

Author(s):

S. Moshkelani ◽

M. Javaheri Koupaei ◽

S. Rabiei ◽

A. Doosti

Keyword(s):

Real Time ◽

Brucella Abortus ◽

Real Time Pcr ◽

Pcr Assay ◽

Milk Samples ◽

Bulk Milk

Download Full-text

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

European Journal of Inflammation ◽

10.1177/1721727x1201000308 ◽

2012 ◽

Vol 10 (3) ◽

pp. 329-334 ◽

Cited By ~ 2

Author(s):

D.M. Valero-Hervás ◽

P. Morales ◽

M.J. Castro ◽

P. Varela ◽

M. Castillo-Rama ◽

...

Keyword(s):

High Resolution ◽

Real Time ◽

Real Time Pcr ◽

High Resolution Melting ◽

Low Cost ◽

Complement C3 ◽

Amplification Refractory Mutation System ◽

Rt Pcr ◽

Dna Amount ◽

Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

Download Full-text