High-resolution melting PCR assay as a powerful tool for the epidemiological surveillance of tularemia in Western Europe

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.

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MALDI-TOF mass spectrometry and high-resolution melting PCR for the identification of Mycoplasma bovis isolates

BMC Veterinary Research ◽

10.1186/s12917-021-02870-5 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Aric J. McDaniel ◽

Rachel J. Derscheid

Keyword(s):

Mass Spectrometry ◽

High Resolution ◽

High Resolution Melting ◽

Clinical Manifestations ◽

Mycoplasma Bovis ◽

Pcr Assay ◽

Accurate Identification ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Abstract Background Mycoplasma bovis is an important pathogen of cattle worldwide. Many different clinical manifestations of infection can occur, including respiratory disease, arthritis, and mastitis, causing heavy losses to beef and dairy industries. Because Mycoplasma species are slow-growing and fastidious, traditional identification methods are not cost- or time-effective, and improved methods are sought to streamline laboratory processes. High-resolution melting PCR (HRM-PCR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are 2 relatively recent tools that are rapid and inexpensive to use; we tested 9 isolates of M. bovis using both assays. The HRM-PCR assay used universal mycoplasma primers for the 16S–23S intergenic spacer region (IGSR). Results The resulting melting profiles of the field isolates were indistinguishable from the reference strain, indicating accurate identification. For the MALDI-TOF MS, each M. bovis isolate was accurately identified. Mycoplasma arginini and Mycoplasma alkalescens isolates did not identify as M. bovis when tested by either assay. Conclusions Our work shows that either assay could be used to identify unknown M. bovis isolates. For future work, the MALDI-TOF MS library should be expanded to include more mycoplasmas, and the HRM-PCR assay should be tested on additional mycoplasmas to ensure that the melting profiles are sufficiently distinctive.

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A real time high-resolution melting PCR assay for detection and differentiation among sheep pox virus, goat pox virus, field and vaccine strains of lumpy skin disease virus

Molecular and Cellular Probes ◽

10.1016/j.mcp.2018.08.003 ◽

2018 ◽

Vol 41 ◽

pp. 57-60 ◽

Cited By ~ 3

Author(s):

Yana Pestova ◽

Olga Byadovskaya ◽

Alexander Kononov ◽

Alexander Sprygin

Keyword(s):

High Resolution ◽

Real Time ◽

Disease Virus ◽

Skin Disease ◽

High Resolution Melting ◽

Pcr Assay ◽

Lumpy Skin Disease ◽

Lumpy Skin Disease Virus ◽

Goat Pox Virus

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Development of high-resolution melting PCR (HRM-PCR) assay to identify native fungal species associated with the wheat endosphere

Journal of Applied Genetics ◽

10.1007/s13353-020-00578-0 ◽

2020 ◽

Vol 61 (4) ◽

pp. 629-635

Author(s):

Tomasz Cłapa ◽

Katarzyna Mikołajczak ◽

Lidia Błaszczyk ◽

Dorota Narożna

Keyword(s):

High Resolution ◽

Large Scale ◽

High Resolution Melting ◽

Low Cost ◽

Fungal Species ◽

Molecular Techniques ◽

Pcr Assay ◽

Fungal Isolates ◽

Large Scale Screening

Abstract Understanding the complexity and biodiversity of fungal communities associated with the wheat endosphere can facilitate the identification of novel strains that might be beneficial to the host plant. However, the differentiation and taxonomic classification of the endosphere-associated fungi with respect to various cultivars and plant organs are challenging, time-consuming, and expensive, even with the use of molecular techniques. In the present work, we describe a fast, simple, and low-cost method based on high-resolution melting PCR (HRM-PCR) for the identification and differentiation of wheat endogenous fungal isolates. Using this approach, we differentiated 28 fungal isolates, which belonged to five different genera, namely Alternaria, Penicillium, Epicoccum, Fusarium, and Trichoderma. Furthermore, the results of the study revealed that this method can allow large-scale screening of cultured samples.

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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.

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