A multiplex molecular assay for detection of six penA codons to predict decreased susceptibility to cephalosporins in Neisseria gonorrhoeae

The emerging cephalosporin-resistant Neisseria gonorrhoeae poses an urgent threat to the continued efficacy of the last-line monotherapy for gonorrhea. Consequently, high-throughput, accurate, and reasonable molecular assays are urgently needed for strengthening antimicrobial-resistance surveillance in N. gonorrhoeae . In this study, we designed a high-throughput multiplex method that incorporates high-resolution melting technology and is based on a 6-codon assay (among the most parsimonious assays) developed following comprehensive and systematic reviews. The results showed that our method can precisely distinguish specific single-nucleotide polymorphisms in resistance-associated genes with a specificity and sensitivity of 100% and a detection limit as low as 10 copies per reaction. This method can be directly applied to clinical samples without cumbersome culture and successfully predicted all cephalosporin-resistant isolates (sensitivity: 100%). The method presented here represents a technique for rapid testing of antimicrobial resistance and will serve as a valuable tool for tailor-made antimicrobial therapy and for monitoring the transmission of cephalosporin-resistant strains.

Development of high-resolution melting (HRM) assay to differentiate the species of Shigella isolates from stool and food samples

Shigella species, a group of intracellular foodborne pathogens, are the main causes of bacillary dysentery and shigellosis in humans worldwide. It is essential to determine the species of Shigella in outbreaks and food safety surveillance systems. The available immunological and molecular methods for identifying Shigella species are relatively complicated, expensive and time-consuming. High resolution melting (HRM) assay is a rapid, cost-effective, and easy to perform PCR-based method that has recently been used for the differentiation of bacterial species. In this study, we designed and developed a PCR-HRM assay targeting rrsA gene to distinguish four species of 49 Shigella isolates from clinical and food samples and evaluated the sensitivity and specificity of the assay. The assay demonstrated a good analytical sensitivity with 0.01–0.1 ng of input DNA template and an analytical specificity of 100% to differentiate the Shigella species. The PCR-HRM assay also was able to identify the species of all 49 Shigella isolates from clinical and food samples correctly. Consequently, this rapid and user-friendly method demonstrated good sensitivity and specificity to differentiate species of the Shigella isolates from naturally contaminated samples and has the potential to be implemented in public health and food safety surveillance systems.

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia

Background Leishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia. Methods Using in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers. Results The results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples. Conclusions The results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions. Graphical Abstract

One-shot high-resolution melting curve analysis for KRAS point-mutation discrimination on a digital microfluidics platform

Proposed high-accuracy one-shot melting curve analysis on a digital microfluidics platform, enabling KRAS gene discrimination with single-nucleotide mutations in 3 seconds.

High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients

<b><i>Background:</i></b> High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. <b><i>Methods:</i></b> Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of <i>KEL</i>*<i>01</i> and <i>KEL</i>*<i>02</i>, <i>JK</i>*<i>01</i> and <i>JK</i>*<i>02</i>, <i>FY</i>*<i>01</i>, <i>FY</i>*<i>02</i>, and <i>FY</i>*<i>02 N.01</i>, <i>DI</i>*<i>01</i> and <i>DI</i>*<i>02</i>, <i>GYPB</i>*<i>03</i> and <i>GYPB</i>*<i>04</i>, <i>RHCE</i>*<i>E</i> and <i>RHCE</i>*<i>e,</i> and <i>DO</i>*<i>01</i> and <i>DO</i>*<i>02.</i> Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. <b><i>Results:</i></b> The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. <b><i>Conclusions:</i></b> We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

Detection of Species Substitution in the Meat Value Chain by High-Resolution Melting Analysis of Mitochondrial PCR Products

Substituting high commercial-value meats with similar cheaper or undesirable species is a common form of food fraud that raises ethical, religious, and dietary concerns. Measures to monitor meat substitution are being put in place in many developed countries. However, information about similar efforts in sub-Saharan Africa is sparse. We used PCR coupled with high-resolution melting (PCR-HRM) analysis targeting three mitochondrial genes—cytochrome oxidase 1 (CO1), cytochrome b (cyt b), and 16S rRNA—to detect species substitution in meat sold to consumers in Nairobi, Kenya. Out of 107 meat samples representing seven livestock animals, 11 (10.3%) had been substituted, with the highest rate being observed in samples sold as goat. Our results indicate that PCR-HRM analysis is a cost- and time-effective technique that can be employed to detect species substitution. The combined use of the three mitochondrial markers produced PCR-HRM profiles that successfully allowed for the consistent distinction of species in the analysis of raw, cooked, dried, and rotten meat samples, as well as of meat admixtures. We propose that this approach has broad applications in the protection of consumers against food fraud in the meat industry in low- and middle-income countries such as Kenya, as well as in developed countries.