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Difengpi, derived from the air-dried stem bark of Illicium difengpi and enlisted in the Chinese Pharmacopoeia for its therapeutic effect against common ailments, confronts challenges due to dwindling wild resources and intentional substitution with potentially harmful botanical relatives. The imperative need to authenticate this herbal remedy has led to the development of robust methods. Here, we integrated chloroplast mini-barcoding and high resolution melting (HRM) analysis to distinguish Difengpi from the reported adulterants. We assembled the complete chloroplast (cp) genomes of I. difengpi and substituted close relatives I. jiadifengpi and I. majus, and conducted an in-depth comparative analysis to screen divergent regions for exploiting as DNA mini-barcodes. Despite the conservativeness characterizing the whole cp genomes among these Illicium species, we identified some highly variable regions with promising potential as molecular markers for species identification. Subsequently, we designed DNA mini-barcodes and subjected them to HRM analysis to assess their efficacy in species discrimination. Melting profiles unveiled that mini-barcodes designed from four divergence regions trnL–trnF, trnF–ndhJ, ycf1–ndhF and rpl32–trnL exhibited substantial discriminatory power, distinctly differentiated I. difengpi from I. jiadifengpi, I. majus and I. verum. We tested ten commercially available Difengpi products from online stores and local traditional markets using these four mini-barcodes. All ten samples clustered closely with the reference I. difengpi with genotype confidence higher than 93 %, indicating the presence of the claimed species in these samples, sans any reported toxic adulterants. Consequently, we simulated Difengpi mimic samples utilizing I. jiadifengpi, I. majus and I. verum and subjected them to evaluate the practicability of these mini-barcodes. The outcomes confirmed the precision of the four mini-barcodes in accurately discerning the mimic samples. In conclusion, integrating taxon-specific DNA mini-barcodes with HRM analysis is an efficient strategy for the authentication of species identity within commercial herbal products.

Lumpy Skin disease (LSD) is an economically important disease in cattle caused by the LSD virus (LSDV) of the genus Capripoxvirus, while pseudocowpox (PCP) is a widely distributed zoonotic cattle disease caused by the PCP virus (PCPV) of the genus Parapoxvirus. Though both viral pox infections are reportedly present in Nigeria, similarities in their clinical presentation and limited access to laboratories often lead to misdiagnosis in the field. This study investigated suspected LSD outbreaks in organized and transhumance cattle herds in Nigeria in 2020. A total of 42 scab/skin biopsy samples were collected from 16 outbreaks of suspected LSD in five northern States of Nigeria. The samples were analyzed using a high-resolution multiplex melting (HRM) assay to differentiate poxviruses belonging to Orthopoxvirus, Capripoxvirus, and Parapoxvirus genera. LSDV was characterized using four gene segments, namely the RNA polymerase 30 kDa subunit (RPO30), G-protein-coupled receptor (GPCR), the extracellular enveloped virus (EEV) glycoprotein and CaPV homolog of the variola virus B22R. Likewise, the partial B2L gene of PCPV was also analyzed. Nineteen samples (45.2%) were positive according to the HRM assay for LSDV, and five (11.9%) were co-infected with LSDV and PCPV. The multiple sequence alignments of the GPCR, EEV, and B22R showed 100% similarity among the Nigerian LSDV samples, unlike the RPO30 phylogeny, which showed two clusters. Some of the Nigerian LSDVs clustered within LSDV SG II were with commonly circulating LSDV field isolates in Africa, the Middle East, and Europe, while the remaining Nigerian LSDVs produced a unique sub-group. The B2L sequences of Nigerian PCPVs were 100% identical and clustered within the PCPV group containing cattle/Reindeer isolates, close to PCPVs from Zambia and Botswana. The results show the diversity of Nigerian LSDV strains. This paper also reports the first documented co-infection of LSDV and PCPV in Nigeria. [ABSTRACT FROM AUTHOR]

Abstract Background The study describes the application of the multiplex high-resolution melting curve (MHRM) assay for the simultaneous detection of five common bacterial pathogens (Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Escherichia coli) directly from bronchoalveolar lavage samples. Results Our MHRM assay successfully identified all five respiratory pathogens in less than 5 h, with five separate melting curves with specific melt peak temperatures (Tm). The different Tm were characterized by peaks of 78.1 ± 0.4 °C for S. aureus, 83.3 ± 0.1 °C for A. baumannii, 86.7 ± 0.2 °C for E. coli, 90.5 ± 0.1 °C for K. pneumoniae, 94.5 ± 0.2 °C for P. aeruginosa. The overall sensitivity and specificity of MHRM were 100% and 88.8–100%, respectively. Conclusions Our MHRM assay offers a simple and fast alternative to culture approach for simultaneous detection of five major bacterial lower respiratory tract infection pathogens. Utilization of this assay can help clinicians initiate prompt and appropriate antimicrobial treatment, towards reducing the morbidity and mortality of severe respiratory infections.

The diagnosis of urinary tract infections (UTIs) is usually based on the results of urine culture, but it is time-consuming, labor-intensive and has a low sensitivity. The aim of this study was to develop multiplex high-resolution melting assay (MHRM) for the simultaneous detection of five common bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, and group B streptococci (GBS)) directly from urine samples. A total of 287 urine specimens were evaluated by HRM assay and the results were compared with the conventional culture method. Five different melt curves generated and differentiated five bacterial pathogens. The detection limit of the MHRM assay was 1.5 × 103CFU/ml for E. coli and K. pneumoniae and 1.5 × 102CFU/ml for S. saprophyticus, E. faecalis and GBS. Compared to culture, the specificity of the MHRM assay ranged from 99.3 to 100%, and sensitivity 100% for all test pathogens. The MHRM assay developed in the current study might be functional tool for the diagnosis of UTIs and has the potential for direct detection of the organism in the clinical samples. Additionally, it creates results in less than 5 h, helping clinicians to start treatment with appropriate antimicrobial agents. This method could be a useful supplement to urine culture. [ABSTRACT FROM AUTHOR]

The diagnosis of urinary tract infections (UTIs) is usually based on the results of urine culture, but it is time-consuming, labor-intensive and has a low sensitivity. The aim of this study was to develop multiplex high-resolution melting assay (MHRM) for the simultaneous detection of five common bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, and group B streptococci (GBS)) directly from urine samples. A total of 287 urine specimens were evaluated by HRM assay and the results were compared with the conventional culture method. Five different melt curves generated and differentiated five bacterial pathogens. The detection limit of the MHRM assay was 1.5 × 103 CFU/ml for E. coli and K. pneumoniae and 1.5 × 102 CFU/ml for S. saprophyticus, E. faecalis and GBS. Compared to culture, the specificity of the MHRM assay ranged from 99.3 to 100%, and sensitivity 100% for all test pathogens. The MHRM assay developed in the current study might be functional tool for the diagnosis of UTIs and has the potential for direct detection of the organism in the clinical samples. Additionally, it creates results in less than 5 h, helping clinicians to start treatment with appropriate antimicrobial agents. This method could be a useful supplement to urine culture.

Background: The study describes the application of the multiplex high-resolution melting curve (MHRM) assay for the simultaneous detection of five common bacterial pathogens (Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Escherichia coli) directly from bronchoalveolar lavage samples. Results: Our MHRM assay successfully identified all five respiratory pathogens in less than 5 h, with five separate melting curves with specific melt peak temperatures (Tm). The different Tm were characterized by peaks of 78.1 ± 0.4 °C for S. aureus, 83.3 ± 0.1 °C for A. baumannii, 86.7 ± 0.2 °C for E. coli, 90.5 ± 0.1 °C for K. pneumoniae, 94.5 ± 0.2 °C for P. aeruginosa. The overall sensitivity and specificity of MHRM were 100% and 88.8–100%, respectively. Conclusions: Our MHRM assay offers a simple and fast alternative to culture approach for simultaneous detection of five major bacterial lower respiratory tract infection pathogens. Utilization of this assay can help clinicians initiate prompt and appropriate antimicrobial treatment, towards reducing the morbidity and mortality of severe respiratory infections. [ABSTRACT FROM AUTHOR]

Abstract Background Pseudocowpox virus (PCPV) of the genus Parapoxvirus in the family Poxviridae causes pseudocowpox in cattle worldwide and presents a zoonotic concern. Most poxviruses produce diseases of similar clinical signs in affected animals, which are impossible to differentiate clinically or by serology. It is, therefore, vital to use molecular assays to rapidly identify the causative agents of poxvirus infections. This study aimed to detect, diagnose, and characterize the causative agent of pox-like skin lesions in a cattle herd in Zambia, initially suspected to be infected with Lumpy Skin Disease virus. Methods We used a High-Resolution Melting (HRM) analysis assay to detect the PCPV genome and sequenced the major envelope protein (B2L gene) for comparative sequence and phylogenetic analysis. Results Our field investigations showed cattle presenting atypical skin lesions and high morbidity within the herd. The laboratory diagnosis, based on the HRM assay revealed PCPV DNA in the samples. Phylogenetic and comparative sequence analyses confirmed PCPV in the samples and revealed genomic differences between samples collected in 2017 and 2018 from the same farm. Conclusion Our work is the first documented report of PCPV in Zambia. It shows the strength of molecular methods to diagnose pox-like infections in cattle and discriminate between diseases causing similar clinical signs. This rapid and accurate diagnosis improves the response time for more accurate veterinary interventions.

Difengpi, derived from the air-dried stem bark of Illicium difengpi and enlisted in the Chinese Pharmacopoeia for its therapeutic effect against common ailments, confronts challenges due to dwindling wild resources and intentional substitution with potentially harmful botanical relatives. The imperative need to authenticate this herbal remedy has led to the development of robust methods. Here, we integrated chloroplast mini-barcoding and high resolution melting (HRM) analysis to distinguish Difengpi from the reported adulterants. We assembled the complete chloroplast (cp) genomes of I. difengpi and substituted close relatives I. jiadifengpi and I. majus , and conducted an in-depth comparative analysis to screen divergent regions for exploiting as DNA mini-barcodes. Despite the conservativeness characterizing the whole cp genomes among these Illicium species, we identified some highly variable regions with promising potential as molecular markers for species identification. Subsequently, we designed DNA mini-barcodes and subjected them to HRM analysis to assess their efficacy in species discrimination. Melting profiles unveiled that mini-barcodes designed from four divergence regions trnL - trnF , trnF - ndhJ , ycf1 - ndhF and rpl32 - trnL exhibited substantial discriminatory power, distinctly differentiated I. difengpi from I. jiadifengpi , I. majus and I. verum . We tested ten commercially available Difengpi products from online stores and local traditional markets using these four mini-barcodes. All ten samples clustered closely with the reference I. difengpi with genotype confidence higher than 93 %, indicating the presence of the claimed species in these samples, sans any reported toxic adulterants. Consequently, we simulated Difengpi mimic samples utilizing I. jiadifengpi , I. majus and I . verum and subjected them to evaluate the practicability of these mini-barcodes. The outcomes confirmed the precision of the four mini-barcodes in accurately discerning the mimic samples. In conclusion, integrating taxon-specific DNA mini-barcodes with HRM analysis is an efficient strategy for the authentication of species identity within commercial herbal products., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

Background: Pseudocowpox virus (PCPV) of the genus Parapoxvirus in the family Poxviridae causes pseudocowpox in cattle worldwide and presents a zoonotic concern. Most poxviruses produce diseases of similar clinical signs in affected animals, which are impossible to differentiate clinically or by serology. It is, therefore, vital to use molecular assays to rapidly identify the causative agents of poxvirus infections. This study aimed to detect, diagnose, and characterize the causative agent of pox-like skin lesions in a cattle herd in Zambia, initially suspected to be infected with Lumpy Skin Disease virus. Methods: We used a High-Resolution Melting (HRM) analysis assay to detect the PCPV genome and sequenced the major envelope protein (B2L gene) for comparative sequence and phylogenetic analysis. Results: Our field investigations showed cattle presenting atypical skin lesions and high morbidity within the herd. The laboratory diagnosis, based on the HRM assay revealed PCPV DNA in the samples. Phylogenetic and comparative sequence analyses confirmed PCPV in the samples and revealed genomic differences between samples collected in 2017 and 2018 from the same farm. Conclusion: Our work is the first documented report of PCPV in Zambia. It shows the strength of molecular methods to diagnose pox-like infections in cattle and discriminate between diseases causing similar clinical signs. This rapid and accurate diagnosis improves the response time for more accurate veterinary interventions. [ABSTRACT FROM AUTHOR]

Infections caused by non-tuberculous mycobacteria (NTM) is increasing wordwide. Due to the difference in treatment of NTM infections and tuberculosis, rapid species identification of mycobacterial clinical isolates is necessary for the effective management of mycobacterial diseases treatment and their control strategy. In this study, a cost-effective technique, real-time PCR coupled with high-resolution melting (HRM) analysis, was developed for the differentiation of Mycobacterial species using a novel rpoBC sequence. A total of 107 mycobacterial isolates (nine references and 98 clinical isolates) were subjected to differentiation using rpoBC locus sequence in a real-time PCR-HRM assay scheme. From 98 Mycobacterium clinical isolates, 88 species (89.7%), were identified at the species level by rpoBC locus sequence analysis as a gold standard method. M. simiae was the most frequently encountered species (41 isolates), followed by M. fortuitum (20 isolates), M. tuberculosis (15 isolates), M. kansassi (10 isolates), M. abscessus group (5 isolates), M. avium (5 isolates), and M. chelonae and M. intracellulare one isolate each. The HRM analysis generated six unique specific groups representing M. tuberculosis complex, M. kansasii, M. simiae, M. fortuitum, M. abscessus–M. chelonae group, and M. avium complex. In conclusion, this study showed that the rpoBC-based real-time PCR followed by HRM analysis could differentiate the majority of mycobacterial species that are commonly encountered in clinical specimens.

Infections caused by non-tuberculous mycobacteria (NTM) is increasing wordwide. Due to the difference in treatment of NTM infections and tuberculosis, rapid species identification of mycobacterial clinical isolates is necessary for the effective management of mycobacterial diseases treatment and their control strategy. In this study, a cost-effective technique, real-time PCR coupled with high-resolution melting (HRM) analysis, was developed for the differentiation of Mycobacterial species using a novel rpoBC sequence. A total of 107 mycobacterial isolates (nine references and 98 clinical isolates) were subjected to differentiation using rpoBC locus sequence in a real-time PCR-HRM assay scheme. From 98 Mycobacterium clinical isolates, 88 species (89.7%), were identified at the species level by rpoBC locus sequence analysis as a gold standard method. M. simiae was the most frequently encountered species (41 isolates), followed by M. fortuitum (20 isolates), M. tuberculosis (15 isolates), M. kansassi (10 isolates), M. abscessus group (5 isolates), M. avium (5 isolates), and M. chelonae and M. intracellulare one isolate each. The HRM analysis generated six unique specific groups representing M. tuberculosis complex, M. kansasii, M. simiae, M. fortuitum, M. abscessus-M. chelonae group, and M. avium complex. In conclusion, this study showed that the rpoBC-based real-time PCR followed by HRM analysis could differentiate the majority of mycobacterial species that are commonly encountered in clinical specimens. [ABSTRACT FROM AUTHOR]