scholarly journals High resolution melting analysis of the 18S rRNA gene for the rapid diagnosis of bovine babesiosis

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Jinming Wang ◽  
Aihong Liu ◽  
Shangdi Zhang ◽  
Shandian Gao ◽  
Muhammad Rashid ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Simultaneous Detection ◽  
Babesia Bovis ◽  
Rrna Gene ◽  
Infected Cattle ◽  
Rt Pcr ◽  
Bovine Babesiosis ◽  
Blood Samples ◽  
Hrm Analysis

Abstract Background Bovine babesiosis is caused by protozoan parasites of the genus Babesia and presents a wide spectrum of clinical manifestations. Disease severity depends on the type of Babesia species infection. Generally, B. bovis and B. bigemina are considered as the causative agents of bovine babesiosis; in addition, Babesia ovata and B. major are a group of benign bovine piroplasms. Therefore, species identification is important for diagnosis, epidemiological investigations and follow-up management. Methods Real-time PCR combined with high resolution melting (RT-PCR-HRM) analysis was used to detect and discriminate four Babesia species infective to cattle, including Babesia bovis, B. bigemina, B. major and B. ovata. The melting profiles and melting temperatures (Tm) of the amplicon targeting 18S rRNA revealed differences that can discriminate the four Babesia spp. Sensitivity and specificity of the analytical method were evaluated using 50 blood samples collected from experimentally infected cattle and 240 blood samples from areas where bovine babesiosis is an issue. Results RT-PCR-HRM analysis allowed to detect and discriminate four Babesia spp. (B. bovis, B. bigemina, B. major and B. ovata), which were responsible for bovine babesiosis in China. The protocol was validated with DNA samples from experimentally infected cattle and field infection in cattle. Conclusions Our results indicate that RT-PCR-HRM is a fast and robust tool for the simultaneous detection and discrimination of four Babesia species that are responsible for bovine babesiosis in China. This approach is applicable for both field and experimental samples, thus it could be useful in epidemiological investigations and diagnoses of bovine babesiosis.

scholarly journals Simultaneous Detection of BovineTheileria and Babesia Species by Reverse Line Blot Hybridization

1999 ◽  
Vol 37 (6) ◽  
pp. 1782-1789 ◽  
Author(s):  
J. M. Gubbels ◽  
A. P. de Vos ◽  
M. van der Weide ◽  
J. Viseras ◽  
L. M. Schouls ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Bos Taurus ◽  
Blot Hybridization ◽  
Simultaneous Detection ◽  
Carrier State ◽  
Reverse Line Blot ◽  
Babesia Bovis ◽  
Rrna Gene ◽  
Blood Samples ◽  
Species Specific

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species ofTheileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to theT. sergenti-T. buffeli-T. orientalis group. TheBabesia species included were Babesia bovis,B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria andBabesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences fromBos taurus or other hemoparasites (Trypanosomaspecies, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigeminawere identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species.

scholarly journals High resolution melting analysis for the differentiation of Mycobacterium species

2014 ◽  
Vol 63 (10) ◽  
pp. 1284-1287 ◽  
Author(s):  
Rahizan Issa ◽  
Hatijah Abdul ◽  
Siti Hasmah Hashim ◽  
Valentinus H. Seradja ◽  
Nurul ‘Aishah Shaili ◽  
...  
Keyword(s):  
High Resolution ◽  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Rrna Gene ◽  
Hrm Analysis ◽  
Suitable Target ◽  
Effective Diagnosis ◽  
Dna Template

A quantitative real-time PCR (qPCR) followed by high resolution melting (HRM) analysis was developed for the differentiation of Mycobacterium species. Rapid differentiation of Mycobacterium species is necessary for the effective diagnosis and management of tuberculosis. In this study, the 16S rRNA gene was tested as the target since this has been identified as a suitable target for the identification of mycobacteria species. During the temperature gradient and primer optimization process, the melting peak (Tm) analysis was determined at a concentration of 50 ng DNA template and 0.3, 0.4 and 0.5 µM primer. The qPCR assay for the detection of other mycobacterial species was done at the Tm and primer concentration of 62 °C and 0.4 µM, respectively. The HRM analysis generated cluster patterns that were specific and sensitive to distinguished small sequence differences of the Mycobacterium species. This study suggests that the 16S rRNA-based real-time PCR followed by HRM analysis produced unique cluster patterns for species of Mycobacterium and could differentiate the closely related mycobacteria species.

scholarly journals High-Resolution Melting Analysis in Comparison with Microscopic Method: An Experimental Study to Diagnosis of Plasmodium Species Infections in Human

2020 ◽  
Author(s):  
Mahya ALLAHMORADI ◽  
Afsaneh MOTEVALLI HAGHI ◽  
Mehdi NATEGHPOUR ◽  
Mehdi MOHEBALI ◽  
Ahmad RAEISI ◽  
...  
Keyword(s):  
High Resolution ◽  
Mixed Infection ◽  
High Resolution Melting ◽  
Clinical Symptoms ◽  
National Laboratory ◽  
Simultaneous Detection ◽  
Plasmodium Species ◽  
Medical Sciences ◽  
Hrm Analysis ◽  
Melting Analysis

Background: Among the human parasitic diseases, malaria is the main cause of morbidity and mortality. To prevent the high mortality and tracking malaria elimination efforts, a prompt and sensitive diagnosis is essential.  This study aimed to compare High-Resolution Melting (HRM) and microscopic methods to diagnose Plasmodium falciparum and P. vivax. Methods: Eighty-one blood samples were collected from patients with clinical symptoms who were suspect to malaria in Chabahar district, southeastern Iran and also, from those who were referred to Malaria National Laboratory in the Tehran University of Medical Sciences, Tehran, Iran. Microscopic examination and HRM method were used to the diagnosis of Plasmodium parasites simultaneously. Results: Microscopic results revealed 45 positive cases (12 P. falciparum and 33 P. vivax) out of 81 collected samples while according to HRM analysis results 11 and 33 samples were identified as P. falciparum and P. vivax, respectively. HRM analysis also revealed 1 mixed infection of P. falciparum and P. malariae. Conclusion: HRM analysis provides a promising mean for simultaneous detection and discrimination of the Plasmodium spp. especially in mixed infection cases.

scholarly journals Differentiation of Mitragyna speciosa, a narcotic plant, from allied Mitragyna species using DNA barcoding-high-resolution melting (Bar-HRM) analysis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chayapol Tungphatthong ◽  
Santhosh Kumar J. Urumarudappa ◽  
Supita Awachai ◽  
Thongchai Sooksawate ◽  
Suchada Sukrong
Keyword(s):  
High Resolution ◽  
Dna Barcoding ◽  
High Resolution Melting ◽  
Dna Barcode ◽  
Herbal Medicines ◽  
Efficient Tool ◽  
Hrm Analysis ◽  
Mitragyna Speciosa ◽  
Leaf Powder

AbstractMitragyna speciosa (Korth.) Havil. [MS], or “kratom” in Thai, is the only narcotic species among the four species of Mitragyna in Thailand, which also include Mitragyna diversifolia (Wall. ex G. Don) Havil. [MD], Mitragyna hirsuta Havil. [MH], and Mitragyna rotundifolia (Roxb.) O. Kuntze [MR]. M. speciosa is a tropical tree belonging to the Rubiaceae family and has been prohibited by law in Thailand. However, it has been extensively covered in national and international news, as its abuse has become more popular. M. speciosa is a narcotic plant and has been used as an opium substitute and traditionally used for the treatment of chronic pain and various illnesses. Due to morphological disparities in the genus, the identification of plants in various forms, including fresh leaves, dried leaf powder, and finished products, is difficult. In this study, DNA barcoding combined with high-resolution melting (Bar-HRM) analysis was performed to differentiate M. speciosa from allied Mitragyna and to assess the capability of Bar-HRM assays to identify M. speciosa in suspected kratom or M. speciosa-containing samples. Bar-HRM analysis of PCR amplicons was based on the ITS2, rbcL, trnH-psbA, and matK DNA barcode regions. The melting profiles of ITS2 amplicons were clearly distinct, which enabled the authentication and differentiation of Mitragyna species from allied species. This study reveals that DNA barcoding coupled with HRM is an efficient tool with which to identify M. speciosa and M. speciosa-containing samples and ensure the safety and quality of traditional Thai herbal medicines.

scholarly journals A Forensic Detection Method for Hallucinogenic Mushrooms via High-Resolution Melting (HRM) Analysis

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 199
Author(s):  
Xiaochun Zhang ◽  
Huan Yu ◽  
Qi Yang ◽  
Ziwei Wang ◽  
Ruocheng Xia ◽  
...  
Keyword(s):  
High Resolution ◽  
Dna Barcoding ◽  
High Resolution Melting ◽  
Its Region ◽  
Its Sequencing ◽  
Melting Temperatures ◽  
Hrm Analysis ◽  
National Drug ◽  
Species Specific ◽  
Its1 And Its2

In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus Psilocybe for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of Psilocybe cubensis were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of Psilocybe cubensis and other fungi, which can be utilized for drug trafficking cases and forensic science.

scholarly journals Genotyping of GATA4 Gene Variant (G296S) in Malaysian Congenital Heart Disease Subjects by Real-Time PCR High Resolution Melting Analysis

2013 ◽  
Vol 32 (2) ◽  
pp. 152-157
Author(s):  
Nora Fawzi ◽  
Ramachandran Vasudevan ◽  
Patimah Ismail ◽  
Mazeni Alwi ◽  
Ahmad Fazli Abdul Aziz ◽  
...  
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
High Resolution ◽  
Congenital Heart ◽  
High Resolution Melting ◽  
New Technology ◽  
Cost Effective ◽  
Study Cohort ◽  
Hrm Analysis ◽  
Melting Analysis

Summary Background: Congenital heart disease (CHD) is the most common birth defect; however, the underlying etiology is unrecognized in the majority of cases. GATA-binding protein 4 (GATA4), a cardiac transcription factor gene, has a crucial role in the cardiogenesis process; hence, a number of heterozygote sequence variations were identified as a cause of CHD. G296S heterozygote variant is the most frequently reported GATA4 gene sequence alteration. This study aims to investigate the role of G296S variant of the GATA4 gene in Malaysian CHD subjects. Methods: We have investigated 86 Malaysian CHD subjects with cardiac septation defects for the presence of the GATA4 gene heterozygote variant (G296S) by the new technology of high resolution melting (HRM) analysis. Results: Genotyping of G296S (c.886G>A) by HRM analysis shows that all the sample genotypes were of the wild GG type genotype and the heterozygote mutant GA genotype was totally absent from this study cohort. Conclusions: The results of our study showed that the G296S variant of the GATA4 gene was not associated with the development of CHD in Malaysian subjects. The use of HRM analysis proved a cost-effective, high-throughput, specific and sensitive genotyping technique which eliminates the need for unnecessary sequencing.

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

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
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.

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