A polymerase chain reaction-coupled high-resolution melting curve analytical approach for the monitoring of monospecificity of avianEimeriaspecies

Objective: The diagnostic sensitivity and specificity of conventional methods for superficial lymph node tuberculosis (LNTB) are not ideal. We evaluated several novel methods including Xpert Mycobacterium tuberculosis/rifampicin (Xpert MTB/RIF) technology, quantitative fluorescence Polymerase Chain Reaction (qPCR) and High-Resolution Melting Curve (HRMC) in the diagnosis of superficial lymph node TB. Methods: Specimens from eighty-one consecutive patients with suspected LNTB and thirteen cases with other lymph node disease were analyzed by Xpert MTB/RIF, qPCR, and HRMC. Results: Among 81 patients with clinical suspicion of LNTB, there were 74 (91.4%) cases positive Mycobacterium tuberculosis Complex (MTBC) of Xpert MTB/RIF, 60 (74%) positive of qPCR, 24 (29.6%) of positive of BACTEC MGIT960 culture, and 13 (16%) cases positive of Roche culture. 38 cases (46.9%) were diagnosed with LNTB. All test methods showed a diagnostic specificity of 100% for LNTB. The sensitivity of molecular biology techniques was significantly higher than that of the traditional diagnostic methods, and Xpert MTB/RIF was the most sensitive diagnostic assay. On Rifampinresistant detection, Xpert MTB/RIF detected three cases (3.7%) with rpoB gene mutation, and Mycobacterium tuberculosis susceptibility testing detected 2 rifampicin-resistant cases (2.4%) which were consistent with Xpert MTB/RIF results. In the Isoniazid-resistant, 7 cases (8.1) of isoniazid resistance mutations (8.1%) were detected by HNC and 1 case was confirmed by Isoniazid susceptibility test. Conclusion: Molecular detection increased the diagnostic sensitivity of LNTB and improved the detection sensitivity for rifampin and isoniazid resistance strain.

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Identification and differentiation of Campylobacter isolated from chicken meat using real-time polymerase chain reaction and high resolution melting analysis of hipO and glyA genes

Veterinary World ◽

10.14202/vetworld.2020.1875-1883 ◽

2020 ◽

Vol 13 (9) ◽

pp. 1875-1883

Author(s):

Ika Kartika Syarifah ◽

Hadri Latif ◽

Chaerul Basri ◽

Puji Rahayu

Keyword(s):

Polymerase Chain Reaction ◽

High Resolution ◽

Real Time ◽

High Resolution Melting ◽

Melting Curve ◽

Chicken Meat ◽

Chain Reaction ◽

Polymerase Chain ◽

Curve Patterns ◽

Campylobacter Spp

Background and Aim: Campylobacter species have been recognized as the most frequently identified bacterial cause of human gastroenteritis. The aims of this study were to identify Campylobacter jejuni and Campylobacter coli species isolated from chicken meat and to analyze the differences in the melting curve patterns of both species. Materials and Methods: A total of 105 chicken meat samples collected from slaughterhouses and retailers in six provinces in Indonesia were examined for the isolation and identification of Campylobacter spp. A total of 56 positive isolates of Campylobacter spp. were analyzed using the quantitative real-time polymerase chain reaction and high resolution melting method. Results: The prevalence of Campylobacter spp. in chicken meat was found to be 61.9%. Regarding the identification, 23 isolates (41.07%) were C. jejuni, 22 (39.29%) were C. coli, six (10.71%) were a mix between C. jejuni and C. coli, and five isolates (8.93%) were Campylobacter spp. All the C. jejuni and C. coli isolates produced varied melting curve patterns. Conclusion: The high prevalence of C. jejuni and C. coli in chicken meat in Indonesia indicates a high risk of the incidence of campylobacteriosis in humans.

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High-Resolution Melting-Based Quantitative Analysis of RASSF1 Methylation in Breast Cancer

Medicina ◽

10.3390/medicina49020014 ◽

2013 ◽

Vol 49 (2) ◽

pp. 14 ◽

Cited By ~ 1

Author(s):

Kristina Stuopelytė ◽

Kristina Daniūnaitė ◽

Aida Laurinavičienė ◽

Valerijus Ostapenko ◽

Sonata Jarmalaitė

Keyword(s):

Breast Cancer ◽

Polymerase Chain Reaction ◽

High Resolution ◽

Quantitative Methods ◽

High Resolution Melting ◽

Cost Effective ◽

Chain Reaction ◽

Specific Polymerase Chain Reaction ◽

Polymerase Chain ◽

Breast Tissues

Background and Objective. Breast cancer is the leading cause of death from cancer among women worldwide. The aberrant promoter methylation of tumor suppressor genes is a typical epigenetic alteration for breast cancer and can be detected in early carcinogenesis. High-throughput and cost-effective methods are needed for the early and sensitive detection of epigenetic changes in clinical material. The main purpose of our study was to optimize a high-resolution melting (HRM) assay for the reliable and quantitative assessment of RASSF1 gene methylation, which is considered one of the earliest epigenetic alterations in breast cancer. Material and Methods. A total of 76 breast carcinomas and 10 noncancerous breast tissues were studied by means of HRM and compared with the results obtained by means of quantitative methylation-specific polymerase chain reaction (QMSP) and methylation-specific polymerase chain reaction (MSP). Results. Both quantitative methods, HRM and QMSP, showed a similar specificity and sensitivity for the detection of RASSF1 methylation in breast cancer (about 80% and 70%, respectively). In breast cancer, the mean methylation intensity of RASSF1 was 42.5% and 48.6% according to HRM and QMSP, respectively. Both methods detected low levels of methylation (less than 5%) in noncancerous breast tissues. In comparison with quantitative methods, MSP showed a lower sensitivity (70%), but a higher specificity (80%) for the detection of RASSF1 methylation in breast cancer. Conclusions. HRM is as a simple, cost-effective method for the reliable high-throughput quantification of DNA methylation in clinical material.

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