Rapid Identification of Clinically Relevant
            <i>Mycobacterium</i>
            Species by Multicolor Melting Curve Analysis

Xu, Ye; Liang, Bin; Du, Chen; Tian, Xueshan; Cai, Xianhua; Hou, Yang; Li, Hui; Zheng, Rongrong; Li, Junlian; Liu, Yuqin; Wang, Kaili; Athar, Muhammad Ammar; Tan, Yi; Li, Qingge

doi:10.1128/jcm.01096-18

Cited by 36 publications

(29 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In clinical samples, it is difficult to detect mixed infections of NTM species and to identify the species involved. The current approach, using molecular probes specific for a single gene target, has limitations, especially when it comes to species identification [ 16 , 17 ]. The presence of more than one species of NTM in cultured specimens might not be apparent from colonial morphologies.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, there is no gold standard for detection and identification of multiple species infection of NTM. LPA and multiplex real-time PCR/melting curve analysis have been applied to detect mixed infections with different NTM species based on the 16S-23S rRNA intergenic transcribed spacer (ITS) region and achieved good performance using material directly from clinical samples [ 16 , 17 , 18 ]. However, the resolution of these techniques is still low because not all NTM species, especially newly discovered species, have not been included in assay development.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Whole-Genome Sequencing Analysis to Identify Infection with Multiple Species of Nontuberculous Mycobacteria

et al. 2021

View full text Add to dashboard Cite

Mixed infection with multiple species of nontuberculous mycobacteria (NTM) is difficult to identify and to treat. Current conventional molecular-based methods for identifying mixed infections are limited due to low specificity. Here, we evaluated the utility of whole-genome sequencing (WGS) analysis to detect and identify mixed NTM infections. Analytical tools used included PubMLST, MetaPhlAn3, Kraken2, Mykrobe-Predictor and analysis of heterozygous SNP frequencies. The ability of each to identify mixed infections of NTM species was compared. Sensitivity was tested using 101 samples (sequence sets) including 100 in-silico simulated mixed samples with various proportions of known NTM species and one sample of known mixed NTM species from a public database. Single-species NTM control samples (155 WGS samples from public databases and 15 samples from simulated reads) were tested for specificity. Kraken2 exhibited 100% sensitivity and 98.23% specificity for detection and identification of mixed NTM species with accurate estimation of relative abundance of each species in the mixture. PubMLST (99% and 96.47%) and MetaPhlAn3 (95.04% and 83.52%) had slightly lower sensitivity and specificity. Mykrobe-Predictor had the lowest sensitivity (57.42%). Analysis of read frequencies supporting single nucleotide polymorphisms (SNPs) could not detect mixed NTM samples. Clinical NTM samples (n = 16), suspected on the basis of a 16S–23S rRNA gene sequence-based line-probe assay (LPA) to contain more than one NTM species, were investigated using WGS-analysis tools. This identified only a small proportion (37.5%, 6/16 samples) of the samples as mixed infections and exhibited only partial agreement with LPA results. LPAs seem to be inadequate for detecting mixed NTM species infection. This study demonstrated that WGS-analysis tools can be used for diagnosis of mixed infections with different species of NTM.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Whole-Genome Sequencing Analysis to Identify Infection with Multiple Species of Nontuberculous Mycobacteria

et al. 2021

View full text Add to dashboard Cite

show abstract

“…However, in their analysis system, the prevalent mutation IVS6+5G>A was not included, and the other three prevalent mutations were detected in three PCR reactions. In other previous studies, the real-time PCR-high resolution melting analysis was reported to be suitable for screening the presence of SLC25A13 mutations, but required further sequencing to identify the mutation types ( 25 , 26 ). Tokuhara et al ( 29 ) reported that citrin was deficient in lymphocytes among patients with citrin deficiency and Western blot analysis of citrin protein in lymphocytes isolated from peripheral blood was established as an alternative diagnostic method for citrin deficiency even in patients without known genetic mutations.…”

Section: Discussionmentioning

confidence: 99%

“…Real-time-PCR-based multicolor melting curve analysis (MMCA) is a recently described detection method that allows the detection of multiple mutations in a single reaction, and has the benefits of speed, ease-of-use and cross-platform compatibility (22). The successful use of MMCA has been reported previously for the detection of genetic disease and pathogen typing (23)(24)(25). In this study, a novel MMCA method was developed that allows rapid screening of the four prevalent SLC25A13 mutations in one closed-tube reaction.…”

Section: Introductionmentioning

confidence: 99%

Rapid Genetic Diagnosis of Citrin Deficiency by Multicolor Melting Curve Analysis

Zeng¹,

Yang²,

Luo³

et al. 2021

Front. Pediatr.

View full text Add to dashboard Cite

Citrin deficiency caused by SLC25A13 genetic mutations is an autosomal recessive disease, and four prevalent mutations including c.851_854del, c.1638_1660dup, IVS6+5G>A, and IVS16ins3kb make up >80% of total pathogenic mutations within the Chinese population. However, suitable assays for detection of these mutations have not yet been developed for use in routine clinical practice. In the current study, a real-time PCR-based multicolor melting curve analysis (MMCA) was developed to detect the four prevalent mutations in one closed-tube reaction. The analytical and clinical performances were evaluated using artificial templates and clinical samples. All four mutations in the test samples were accurately genotyped via their labeling fluorophores and Tm values, and the standard deviations of Tm values were indicated to be <0.2°C. The limit of detection was estimated to be 500 diploid human genomes per reaction. The MMCA assay of 5,332 healthy newborns from southern China identified a total of 107 SLC25A13-mutation carriers, indicating a carrier rate of 2%. The genotypes of 107 carriers and 112 random non-carriers were validated using direct sequencing and Long-range PCR with 100% concordance. In conclusion, the assay developed in this study may potentially serve as a rapid genetic diagnostic tool for citrin deficiency.

show abstract

“…Numerous researchers have devoted their efforts in developing molecular methods to solve the problem of multiple detection in a reaction [33,34]. Currently, there are various studies focusing on the multiplex real-time PCR dissolution curve method [35,36], because the PCR dissolution curve method based on EvaGreen dye is rapid, efficient, sensitive, specific, and low cost [24,30]. e noteworthy characteristic of this methodology is the implementation of well-designed primers to make each detection target have different T m values.…”

Section: Discussionmentioning

confidence: 99%

Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis

Han

Tong

et al. 2020

Canadian Journal of Infectious Diseases and Medical Microbiolog

View full text Add to dashboard Cite

Background and Objective. Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis are primary respiratory bacterial pathogens contributing to morbidity and mortality in developing countries. This study evaluated the diagnostic performance of multiplex real-time PCR with fluorescence melting curve analysis (MCA) assay, which was used to detect eight respiratory bacterial pathogens simultaneously. Methods. A total of 157 sputum specimens were examined by multiplex real-time with fluorescence MCA, and the results were compared with the conventional culture method. Results. Multiplex real-time PCR with fluorescence MCA specifically detected and differentiated eight respiratory bacterial pathogens by different melting curve peaks for each amplification product within 2 hours and exhibited high repeatability. The limit of detection ranged from 64 to 102 CFU/mL in the multiplex PCR system. Multiplex real-time PCR with fluorescence MCA showed a sensitivity greater than 80% and a 100% specificity for each pathogen. The kappa correlation of eight bacteria ranged from 0.89 to 1.00, and the coefficient of variation ranged from 0.05% to 0.80%. Conclusions. Multiplex real-time PCR with fluorescence MCA assay is a sensitive, specific, high-throughput, and cost-effective method to detect multiple bacterial pathogens simultaneously.

show abstract

Rapid Identification of Clinically Relevant Mycobacterium Species by Multicolor Melting Curve Analysis

Cited by 36 publications

References 35 publications

Whole-Genome Sequencing Analysis to Identify Infection with Multiple Species of Nontuberculous Mycobacteria

Whole-Genome Sequencing Analysis to Identify Infection with Multiple Species of Nontuberculous Mycobacteria

Rapid Genetic Diagnosis of Citrin Deficiency by Multicolor Melting Curve Analysis

Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis

Contact Info

Product

Resources

About