RESUMEN
BACKGROUND AND OBJECTIVE: Coxiella burnetii is the bacterial causative agent of Q fever in humans. Because Q fever can establish itself with an initial inoculation of fewer than ten C. burnetii cells, a sensitive detection method for C. burnetii infection is needed for early detection. We aimed to evaluate the effectiveness of a complementary locked primer (CLP)-based real-time PCR method for sensitive detection of C. burnetii infection. METHODS: To evaluate the ability of CLPs to enhance the efficiency of the real-time PCR assay for the C. burnetii IS1111 insertion sequence, the mean threshold cycle values from 20 real-time PCR replicates with either CLPs or conventional primers were determined using tenfold serial dilutions (102-108) of purified C. burnetii Nine Mile genomic DNA. In addition, the cross-reactivity between C. burnetii and 31 non-Coxiella species was examined. RESULTS: The CLP-based real-time PCR allowed specific and reliable detection of as few as 59 copies of the IS1111 element present in the genome of C. burnetii, which represents approximately 2.96 genome equivalents or three cells of C. burnetii. These results demonstrate the effectiveness of CLP-based real-time PCR for sensitive detection of C. burnetii infection. CONCLUSION: It can be concluded that the CLP-based real-time PCR assay is a more appropriate method for sensitive detection and quantification of C. burnetii than previously reported methods.
Asunto(s)
Coxiella burnetii/genética , Reacción en Cadena de la Polimerasa , Fiebre Q/diagnóstico , ADN Bacteriano/genética , Humanos , Sensibilidad y EspecificidadRESUMEN
Charcot-Marie-Tooth (CMT) disease and hereditary neuropathy with liability to pressure palsies (HNPP) are frequent forms of genetically heterogeneous peripheral neuropathies. Reciprocal unequal crossover between flanking CMT1A-REPs on chromosome 17p11.2-p12 is a major cause of CMT type 1A (CMT1A) and HNPP. The importance of a sensitive and rapid method for identifying the CMT1A duplication and HNPP deletion is being emphasized. In the present study, we established a molecular diagnostic method for the CMT1A duplication and HNPP deletion based on hexaplex PCR of 6 microsatellite markers (D17S921, D17S9B, D17S9A, D17S918, D17S4A and D17S2230). The method is highly time-, cost- and sample-saving because the six markers are amplified by a single PCR reaction and resolved with a single capillary in 3 h. Several statistical and forensic estimates indicated that most of these markers are likely to be useful for diagnosing the peripheral neuropathies. Reproducibility, as determined by concordance between independent tests, was estimated to be 100%. The likelihood that genotypes of all six markers are homozygous in randomly selected individuals was calculated to be 1.6 x 10(-4) which indicates that the statistical error rate for this diagnosis of HNPP deletion is only 0.016%.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/diagnóstico , Enfermedad de Charcot-Marie-Tooth/genética , Eliminación de Gen , Duplicación de Gen , Repeticiones de Microsatélite/genética , Enfermedades del Sistema Nervioso Periférico/genética , Reacción en Cadena de la Polimerasa/métodos , Alelos , Cromosomas Humanos Par 17/genética , Dosificación de Gen , Frecuencia de los Genes , Genoma Humano/genética , Humanos , Linaje , Fenotipo , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
Mutations of the KCNJ2 gene are a major underlying cause of Andersen-Tawil syndrome (ATS), a rare autosomal dominant inherited disorder that is characterized by periodic paralysis, cardiac arrhythmias, and developmental dysmorphic features. The KCNJ2 gene encodes an inward rectifying K(+) channel protein, Kir2.1, which plays an important role in maintaining the homeostasis of channel current in various cell types. We have identified two missense mutations of KCNJ2 (R218Q and M307I) in two Korean families diagnosed with ATS. The M307I mutation is a novel mutation, located at the intracellular C-terminal domain, which is known to be concerned with putative phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding and channel trafficking.
Asunto(s)
Síndrome de Andersen/genética , Pueblo Asiatico/genética , Predisposición Genética a la Enfermedad , Mutación/genética , Canales de Potasio de Rectificación Interna/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Análisis Mutacional de ADN , Cara/anomalías , Humanos , Corea (Geográfico)/etnología , Masculino , Datos de Secuencia Molecular , Linaje , Canales de Potasio de Rectificación Interna/químicaRESUMEN
The population genetic data of 18 X-chromosomal short tandem repeat (STR) markers DXS6807, DXS8378, DXS9895, DXS9902, DXS6810, DXS7132, DXS981, DXS6800, DXS9898, DXS6789, DXS101, DXS6797, GATA172D05, GATA165B12, HPRTB, GATA31E08, DXS8377, and DXS7423 were analyzed in samples of unrelated 220 males and 181 females from Korean population. The exact test for genotype distribution of the markers showed no significant deviation from the Hardy-Weinberg equilibrium. Allele frequencies between male and female samples were not significantly different in all examined markers. All examined males and females showed different hemizygotic haplotype and combined genotypes, respectively. Four cases of mutation were found in GATA172D05, GATA31E08, DXS7132, and HPRTB from the analysis of 95 father-child-mother trios. Details of X chromosomal STRs in Koreans would be useful in paternity tests and forensic purposes as well as whole X-chromosomal mapping studies.