Genetic Analysis of Dystrophin Gene for Affected Male and Female Carriers with Duchenne/Becker Muscular Dystrophy in Korea

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked recessive disorders caused by mutation in dystrophin gene. We analyzed the results of a genetic test in 29 DMD/BMD patients, their six female relatives, and two myopathic female patients in Korea. As the methods developed, we applied different procedures for dystrophin gene analysis; initially, multiplex polymerase chain reaction was used, followed by multiplex ligation-dependent probe amplification (MLPA). Additionally, we used direct DNA sequencing for some patients who had negative results using the above methods. The overall mutation detection rate was 72.4% (21/29) in DMD/BMD patients, identifying deletions in 58.6% (17/29). Most of the deletions were confined to the central hot spot region between exons 44 and 55 (52.9%, 7/19). The percentage of deletions and duplications revealed by MLPA was 45.5% (5/11) and 27.2% (3/11), respectively. Using the MLPA method, we detected mutations confirming their carrier status in all female relatives and symptomatic female patients. In one patient in whom MLPA revealed a single exon deletion of the dystrophin gene, subsequent DNA sequencing analysis identified a novel nonsense mutation (c.4558G > T; Gln1520X). The MLPA assay is a useful quantitative method for detecting mutation in asymptomatic or symptomatic carriers as well as DMD/BMD patients.

Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction

OBJECTIVES: Detection of mutations associated to nucleos(t)ide analogs and hepatitis B virus (HBV) genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR) assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients. METHODS: HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples. RESULTS: The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1 percent) with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100 percent) at codon 180 and codon 204. Concordant results were observed for 99.4 percent of the 158 samples at codon 181 and 98.7 percent at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1 percent mutant plasmid in a background of wild-type plasmid. CONCLUSION: The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.

Assessment of rose bengal test in diagnosing Egyptian human brucellosis

A total of 30 patients suffering from brucellosis were suspected based on history taking, clinical manifestations and positive serum tube agglutination test [at titer >/= 1/160]. The followings were done for all cases; complete blood picture [differential leucocytic count] and liver function tests, serodiagnosis of Brucella [serum tube agglutination test [STAT] as well as Rose Bengal test [RBT] and PCR. The study aimed to analyze the diagnostic value of RBT as compared to STAT and PCR for human brucellosis, and to evaluate the sensitivity, specificity, accuracy, the cost and the time consuming of RBT as compared to STAT and PCR. There was a significant difference between diagnosis by RBT and both STAT >/= 1/640, and STAT >/= 1/1280. Also, there was a significant difference between PCR and both STAT>1/640, and STAT >/= 1/1280. No significant difference was detected between RBT in diagnosing acute and chronic infection. STAT >/= 1/320 proved to be better than STAT at other titers and RBT in diagnosis of brucellosis. RBT proved to be suitable as screening test regarding time [faster] and cost. But, STAT >/= 1/320 from a practical and economic point of views proved to be the best one in diagnosing human brucellosis

Establishment of the multiplex quantitative ligase chain reaction for detecting mutations of deafness genes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a low-cost, convenient and accurate multiplex quantitative ligase chain reaction (MQ-LCR) technique to detect the five common mutations in Chinese patients with deafness.</p><p><b>METHODS</b>Primers and probes for 5 common mutations of deafness genes, i.e., GJB2 gene 235delC and 299-300delAT, mtDNA A1555G, SLC26A4 gene IVS7-2 A>G and 2168A>G, were designed and synthesized. The technique for those mutations was established, and the reliability of the technique was tested in 98 patients with impaired hearing and 30 children with normal hearing, who were randomly selected from the ENT in Children's Hospital of Fudan University. The subjects were detected by MQ-LCR and direct DNA sequencing of PCR products, following a double-blind approach. Finally the results from the two methods were compared.</p><p><b>RESULTS</b>The results revealed 48 cases carried two mutations, 31 cases carried heterozygous mutations in the 98 deaf children, and 3 had heterozygous mutation in 30 normal controls. These results were consistent with that from DNA sequencing. No false positive and false negative result was obtained.</p><p><b>CONCLUSION</b>The MQ-LCR technique established in this study is of low-cost, convenience, accuracy, high sensitivity and high specificity. It is suitable for large-scale detection and preventive diagnosis of mutations in deafness.</p>

Studies on the molecular diagnosis and prenatal diagnosis of the spinal muscular atrophy carriers by multiplex ligation-dependent probe / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the application of the multiplex ligation-dependent probe amplification (MLPA) method in genetic and prenatal diagnosis for spinal muscular atrophy (SMA).</p><p><b>METHODS</b>Four patients, 16 parents and 4 fetuses from 8 SMA pedigrees were included. MLPA was performed for molecular analysis, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for the mutation detection of the 4 patients.</p><p><b>RESULTS</b>For all the four patients, the same homozygous deletion of the exons 7 and 8 of the survival motor neuron 1 (SMN1) gene, was detected by PCR-RFLP and MLPA. All fourteen parents from the 8 pedigrees were carriers of the SMN1 gene heterozygous deletion, except the mothers in pedigrees 1 and 4 in whom the mutations were different.</p><p><b>CONCLUSION</b>MLPA is a simple and efficient quantitative method for copy number analysis of the SMN genes. It can be used for the genetic diagnosis and prenatal diagnosis of the SMA patients and carriers.</p>

Comparison of real-time PCR and conventional PCR for detection of Leishmania (Leishmania) infantum infection: a mini-review

In recent years, the polymerase chain reaction (PCR) technique has significantly advanced towards expanding its use and versatility by working with quantitative real-time PCR (qPCR). Data from the literature show that both methods present interesting characteristics for the diagnosis of visceral leishmaniasis. The benefits of qPCR in relation to conventional PCR include speed, reproducibility and quantitative ability. In addition to operational advantages, qPCR is more sensitive and reproducible and may replace conventional PCR in diagnostic routines. Regarding visceral leishmaniasis, the possibility of deployment of real-time PCR in highly complex diagnoses (reference services) in endemic areas will facilitate a swift and safe return for patients. Moreover, the use of a technique that possesses elevated diagnostic sensitivity, and can monitor therapy and prevent relapses promotes broader prospects for the disease control.

Red-pink colony-producing Pseudomonas sp. is the causative agent of mass mortality in larvae and post-larvae of Litopenaeus vannamei raised in hatcheries in south Iran

This study aimed to identify the causative of the mass mortality observed in zoeal to post-larval shrimp raised in hatcheries in south Iran. For three consecutive months, samples of nauplii and zoea of Litopenaeus vannamei were collected from an affected hatchery located in the province of Bushehr. Upon culture on marine agar, bacterial colonies that produced white, orange, yellow and red pigments were identified. In the hatcheries in which mass mortality was observed, the water columns of the affected tanks exhibited a red-pink color. Therefore, the bacteria that produced red pigment were selected for further phenotypic characterization using polymerase chain reaction [PCR] and virulence bioassays. Our results indicate that this bacterium belonged the genus Pseudomonas and that it was identical to P. mesophilica and/ anguilliseptica. PCR analysis of this bacterium revealed the production of a 150-bp band that was consistent with the Pseudomonas genus. To determine the pathogenicity of the isolated bacteria in nauplii and post-larvae of L vannamei, we performed bioassay experiments by bath immersion at 27-28°C. Our results showed that culture of nauplii and post-larvae of L. vannamei with the bacteria at a concentration of 1.5-2.0[x] 10[5] CFU/mL in marine broth resulted in a 100% mortality rate 24-48 h post-challenge. In contrast, there was no mortality in the nauplii and post-larvae that were cultured in the absence of bacteria. Upon pathological examination, we found that the color of the larvae was abnormal and pink, with acute necrosis of the entire body 48 h post-challenge

Detection of microdeletion in Williams syndrome by multiplex ligation-dependent probe amplification / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a method of multiplex ligation-dependent probe amplification (MLPA) for clinical screening of Williams syndrome (WS) and for routine use in WS diagnosis.</p><p><b>METHODS</b>Probes for MLPA were designed according to the frequent deletion regions, and used to screen the two patients suspected with Williams syndrome, and the density of the bands were analyzed with software. Linkage analysis using polymorphic markers was performed to confirm the positive result of MLPA.</p><p><b>RESULTS</b>The MLPA data indicated that the two children had possible microdeletions in the WS critical region. The deletions were confirmed and both were maternal origin by polymorphism analysis.</p><p><b>CONCLUSION</b>MLPA is a quick and convenient method for detecting deletion or duplication mutations. It can provide reliable and helpful information for clinical diagnose of Williams syndrome.</p>

Detection of the genetic abnormalities in patients with mental retardation using multiplex ligation-dependent probe amplification assay / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between subtelomeric rearrangements and idiopathic mental retardation (MR).</p><p><b>METHODS</b>Thirty unrelated patients were recruited using strict selection criteria. Patients were screened by multiplex ligation-dependent probe amplification (MLPA) for subtelomeric imbalance.</p><p><b>RESULTS</b>Five subtelomeric deletions/duplications were identified. They were: 4p deletion, 21p duplication, 10p duplication combined with 4p deletion, 15p duplication, and 9p deletion combined with 3p duplication. These subtelomeric rearrangements were previously unidentified by conventional technique.</p><p><b>CONCLUSION</b>Children with unexplained mental retardation are related with subtelomeric rearrangements. MLPA is a rapid and an effective technique for detecting genetic abnormalities in patients with idiopathic MR.</p>

Hepatitis B viral load in various categories of chronic hepatitis B carriers

To determine serum hepatitis B virus [HBV] DNA levels by Real-time Polymerase chain reaction [PCR] in different categories of treatment-naive patients with chronic HBV infection in context with Hepatitis B serology and serum Alanine aminotransferase [ALT] levels. Cross-sectional study. A total of 122 chronic hepatitis B carriers, including 79 low grade carriers [Anti-HBe positive HBeAg negative], 40 high grade carriers [HBeAg positive, Anti-HBe negative] and 3 intermediate grade carriers [Both HBeAg and Anti-HBe negative] were evaluated for HBV DNA levels and serum ALT levels. The serum HBV DNA levels of the low grade carriers with normal ALT levels [<40 IU/L] were significantly lower than the low-grade carriers with raised ALT levels [mean viral load 3x10[3] vs. 1.6x10[6] copies/mL; p=0.0003]. The HBV DNA levels of the high grade carriers were significantly higher than those of the low grade carriers with normal ALT levels [mean viral load 6.4x10[7]vs. 3x10[3] copies/mL; p=0.0007] and than those of low grade carriers with raised ALT levels [mean viral load 6.4x10[7] vs. 1.6x10[6] copies/ mL; p=0.03]. The results show that HBV DNA levels vary in different categories of chronic hepatitis B carriers and when evaluated by a sensitive quantitative PCR assay the HBV DNA levels can be used for differentiation between HBeAg-negative chronic hepatitis B and inactive hepatitis B surface antigen carrier state

Detección de Chlamydia trachomatis en orina por LCR: aplicación del método de mezcla de muestras biológicas / Ligase chain reaction testing of pooled urine specimens to diagnose Chlamydia trachomatis infection

Objective. The purpose of this study was to assess the utility and validity of pooling urine samples for molecular diagnosis of Chlamydia trachomatis infection. Material and methods. Of 1,220 urine samples collected from Mexican female and male adolescents, 305 pools were composed of fourth individual samples each, based on a calculation of optimal pool size. These were processed by ligase chain reaction (LCR) for the detection of C. trachomatis. Positive and gray-zone pools were reanalyzed individually. Cost savings were calculated comparing actual costs of testing to the cost that would have been incurred testing all 1,220 samples individually. Results.Pools results were: 56 positive, 19 gray-zones and 230 negative. Following individual retesting of positive and gray-zone pools, 59 cases of C. trachomatis infection were identified (4.8% prevalence). Thus, a total of 601 LCR tests were performed, for a 50.4% savings considering only the direct cost of the test. Conclusions.Our experience shows that sample pooling is both a reliable and convenient tool for CT surveillance in our setting. It should be considered in other similar settings where limited resources constraint surveillance of STIs.

Objetivo. Evaluar la validez y conveniencia de la estrategia de la mezcla de muestras de orinas para el diagnóstico molecular de Chlamydia trachomatis (CT). Material y métodos. A partir de 1,220 muestras de orina recolectadas de jóvenes de uno y otro sexos, se conformaron 305 mezclas con cuatro alícuotas de muestras individuales, previo cálculo del tamaño óptimo de la mezcla. A continuación se determinó la presencia de ácidos nucleicos de clamidia en esas mezclas, mediante el método de reacción en cadena de la ligasa. Las mezclas positivas o en zona gris fueron reanalizadas de manera individual (cuatro pruebas adicionales). El número final de pruebas realizadas se comparó con el total de pruebas que se habrían efectuado individualmente. Resultados. Del total de mezclas analizadas, 230 resultaron negativas, 56 fueron positivas y 19 más se ubicaron en zona gris. Una vez reanalizadas de manera individual las mezclas positivas y las de zona gris, se obtuvieron 59 muestras de orina positivas a clamidia (prevalencia de 4.81%). De esta manera, el número total de pruebas efectuadas fue de 605 en contraste con las 1,220 que tendrían que haberse hecho si se hubieran procesado las muestras individualmente, es decir, que se logró un ahorro de 50.5% del costo directo del reactivo de diagnóstico. Conclusiones. La metodología aplicada mostró ser tanto confiable como conveniente en el entorno mexicano para llevar a cabo vigilancia epidemiológica de la infección por CT. Dado lo anterior, esta metodología podría ser considerada en otros entornos en los que la falta de recursos limita la vigilancia de las infecciones de transmisión sexual.

Evaluation of a commercial test based on ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens

O método de amplificação de DNA baseado na reação em cadeia da ligase (Abbott LCx MTB) foi avaliado para detecção do Mycobacterium tuberculosis em espécimes pulmonares. Os resultados do LCx MTB foram comparados aos resultados de baciloscopia, cultura e diagnóstico clínico para cada paciente. Um total de 297 espécimes (escarro e lavado broncoalveolar) de 189 pacientes foram testadas. Os valores de sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo do LCX vs cultura foram 92,7%, 93%, 67,8% e 98,7%, respectivamente. Quando comparados ao diagnóstico clínico, os valores de sensibilidade, especificidade, VPP e VPN para o LCx foram 88,9%, 96,8%, 86,5% e 97,4%, respectivamente. A sensibilidade do LCx MTB foi de 75% para as amostras com baciloscopia negativa e cultura positiva. Os resultados indicam que o teste LCx MTB é simples, rápido, eficiente e pode ser utilizado como um recurso complementar para o diagnóstico da tuberculose.

Development of a gap ligase chain reaction for detection of Chlamydia trachomatis in newborn infants / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish a gap ligase chain reaction (G-LCR) assay for the detection of Chlamydia trachomatis (Ct) in neonates with pneumonia.</p><p><b>METHODS</b>A G-LCR DNA amplification assay that targeted the outer major membrane protein gene (omp1) of Ct was developed to detect Ct. The sensitivity and specificity of the G-LCR test was examined by the use of highly purified elementary bodies (EBs). Nasopharyngeal swabs taken from 328 neonates with pneumonia were analyzed by Gap-LCR and cell culture.</p><p><b>RESULTS</b>The detection limit of G-LCR was 2 EBs. G-LCR could detect five species of Ct and was not cross-reacted with C psittaci and other bacteria. The prevalence of Ct in 328 neonates with pneumonia, using an expanded gold standard of a positive cell culture or two confirmed positive non-culture tests, was 21% (69/328). After analysis of discrepant results, the sensitivity, specificity, and positive and negative predictive values for the G-LCR were 98.6%, 100%, 100% and 99.6%, respectively; whereas those for culture were 86.9%, 100%, 100% and 96.6%, respectively.</p><p><b>CONCLUSION</b>This study demonstrated that the G-LCR was a highly sensitive nonculture technique and good alternative test for the detection of chlamydial infections.</p>

Direct Detection of Mycobacterium Tuberculosis in Respiratory Specimens by Ligase Chain Reaction / 대한임상병리학회지

BACKGROUND: The most common clinical manifestation of tuberculosis is respiratory tract infections. Currently, respiratory tract tuberculosis is diagnosed by using X-ray, acid-fast smear, culture, or DNA probe technology. The nucleic acid amplification technologies include the polymerase chain reaction (PCR) and the ligase chain reaction (LCR). The potential utility of LCx (Abbott Lab.) kit for the detection and identification of Mycobacterium tuberculosis in respiratory specimens has been measured. METHODS: Four different methods such as acid-fast smear, culture, PCR, and LCR were evaluated using 58 specimens isolated from patients. The IS6110 sequences for Mycobacterium tuberculosis synthesized and provided by Applied Biosystems were used for PCR procedure. The LCR assay using LCx kit was performed according to the manufacturer's instruction (Abbott Lab., U.S.A.). RESULTS: Sensitivity, specificity, and positive and negative predicative values for acid-fast smear method were 72, 100, 100 and 89%, respectively and were 89, 100, 100 and 95%, respectively for culture method. Whereas those values for PCR method were 78, 100, 100, and 91% respectively, and those for LCR were 100, 95, 90 and 100%, respectively. CONCLUSIONS: The LCR assay performed on respiratory specimens for the detection of Mycobacterium tuberculosis has been evaluated as a highly effective method among 4 different identification systems.