The preliminary establishment and application of a new method for evaluating K-ras mutations based fluid chip / 中华检验医学杂志

Objective A new method for detecting K-ras mutations based liquid chip was used to evaluate K-ras mutations associated with non-small cell lung cancer (NSCLC) patients,to direct the personalized treatment and prognosis evaluation.Methods Take the diagnosis technology research methods,the sensitivity and repeatability of the liquid chip K-ras gene mutation detection method were assessed.A total of 100 NSCLC patients from Nov 2011 to Feb 2012 in Shanghai Chest hospital were included in this study,the fresh tumor tissues were collected for DNA extraction.The 2nd exon 12 and 13 codons,containing 8 K-ras mutations occuring in high frequency were amplified by polymerase chain reaction (PCR),followed by ligation of the PCR products to a series of special probes using ligase detection reaction (LDR),then the PCR-LDR products were analyzed by liquid chip platform.Direct sequencing was applied to compare with the detection results.Results The sensitivity of liquid chip technology detection was 10％-20％,higher than the traditional sequencing method by 1％.Average CV value was 4％-15％ and showed good repeatability.5 K-ras mutations in 100 patients (5％) were detected using multiplex PCR-LDR combined fluid chip methods,including 3 Glyl2Val and 2 Gly12Asp mutations in exon 2.The 5 K-ras mutations were verified accurately by direct sequencing.Conclusions The novel detection method of K-ras mutations based PCRLDR and fluid chip shows high throughput,high sensitivity,good repeatability and the results are reliable and accurate.This method can be used to accurately identified K-ras mutations for NSCLC patients prior to their targeted therapy with TKIs.

Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation- dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics.

Comparison of Acid-Fast staining, PCR, LCR, PCR=Hybridization for dection of mycobacterum tuberculosis in clinical specimens / 결핵

BACKGROUND: Mycobacterial culture is a confirmatory test to detect M.tuberculosis, but it takes at least 6 weeks to diagnose. PCR is a rapid and sensitive method, but it is known that PCR has a high false positive rate due to contamination, and a high false negative rate due to inhibitors. It is also known that LCR and PCR-Hybridization, recently developed methods, are more specific methods than PCR in terms of detection M.tuberculosis. In this study, we estimated the clinical utility of in house PCR, LCR and PCR-Hybridization for the detection of M.tuberculosis. METHODS: We evaluated 75 specimens, upon which M.tuberculosis culture based testing was requested, by PCR LCR, and PCR-Hybridization and compared results. Mycobacterial culture was performed on 3% Ogawa media for 8 weeks, and an in house PCR, LCx Mycobacterium tuberculosis assay kit(Abbott Laboratories, North Chicago, III) and the AMPLICOR M.tuberculosis test kit(Roche Molecular Systems, Inc. Branchburg, NJ, USA). RESULTS: In the view of the culture results, the sensitivities of the three tests were 40%, 80%, and 100% and their specificities were 98.6%, 94.3%, and 94.3%. CONCLUSION: LCR and PCR-Hybridization and rapid and sensitive methods for detecting M.tuberculosis in clinical laboratories.

Study on the perinatal Chlamydia Trachomatis infection in Chongqing area using urine Gap-LCR-ELISA / 中华围产医学杂志

Objective To study the prevalence of perinatal chlamydia trachomatis infection in the city of Chongqing. Methods First void urine (FVU) samples and cervical smear from 512 pregnant women (gestational age≥28 weeks) were collected. According to the “expanded gold standard”, the methodologic indices were compared between Gap LCR ELISA using plasmid probes and omp1 probes on FVU and cervical smear, respectively. Results (1) CT infection in pregnancy is usually asymptomatic. Forty two CT positive cases were confirmed by “expanded gold standard” indicating a prevalence of CT infection in pregnant women in Chongqing was 8.20%(42/512). Among these 42 women, 37 (88.1%) were detected from both FVU and cervical smear, while positive results were shown in 4 cases (9.5%) from cervical smear and only one case (1/42) 2.4% from FVU . (2) The sensitivity of Gap LCR ELISA using plasmid probes and ompl probes were 90.48% and 71.43% ( P 0.05). The specificity of all Gap LCR ELISA tests were 100%. Conclusions FVU plasmid Gap LCR ELISA is a noninvasive, highly sensitive and specific method which is suitable for large scale screening for perinatal CT infections in pregnant women in developing countries and regions.

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.

Diagnostic Value of Gap-Ligase Chain Reaction Enzyme Linked Immunosorbent Assay for Chlamydia Trachomatis Pneumonia in Neonatal and Small Infants / 实用儿科临床杂志

Objective To explore the diagnostic value of Gap-ligase chain reaction(LCR)-enzyme linked immunosorbent assay(ELISA)for Chlamydia trachomatis(CT) pneumonia in infants(28 days-3 months old baby was 16.9%(27/160 cases),and the incidence in 3-6 months old baby was 8.1%(12/149 cases).The clinical symptoms included that 25 cases(51.0%) had fever,48 cases(98.0%) with paroxysmal cough,45 cases(91.8%) with rhinocleisis,45 cases(91.8%) with spitting foam,49 cases(100%) with tachypnea,28 cases(57.1%) with lips cyanosis,26 cases(53.1%) with medium and moist rales,24 cases(49.0%) with dry rales,18 cases(36.7%) with phlegm whimper,29 cases(59.2%) with rough breath sounds in both lungs and there were 13 cases(26.5%) with conjunctivitis.Chest film and paper capacitor showed that bilateral extensive interstitial and(or) alveolar infiltration,over-inflation 23 cases(46.9%) had no lobal consolidation or pleural effusion.The WBC counts were normal or slightly high.All children were treated with cephalothin or penicillin before confirmation with CT infection.Eleven cases were treated with azithromycin after diagnosis with CT pneumonia and were cured 9 days after treatment and the others were not treated with azithromycin so their course prolonged from 15 to 44 days,among them there were 11 out-patients who were treated many times because of repeated cough.Conclusions CT is important pathogenic organism to cause pneumonia in neonatal and small infants.It is important to pay attention to the possibility of pneumonia caused by CT and make the diagnosis in early period as soon as possible and treat them with sensitive drug to shorten the course.

Detection of Chlamydia trachomatis DNA by gap ligase chain reaction (G-LCR) / 重庆医科大学学报

Objective:To develop a new nucleic amplication method for detection of Chlamydia trachomatis DNA by gap ligase chain reaction(G-LCR).Methods:A G-LCR DNA amplification assay that targeted the outer major membrane protein gene(omp1)of CT was established to detect CT infection.The sensitivity and specificity of a newly developed G-LCR test was examined by the use of highly purified elementary bodies (EBs).DNA fragments of different species and from other bacteria1 were detected with G-LCR and routine polymerase chain reaction (PCR).Results:Using G-LCR,DNA fragments of 54bp were amplified from five different species.The sensitivity could be improved to detect out 2 chlamydial elementary bodies.G-LCR detected ten-fold EBs than PCR.No signal was observed when C.pneumoniae and other bacteria were used as templates.Conclusion:G-LCR is sensitive,rapid and specific for detection of Chlamydia trachomatis.

Investigation methods of diagnosis of Chlamydia trachomatis by Gap ligase chain reaction / 重庆医科大学学报

Objective:To investigate the method with good sensitivity,specificity,reliability for diagnosis of Chlamydia trachomatis.Methods:Inoculate the nasopharyngeal swabs to detect the Chlamydia trachomatis(CT) with McCoy cell culture and plasmid gene probes labeled with biotin ligase-chain reaction.Then calculate the sensitivity,specificity,positive predicative value(PPV) and negative predicative value(NPV) of both TC and G-LCR respectively.Compare the difference of the two methods.Results:There were 49 positive specimens and 344 negative by enlarged gold standard.There was significant difference with cell culture,G-LCR-PAGE,G-LCR-ELISA by two-related-samples ? 2 tests( P