Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent.

Verification of multiplex ligation-dependent probe amplification probes in the absence of positive samples.

Deletions and duplications of single or multiple exons in specific genes are associated with human diseases. Multiplex ligation-dependant probe amplification (MLPA), a technique recently introduced to clinical laboratories, can detect deletions or duplications at the exon level. MLPA kits have a high multiplexing capability containing mixtures of exon-specific probes that target the gene of interest and control probes that hybridize to other genomic areas before PCR amplification. To verify each probe set, known positive samples with a single-exon deletion or duplication and normal samples are ideally used. Often, positive samples do not exist for each exon and normal samples are not suited to verify the identity of each probe set. We designed a straightforward approach using mixes of exon-specific PCR products as template to unequivocally verify each probe set in MLPA kits. This method can be used to verify the identity of MLPA probes for exons when positive samples are unavailable. Exon-specific probes from 15 MLPA kits were shown to hybridize to the targeted exons of interest. In one kit, this method identified probes that also bind a pseudogene, making them unreliable for clinical analysis. Incorporating this methodology in the analytical validation process will help ensure that MLPA results are interpreted correctly.

Accuracy of marker analysis, quantitative real-time polymerase chain reaction, and multiple ligation-dependent probe amplification to determine SMN2 copy number in patients with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by absence of or mutations in the survival motor neuron1 gene (SMN1). All SMA patients have a highly homologous copy of SMN1, the SMN2 gene. Severe (type I) SMA patients present one or two SMN2 copies, whereas milder chronic forms (type II-III) usually have three or four SMN2 copies. SMN2 dosage is important to stratify patients for motor function tests and clinical trials. Our aim was to compare three methods, marker analysis, real-time quantitative polymerase chain reaction using the LightCycler instrument, and multiple ligation-dependent probe amplification (MLPA), to characterize their accuracy in quantifying SMN2 genes. We studied a group of 62 genetically confirmed SMA patients, 54 with homozygous absence of exons 7 and 8 of SMN1 and 8 with SMN2-SMN1 hybrid genes. A complete correlation using the three methods was observed in 32 patients (51.6%). In the remaining 30 patients, discordances between the three methods were found, including under or overestimation of SMN2 copies by marker analysis with respect to the quantitative methods (LightCycler and MLPA) because of lack of informativeness of markers, 3' deletions of SMN genes, and breakpoints in SMN2-SMN1 hybrid genes. The technical limitations and advantages and disadvantages of these methods are discussed. We conclude that the three methods complement each other in estimating the SMN2 copy number in most cases. However, MLPA offers additional information to characterize SMA cases with particular rearrangements such as partial deletions and hybrid genes.

Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application.

BACKGROUND: Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. METHODS: The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. CONCLUSIONS: The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. SIGNIFICANCE: The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food.

Detection of Chlamydia trachomatis-DNA in synovial fluid: evaluation of the sensitivity of different DNA extraction methods and amplification systems.

INTRODUCTION: Polymerase chain reaction (PCR) and ligase chain reaction (LCR) are used in research for detection of Chlamydia trachomatis (C. tr.) in synovial fluid (SF). However there is no standardized system for diagnostic use in clinical practice, therefore this study aimed at determining the molecular biology method best suited to detect C. tr. from SF. METHODS: SF samples were spiked with C. tr. elementary bodies (EB) and human peripheral blood monocytes (PBMo) persistently infected with C. tr. in vitro to evaluate the sensitivity of different molecular biology methods and assays. Five different DNA-extraction methods were tested: 1) Alkaline lysis, 2) QIAex II Gel Extraction Kit+ CTAB, 3) Chelex-extraction, 4) QIAmp Tissue Kit and 5) QIAmp DNA Stool Kit. All DNA extracts were subjected to 5 different DNA amplification systems to detect C. tr.- DNA in the spiked SF samples: two C. tr. -omp1-- directed PCR, one C. tr.-plasmid-PCR, one C. tr. -16s RNA directed PCR, and one commercially available LCR (LCX), Abbott laboratories). RESULTS: In SF samples spiked with C. tr.-EB and with C. tr.-PBMo, alkaline lysis, detecting 1 C. tr.-EB/ml SF, 0,1 C. tr.-PBMo/ml SF and QIAmp gel extraction kit+ CTAB detecting 0,1 C. tr. -EB/ml SF, 1 C. tr.-PBMo/ml, respectively, allowed most sensitive detection of the organism in combination with the C. tr.- omp1-(152 bp) PCR. Sensitivity decreased in all methods after storage of the DNA of C. tr.- dilution series at -20 degrees C for 4 months by at least one log phase. CONCLUSIONS: The sensitivity to detect C. tr.- DNA from SF is highly dependent on the DNA extraction method and the detection system applied. Alkaline lysis as well as the QIAmp Gel extraction kit + CTAB in combination with C. tr.- omp1 - (152 bp) PCR evolved as the most sensitive methods to identify C. tr. in serial dilutions.

Automatic analysis of multiplex ligation-dependent probe amplification products (exemplified by a commercial kit for prenatal aneuploidy detection).

For use in routine prenatal diagnostics, we developed software and methods for automatic aneuploidy detection based on a commercial multiplex ligation-dependent probe amplification (MLPA) kit. Software and methods ensure a reliable, objective, and fast workflow, and may be applied to other types of MLPA kits. Following CE of MLPA amplification products, the software automatically identified the peak area for each probe, normalized it in relation to the neighboring peak areas of the test sample, computed the ratio relative to a reference created from normal samples, and compensated the ratio for a side effect of the normalization procedure that scaled all chromosomally normal DNA peak areas slightly up or down depending on the kind of aneuploidy present. For the chromosomes 13, 18, 21, X, and Y, probe reliability weighted mean ratio values and corresponding SDs were calculated, and the significance for being outside a reference interval around ratio 1.0 was tested. p < or = 1% suggested aneuploidy and 1 < p < or = 5% suggested potential aneuploidy. Individual peaks, where the normalized area was situated more than 4 SD from the corresponding reference, suggested possible partial deletion or gain. Sample quality was automatically assessed. Control probes were not required. Having used the software and methods for two years, we conclude that a reliable, objective, and fast workflow is obtained.

Usefulness of ligation-mediated PCR genotyping in tracking outbreak-associated Mycobacterium tuberculosis strains.

With the emergence of a multidrug resistant tuberculosis (MDR-TB) outbreak, the availability of a rapid typing method to carry out a nationwide prospective survey for the tracking of newly emerging MDR-TB foci became a priority. For this purpose, we have applied the IS6110 PCR-based genotyping assay, namely, LM-PCR (ligation-mediated PCR). The latter relies on ligation of a synthetic oligonucleotide priming site to a restriction site flanking IS6110. Sequences between the IS element and the restriction site are then amplified using an IS6110 specific outward primer and an oligonucleotide specific to the ligated priming site. Although it was found slightly less discriminative than the standard IS6110 restriction fragment length polymorphism analysis (IS6110 RFLP), LM-PCR allowed for the rapid and prospective identification of new outbreak-related cases within a large pool of circulating M. tuberculosis isolates. In comparison to IS6110 RFLP LM-PCR was found simple enough to justify its implementation in laboratories involved in MDR-TB surveillance at a nationwide scale.

Polymorph count for predicting non-gonococcal urethral infection: a model using Chlamydia trachomatis diagnosed by ligase chain reaction.

BACKGROUND/OBJECTIVES: The criteria for the diagnosis of non-gonococcal urethritis (NGU) on a Gram stained urethral smear are derived from previous studies which used culture as a diagnostic test for Chlamydia trachomatis. Our objectives were (1). to re-assess the relation between urethral polymorph count and C trachomatis infection, using ligase chain reaction (LCR) as the diagnostic test; and (2). to assess other possible predictors of C trachomatis infection such as symptoms, signs, demographic and behavioural variables. METHODS: We collected data from 363 men consecutively attending a genitourinary medicine clinic (excluding those with gonorrhoea and follow up visits) who had a urethral smear and a urethral LCR test for C trachomatis. The sensitivity and specificity of a discrete cut off in urethral polymorphonuclear leucocyte (PMNL) count as a diagnostic test for chlamydia urethritis were calculated. The associations between other variables, such as age and symptoms, and this infection were also estimated. RESULTS: 8% of men had C trachomatis infection and 26% of men had a PMNL count of 5 or more. Of those men with chlamydia 37% did not have NGU; 20% of men with NGU had chlamydia. Adjusted odds ratios for risk of chlamydial infection were significant for age less than 30 relative to 40 years and over (adj OR 13.6; 95% confidence interval 1.69 to 110), a PMNL count of 20 or more (6.56; 2.15 to 20.0), a PMNL count of 5-19 (3.59; 1.41 to 9.15), and the symptom of dysuria (3.27; 1.32 to 8.08). However a PMNL count of 5 or more was only 63% sensitive and 77% specific for C trachomatis infection. No association between sexual behaviour and chlamydial infection was found in this setting. CONCLUSIONS: The PMNL count is associated with presence of chlamydial infection but a large proportion of men with chlamydia have PMNL counts below the recommended cut off for a diagnosis of NSU. Lower age and the presence of symptoms may be as predictive as the urethral polymorph count for chlamydial urethritis and possibly for other urethral infections.

Evaluation of ligase chain reaction for the non-cultural detection of rectal and pharyngeal gonorrhoea in men who have sex with men.

OBJECTIVES: To compare a nucleic acid amplification test (ligase chain reaction) with culture for detecting rectal and pharyngeal gonorrhoea in men who have sex with men (MSM). METHODS: Duplicate rectal and throat swabs from MSM attending a genitourinary medicine clinic were collected for culture on modified New York City medium and detection of gonococcal nucleic acid by the Abbott ligase chain reaction (LCR) utilising probes based on opa 1 gene sequences. LCR positive culture negative specimens were tested by a second LCR utilising probes based on pilin gene sequences. Patients with rectal and/or pharyngeal cultures yielding Gram negative diplococci confirmed as Neisseria gonorrhoeae by biochemical and immunological methods were diagnosed with rectal and/or pharyngeal gonorrhoea. The criteria for diagnosing rectal and pharyngeal infection by LCR included a positive opa LCR with a positive culture from the same site or, in the case of a negative culture, a positive opa LCR and a positive pilin LCR. RESULTS: Duplicate rectal samples were obtained from 227 MSM. The results of LCR and culture were concordant in 219 samples (96.5%). The prevalence of rectal gonorrhoea by LCR and culture was 7.0% (16/227) and 4.0% (9/227), respectively. Duplicate throat samples were obtained from 251 MSM. The results of LCR and culture were concordant in 230 (91.6%) cases. The prevalence of pharyngeal gonorrhoea by LCR and culture was 12.7% (32/251) and 6.0% (15/251), respectively. The specificity of LCR was 99.5% (210/211) for rectal and 98.2% (215/219) for pharyngeal specimens. CONCLUSIONS: The high prevalence and asymptomatic nature of pharyngeal and rectal gonococcal infection suggests that routine screening for infection at these sites by a nucleic acid amplification test method such as LCR should be considered as part of the overall strategy to control gonorrhoea in MSM.

Correlation between culture testing of swabs and ligase chain reaction of first void urine from patients recently treated for Chlamydia trachomatis.

We assessed the correlation between ligase chain reaction (LCR) on first void urine (FVU) and cultures of urethral and cervical swabs to detect chlamydia during three post-treatment follow up visits for 10 men and 19 women with genital chlamydial infections who had been treated with azithromycin or doxcycline.

LCR testing for gonorrhoea and chlamydia in population surveys and other screenings of low prevalence populations: coping with decreased positive predictive value.

OBJECTIVE: Nucleic acid amplification tests have facilitated field based STD studies and increased screening activities. However, even with highly specific tests, the positive predictive value (PPV) of such tests may be lower than desirable in low prevalence populations. We estimated PPVs for a single LCR test in a population survey in which positive specimens were retested. METHODS: The Baltimore STD and Behavior Survey (BSBS) was a population based behavioural survey of adults which included collecting urine specimens to assess the prevalence of gonorrhoea and chlamydial infection. Gonorrhoea and chlamydial infection were diagnosed by ligase chain reaction (LCR). Nearly all positive results were retested by LCR. Because of cost considerations, negative results were not confirmed. Predicted curves for the PPV were calculated for a single testing assuming an LCR test sensitivity of 95%, and test specificities in the range 95.0%-99.9%, for disease prevalences between 1% and 10%. Positive specimens were retested to derive empirical estimates of the PPV of a positive result on a single LCR test. RESULTS: 579 participants age 18-35 provided urine specimens. 20 (3.5%) subjects initially tested positive for chlamydial infection, and 39 (6.7%) tested positive for gonococcal infection. If positive results on the repeat LCR are taken as confirmation of a "true" infection, the observed PPV for the first LCR testing was 89.5% for chlamydial infection and 83.3% for gonorrhoea. This is within the range of theoretical PPVs calculated from the assumed sensitivities and specificities of the LCR assays. CONCLUSIONS: Empirical performance of a single LCR testing approximated the theoretically predicted PPV in this field study. This result demonstrates the need to take account of the lower PPVs obtained when such tests are used in field studies or clinical screening of low prevalence populations. Repeat testing of specimens, preferably with a different assay (for example, polymerase chain reaction), and disclosure of the non-trivial potential for false positive test results would seem appropriate in all such studies.

The ligase chain reaction as a primary screening tool for the detection of culture positive tuberculosis.

BACKGROUND: The ligase chain reaction Mycobacterium tuberculosis assay uses ligase chain reaction technology to detect tuberculous DNA sequences in clinical specimens. A study was undertaken to determine its sensitivity and specificity as a primary screening tool for the detection of culture positive tuberculosis. METHODS: The study was conducted on 2420 clinical specimens (sputum, bronchoalveolar lavage fluid, pleural fluid, urine) submitted for primary screening for Mycobacterium tuberculosis to a regional medical microbiology laboratory. Specimens were tested in parallel with smear, ligase chain reaction, and culture. RESULTS: Thirty nine patients had specimens testing positive by the ligase chain reaction assay. Thirty two patients had newly diagnosed tuberculosis, one had a tuberculosis relapse, three had tuberculosis (on antituberculous therapy when tested), and three had healed tuberculosis. In the newly diagnosed group specimens were smear positive in 21 cases (66%), ligase chain reaction positive in 30 cases (94%), and culture positive in 32 cases (100%). Using a positive culture to diagnose active tuberculosis, the ligase chain reaction assay had a sensitivity of 93.9%, a specificity of 99.8%, a positive predictive value of 83.8%, and a negative predictive value of 99.9%. CONCLUSIONS: This study is the largest clinical trial to date to report the efficacy of the ligase chain reaction as a primary screening tool to detect Mycobacterium tuberculosis infection. The authors conclude that ligase chain reaction is a useful primary screening test for tuberculosis, offering speed and discrimination in the early stages of diagnosis and complementing traditional smear and culture techniques.