Effects of additives on efficiency and specificity of ligase detection reaction.

Ligase detection reaction (LDR) is adaptable to a wide variety of applications ranging from scientific research to clinical diagnosis, especially in the field of nucleotide polymorphism discrimination and analysis. Efficiency and specificity of LDR are the most two important characteristics that influence its application. To improve the specificity or efficiency of ligase, optimization of the design of LDR probes and the reaction of LDR were investigated previously by most researchers. But the effects of additives on LDR have not been reported. In this study, the effects of additives (DMSO, Tween-20, glycerol, formamide, and PEG- 6000) on LDR efficiency and specificity were investigated. The results showed that all of these compounds, except for Tween-20, could improve the specificity of LDR. PEG-6000 was proved to be the best additive among the five tested with an optimal concentration of 5% at which the highest yield was obtained with a relatively improved specificity.

Sensitive detection of HBsAg mutants by a gap ligase chain reaction assay.

Hepatitis B virus (HBV) surface gene variants have been associated with diagnostic escape and immune escape following vaccination. The most common mutation observed in these variants is a glycine-to-arginine substitution at amino acid 145 (G145R). In order to sensitively detect the presence of this mutant in serum, a new molecular detection system was developed; in this new system, a gap ligase chain reaction (gLCR) assay was coupled with electrochemiluminescence detection of reaction products. The gLCR assay could detect approximately 10 copies of mutant DNA and could discriminate low levels of mutant DNA in the presence of excess wild-type DNA. Detection of the G145R mutant in clinical specimens was evaluated by testing 56 suspect serum specimens. The G145R mutation was observed in 18 of 28 HBV-DNA-positive samples. The approximate percentage of mutant present in each specimen was calculated by comparison with a standard curve of an increasing ratio of mutant DNA to wild-type DNA. Most samples contained a very low percentage of mutant virus (approximately 5%), with an observed range of approximately 3 to 74%. The G145R mutation was most frequently observed in specimens producing a diagnostic anomaly or from transplant patients but was also observed in specimens from vaccinated individuals and specimens in which HBsAg diagnostic escape was suspected. Therefore, the gLCR assay is a sensitive and specific method for detection of G145R mutants, which could be modified to include the detection of other HBV mutants.

Accuracy of results obtained by performing a second ligase chain reaction assay and PCR analysis on urine samples with positive or near-cutoff results in the LCx test for Chlamydia trachomatis.

Nucleic acid amplification assays such as the ligase chain reaction and PCR have encountered reproducibility problems. The initial extract and a newly extracted aliquot of urine specimens (n = 120) which had signal-to-cutoff (S/CO) ratios above 0.80 by the LCx Chlamydia assay were retested. Nucleic acid was extracted from an additional urine sample for testing by the AMPLICOR PCR Chlamydia assay. Fifteen percent (18 of 120) of the urine specimens were negative by all repeat tests (initial mean S/CO ratio by the LCx Chlamydia assay, 0.93; S/CO ratio range, 0.80 to 3.30). Repeat testing of the 102 specimens with possible positive results by the LCx Chlamydia assay by use of the initially extracted aliquot confirmed the results for 95 (93.1%) of the specimens; repeat testing of a newly extracted aliquot confirmed the results for 87 (85.3%) of the specimens. Twenty specimens had discordant results by the two repeat LCx Chlamydia assays. A total of 78 of 102 (76.5%) of the specimens were positive by the AMPLICOR PCR, and the AMPLICOR PCR confirmed the results for 82.1% (78 of 95) and 89.6% (78 of 87) of the specimens positive by the two repeat LCx Chlamydia assays, respectively. Some of the discrepancies observed by multiple repeat tests may have been due to specimen mislabeling or contamination during performance of the procedure rather than to the LCx Chlamydia assay. Both assays suffered from a lack of reproducibility on repeat testing with a small proportion of specimens, probably due to the presence of low levels of DNA, the presence of variable amounts of amplification inhibitors, and the loss of DNA during extraction.

The 'real-world' impact of improved diagnostic techniques for Chlamydia trachomatis infection in Glasgow.

In April 1997, the main chlamydia laboratory in Glasgow introduced ligase chain reaction (LCR) as its standard diagnostic test. The diagnostic effectiveness and health economic impact of introduction of LCR testing was assessed. Between April 1996 to March 2000, results of all chlamydia detection tests on genital specimens sent from general practitioners and the two main sexual healthcare providers (Genitourinary Medicine and Family Planning services) were reviewed. A preliminary economic assessment, inclusive of staff, reagents, consumables and laboratory overheads was conducted. Overall, testing activity increased four and a half times between 1996-97 and 1999-2000; the proportionate rise was greatest in general practice. Although chlamydia testing in both genders increased over the review period, testing activity rose disproportionately in women (59%, compared with a 31% increase in men). The overall Chlamydia trachomatis detection rate rose from 4.8% in 1996-97 to 7.8% in 1999-2000. Following introduction of LCR testing, an estimated additional 331 men and 844 women were diagnosed during the study period. The cost per additional diagnosis made was estimated at 162 Pounds for men and 263 Pounds for women. Substantial health gains are likely to be achieved, at both an individual and public health level, as a result of introduction of LCR testing for genital chlamydial infection.

Molecular genetic and traditional methods for detection of Mycobacterium tuberculosis complex (discrepancy analysis).

In the past six and half years, 862 different clinical samples [sputum, bronchoalveolar lavage, thorax puncture, cerebrospinal fluid and skin samples] were tested by Gen-probe amplified Mycobacterium tuberculosis direct test (MTD) or ligase chain reaction (LCR) or polymerase chain reaction (PCR). 239 parallel clinical samples were cultivated, and some samples were stained with Ziehl-Neelsen staining. 1-4 samples were tested per patient. 29 (12.13%) samples were positive and 177 (74.05%) samples were negative with both cultivation and molecular genetic methods. 2 (0.83%) samples were positive only on cultivation, and 31 (12.97%) samples were positive only with the molecular diagnostic methods. The differences are undoubtedly explained by the sensitivity of the molecular diagnostic methods.