Development and evaluation of a multiplex quantitative polymerase chain reaction assay for detecting bacteria associated with lower respiratory tract infection.

OBJECTIVES: This study aimed to establish a multiplex quantitative polymerase chain reaction (MQ-PCR) assay for 12 bacterial pathogens found in lower respiratory tract infection (LRTI) and to evaluate its performance in a cohort of 211 patients with LRTI. METHODS: The study was divided into two stages: a pilot study to establish the methodology and a clinical validation study to evaluate its performance. In the pilot study, we established the MQ-PCR and analyzed its performance regarding limits of detection, reproducibility, specificity, and efficiency. In the clinical validation study, we obtained 211 sputum and/or bronchoalveolar lavage fluid (BALF) samples and detected pathogens by MQ-PCR. The MQ-PCR time was 3 h from sample collection to complete pathogen detection. RESULTS: The limit of detection was 1000 copies/ml, and the maximum efficiency was >95%. When cutoffs of ≥105 copies/ml in sputum and ≥104 copies/ml in BALF were applied, the sensitivity, specificity, and positive and negative predictive values of the MQ-PCR were 77% (95% confidence interval [CI] 67-88%), 94% (95% CI 93-95%), 25% (95% CI 19-31%), and 99% (95% CI 99-100%), respectively. CONCLUSIONS: This study demonstrates that the new MQ-PCR assay is time-saving, more effective and sensitive, and brings us closer to mainstream adoption of quantitative molecular detection of bacteria.

A Microfluidic-Based SNP Genotyping Method for Hereditary Hearing-Loss Detection.

The genotyping of SNPs (single nucleotide polymorphisms) is a prerequisite for the analysis of many genetic diseases, including hereditary hearing-loss. However, the existing methods for SNP detection suffer from a long detection period, tedious operation, and a high risk of carryover contamination. To address these challenges, a microfluidic chip is constructed for rapid and efficient SNP genotyping by dividing the sample into many independent chambers for Kompetitive Allele Specific PCR in this study. Using this strategy, multiple detection can be easily accomplished and the challenge for the establishment of multiplex PCR is fundamentally overcome. The entire detection can be finished within 2 h in a fully sealed manner with this method, which is quite simple compared to SNaPshot and MassArray. After assessment of the basic performance, this chip was applied to screen 15 mutations, including SNPs and InDels (insertion-deletion markers), that can cover more than 80% of cases of hereditary hearing-loss in China. Over 40 clinical samples were analyzed with this microfluidic chip for SNP genotyping, and the results are consistent with that obtained by Sanger sequencing, demonstrating its practicability and potential in the application of genetic disease detection.

Microarray-based genotyping and detection of drug-resistant HBV mutations from 620 Chinese patients with chronic HBV infection

Background: Research has shown that hepatitis B virus (HBV) genotypes are closely linked to the clinical manifestations, treatment, and prognosis of the disease. Objective: To study the association between genotype and drug-resistant HBV mutations in 620 Chinese patients with chronic HBV infection. Methods: HBV DNA levels were determined using real-time quantitative PCR in plasma samples. Microarrays were performed for the simultaneous detection of HBV genotypes (HBV/B, C, and D) and drug-resistance-related hotspot mutations. A portion of the samples analyzed using microarrays was selected randomly and the data were confirmed using direct DNA sequencing. Results: Most samples were genotype C (471/620; 76.0%), followed by genotype B (149/620; 24.0%). Among the 620 patient samples, 17 (2.7%) had nucleotide analogs (NA) resistance-related mutations. Of these, nine and eight patients carried lamivudine (LAM)-/telbivudine (LdT)-resistance mutations (rtL180M, rtM204I/V) and adefovir (ADV)-resistance mutations (rtA181T/V, rtN236T), respectively. No patients had both lamivudine (LAM)- and either ade-fovir (ADV) or entecavir (ETV) resistance mutations. Additionally, out of the 620 patient samples, 64.0% (397/620) were also detected with the precore stop-codon mutation (G1896A) by microarray assay. Conclusion: The results of the current study revealed that the prevalence of nucleotide analogs (NA)-resistance in Chinese hospitalized HBV-positive patients was so low that intensive nucleotide analogs (NA)-resistance testing before nucleotide analog (NA) treatment might not be required. In addition, the present study suggests that chronic HBV patients with genotype C were infected with fitter viruses and had an increased prevalence of nucleotide analogs (NA)-resistance mutations compared to genotype B virus. .

Microarray-based genotyping and detection of drug-resistant HBV mutations from 620 Chinese patients with chronic HBV infection.

BACKGROUND: Research has shown that hepatitis B virus (HBV) genotypes are closely linked to the clinical manifestations, treatment, and prognosis of the disease. OBJECTIVE: To study the association between genotype and drug-resistant HBV mutations in 620 Chinese patients with chronic HBV infection. METHODS: HBV DNA levels were determined using real-time quantitative PCR in plasma samples. Microarrays were performed for the simultaneous detection of HBV genotypes (HBV/B, C, and D) and drug-resistance-related hotspot mutations. A portion of the samples analyzed using microarrays was selected randomly and the data were confirmed using direct DNA sequencing. RESULTS: Most samples were genotype C (471/620; 76.0%), followed by genotype B (149/620; 24.0%). Among the 620 patient samples, 17 (2.7%) had nucleotide analogs (NA) resistance-related mutations. Of these, nine and eight patients carried lamivudine (LAM)-/telbivudine (LdT)-resistance mutations (rtL180M, rtM204I/V) and adefovir (ADV)-resistance mutations (rtA181T/V, rtN236T), respectively. No patients had both lamivudine (LAM)- and either adefovir (ADV) or entecavir (ETV) resistance mutations. Additionally, out of the 620 patient samples, 64.0% (397/620) were also detected with the precore stop-codon mutation (G1896A) by microarray assay. CONCLUSION: The results of the current study revealed that the prevalence of nucleotide analogs (NA)-resistance in Chinese hospitalized HBV-positive patients was so low that intensive nucleotide analogs (NA)-resistance testing before nucleotide analog (NA) treatment might not be required. In addition, the present study suggests that chronic HBV patients with genotype C were infected with fitter viruses and had an increased prevalence of nucleotide analogs (NA)-resistance mutations compared to genotype B virus.