Detection and identification of human metapneumovirus by real time reverse transcription PCR / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a rapid, sensitive and specific real time reverse transcription PCR for detecting and identifying human metapneumovirus.</p><p><b>METHODS</b>The Hmpv-L gene of human metapneumovirus was chosen as target gene, the primers and TaqMan probe were designed, and the PCR reaction was optimized systematically. The total RNA was extracted from respiratory specimens, and reverse transcription was performed through random primer. The cDNA was detected by using real time PCR. The specificity, sensitivity and reproducibility of real time PCR were estimated. The real time PCR was applied to detect 180 clinical respiratory specimens.</p><p><b>RESULTS</b>The human metapneumovirus can be detected using real time reverse transcription PCR accurately and quickly, and the sensitivity was 1 copy/microl. The coefficient of variation of intra-assay and inter-assay was less than 5%. Among those 180 specimens, 28 (15.56%) were positive for human metapneumovirus, the clinical diagnoses for these 28 patients were pneumonia (15.60%, 17/109) and bronchiolitis (15.49%, 11/71). 21 positive specimens were from patients under 2 years of age, and 6 positive specimens were from patients between 2 and 5 years of age, only 1 positive specimens was from patients over 5 years.</p><p><b>CONCLUSION</b>It is demonstrated that real time reverse transcription PCR is a reliable, accurate and feasible assay for human metapneumovirus, which has become one of the most important pathogens induced acute respiratory infections in pediatric patients.</p>

Detection and identification of seven clinical common pathogenic bacteria by oligonucleotide microarray / 中华流行病学杂志

<p><b>OBJECTIVE</b>Using 16S rDNA and 23S rDNA genes as the target sequences to develop a system based on oligonucleotide microarray and to detect the seven clinical pathogenic bacteria, commonly seen.</p><p><b>METHODS</b>Double polymerase chain reaction (PCR) was applied to amplify the segments of 16S rDNA and 23S rDNA genes of the target bacteria. An oligonucleotide microarray was constructed to simultaneously detect EHEC O157:H7, Vibrio parahaemolyticus, Salmonella sp., Vibrio cholerae, Listeria monocytogenes, Campylobacter jejuni and Shigella sp. Specificity, sensitivity and reproducibility of the microarray during detection were checked. And then microarray was used to detect the microbes in stool specimens of 81 patients with diarrhea and vomiting.</p><p><b>RESULTS</b>The double PCR method could simultaneously amplify the target sequences of 16S rDNA and 23S rDNA genes of the seven pathogens. The sensitivity of the developed oligonucleotide microarray could reach 10(3) cfu/ml but no positive results were presented for non-targeted bacteria. The coefficients of differentiation in one lot or among different lots of the microarray slices were 3.89% - 5.81%. The positive detection rate of the stool specimens by oligonucleotide microarray was 39.5% (32/81), with a coincidence of 96.3% (78/81) for the patients and another coincidence of 96.8% (31/32) for bacterial genus or species identification, when comparing to the results by routine bacteriological examinations.</p><p><b>CONCLUSION</b>The established assay in this study based on oligonucleotide microarray to detect the seven pathogenic bacteria has many advantages such as convenient, rapid, accurate, stable and high flux, which is suitable for clinical specimen examination and epidemiological field investigation.</p>