| A novel avian-origin influenza A (H7N9) virus, whose infections mainly resulted in acute respiratory diseases, was firstly discovered in Shanghai and Zhejiang province, China during February to March 2013. Patients suffered in the infections generally presented as flu-like symptoms, such as fever, cough, less sputum, sometimes companied by headache, muscle pain and discomfort. Patients with severe symptoms is characterized by rapidly progressing severe pneumonia, and presented with lasting fever (oral temperature over 39℃), dyspnea, bloody sputum. Most of the cases with H7N9 infections were malignant and rapidly developed into severe pneumonia and acute respiratory distress syndrome (ARDS) that even caused death. The present common laboratory examinations for H7N9 infection include routine blood test, blood biochemistry, etiological related detection and chest imaging examination etc., while etiological diagnosis is considered essential for the screening and determination of H7N9 virus. Viral culture is the’Gold Standard’ diagnosis for etiology with lower sensitivity and specificity while compared with PCR. Based on the sequence analysis on the conservative region of the subtype of avian influenza A and H7N9 virus, the present study designed the high specific primers and Taqman probes respectively, consequently established a quadruple fluorescent quantitative real-time RT-RCR method which can simultaneously detect M gene, the H7, N9 and internal RP genes by one-step. The study could be divided into two parts:Firstly, the establishment of a quadruple fluorescent quantitative real-time RT-RCR method for detecting H7N9; secondly, the clinical application of this method.Part one:Establishment of a quadruple fluorescent quantitative real-time RT-RCR method for detecting a novel avian influenza A H7N9Method:1. Multiple nucleotide sequences of FluA and H7N9 virus across the world were downloaded from NCBI database, and applied for the homologous analysis by using the DNAman software to find their conservative regions. High specific primers and TaqMan probes were designed based on these conservative regions by using Primer Express3.0. The specificity of primers was further verified in the BLAST.4 pairs of primers and probes in the RT-PCR reaction were optimized.2. The reference cDNA standard fragment of each gene was synthesized and cloned into the PmdTM 19-T Simple Vector for subsequent transformation and culture. DNA from the identified plasmid were extracted and detected by NanoDrop ND-2000 spectrophotometer. Copies of DNA were determined as the quantitation standard for sensitivity. According to the requirement of the experiment, concentration gradient dilution method was used for the standard quantitation of each gene from the highest concentration as 107 copies/mL to 102 copies/mL to verify the sensitivity in the parallel fluorescent quantitative real-time RT-RCR reaction.3. Other 21 respiratory pathogen microbes that included seasonal H1N1, H3N2, novel influenza A H1N1 (2009), influenza B virus, respiratory syncytial virus A, B, measles virus, enterovirus EV71, CoxA16, Mycoplasma pneumoniae, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Monilia albicans, avian influenza H5N1, H5N3, H9N2, human parainfluenza virus Ⅰ,Ⅱ,Ⅲ, were added into this reaction system for the detection of specificity.4. The DNA concentration of each plasmid were determined and diluted according to the experiment, concentration gradient dilution method was used from 106 copies/mL to 102 copies/mL to detect the CT values and calculate the standard deviation and its coefficient of variation (CV) for the detection of reaction repeatability. The detection was replicated for 5 times.Results:1. The sensitivity of the quadruple fluorescent quantitative real-time RT-RCR reaction:The method used in this study for detection of the M gene, the H7, N9 and RP genes of the avian influenza A virus was proven as consistent with the method that was recommended by WHO, both of which can amplify the bands at the concentration of 102 copies/mL.2. The specificity of the quadruple fluorescent quantitative real-time RT-RCR reaction:the RNA extraction of the above 21 respiratory pathogen was used as the template for the multiple fluorescent quantitative real-time RT-RCR separately, and all viruses were detected as negative except the influenza virus with positive result.3. The repeatability of the quadruple fluorescent quantitative real-time RT-RCR reaction:The Ct values in the various concentration of nucleic acids were acquired between 0.11-0.37, all CVs were less than 1.62%, showing good repeatability.Conclusion:The established quadruple fluorescent quantitative real-time RT-RCR method can detect the avian influenza A virus, distinguish the subtype of H7N9 rapidly, accurately and is worthy to be widely spread in the clinic.Part two:Clinical application of the quadruple fluorescent quantitative real-time RT-RCR method for detection of H7N9 virusMethod:1.1896 sputum specimens from suspected patients were detected by using the established quadruple fluorescent quantitative real-time RT-RCR method, the acquired results were treated by statistical analysis and compared with the results from the method of the commercial kit produced by Shanghai ZJ Bio-Tech Co., Ltd.2.35 throat swabs and sputum specimens, which were collected from H7N9 confirmed cases for 20 successive days were detected by this method, the effect of various specimens on detection were further investigated.Results:1.1896 sputum specimens from suspected patients were detected by this method, total 235 avian influenza A viruses were screened among which 127 were infected by H7N9 virus, this result was in accordance with the detection result acquired from the method of the commercial kit produced by Shanghai ZJ Bio-Tech Co., Ltd.2. The positive rate of the sputum specimens was obviously higher than the rate of throat swabs, (26.57%vs8.86%,χ2=75.34, P<0.001), and the lasting time of H7N9 virus detected in the sputum specimens was obviously longer than the residual time in the throat swabs (average 6.14 days vs 2.42days).Conclusion:The established fluorescent quantitative real-time RCR method in this study is proven as simple, fast, and with good repeatability, it could not only provide the early diagnosis for the suspected patients with avian influenza A infections, and distinguish high pathogenic subtype of H7N9 virus, but also offer the reference basis for establishing the clinical treatment. Besides, by using this method, the pathogen could be detected from both throat swabs and sputum specimens of the suspected patients, while the results from sputum specimens showed much more stable and reliable. |
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