| Background and objectiveAccording to the World Health Organization,there are approximately 3 million children under the age of five dying every year from acute respiratory infections or diarrheal diseases.Acute respiratory infection is the most common acute infectious disease causing human morbidity or even death worldwide.In recent years,new acute respiratory infections have frequently occurred.On the other hand,diarrhoeal diseases are the second leading cause of death among children under the age of five worldwide.Rapid and accurate detection of respiratory and infectious diarrhea pathogens is the key to the diagnosis,treatment,prevention and control of diseases.Liquid-phase microarray technology is applied in this study to detect children with acute respiratory infections and infectious diarrhea pathogens.Experimental basis is provided for the establishment of a rapid emergency detection platform for major infectious agents.Methodsa.74 clinical samples of children’s respiratory throat swabs and 96 clinical samples of intestinal diarrhea feces were collected from the Second People’s Hospital of Guangdong Province.After pre-treating the samples,the nucleic acid extraction kit is used to extract the nucleic acids of the samples;b.The extracted 74 throat swab specimen nucleic acids and 96 fecal specimen nucleic acids were detected by the Luminex liquid-phase chip kit;c.74 throat swab specimen nucleic acids and 96 fecal specimen nucleic acids were detected by Fluorescence quantitative PCR;d.The multiplex PCR products of the inconsistent detection samples were analyzed by cloning and sequencing.Resultsa.The positive rates of 74 children’s throat swab specimens detected by liquid-phase chip and qPCR method were 77.0%and 68.9%,respectively.The positive detection rates of mixed infection were 33.8%and 25.6%,respectively.A total of eight viruses were detected including enterovirus,rhinovirus,human bocavirus,adenovirus,respiratory syncytial virus,parainfluenza virus,coronavirus and H1N1 virus.Among them,the detection rate of human Boca virus is the highest,followed by enterovirus and rhinovirus.The detection rate of human bocavirus,enterovirus,rhinovirus,adenovirus,respiratory syncytial virus,H1N1 virus by using liquid chip and qPCR method was statistically significant.The agreement rate between the two methods was 79.72%.In general,the positive detection rate of children’s throat swab specimens,the positive detection rate of mixed infections,and the detection rate of each virus with liquid chips were higher than those of qPCR.b.The positive rates of 96 fecal samples detected by Luminex liquid-phase chip and qPCR method were 65.63%and 64.58%,respectively,and the positive detection rates of mixed infection were 26.04%and 24.0%,respectively.Seven pathogenic microorganisms were mainly detected,including intestinal adenovirus,norovirus,rotavirus A,Campylobacter jejuni,Escherichia coli,enterotoxin-producing Escherichia coli and Salmonella.Among them,the detection rate of Salmonella was the highest,followed by Campylobacter jejuni,rotavirus A,Norovirus.The detection rate of Salmonella,Campylobacter jejuni,rotavirus A and Norovirus by using liquid chip and qPCR method was statistically significant.The agreement rate of the two methods was 91.67%.Overall,the positive detection rate of children’s stool samples,the positive detection rate of mixed infections with liquid chips were comparable to those of qPCR.However,the liquid phase chip has a higher detection rate for each fecal pathogen.c.The multiplex PCR products of 15 children’s throat swab clinical specimens and 13 children’s fecal clinical specimens were cloned and sequenced.The sequencing results were basically consistent with the qPCR results.Conclusionsa.The accuracy of liquid-phase microarray technology is higher in detecting pathogens of respiratory and intestinal feces.And it also has the advantages of high throughput,high detection rate,low sample volume and relatively simple experimental operation compared with qPCR.It can be used in respiratory tract and rapid emergency detecting of intestinal pathogens.b.Liquid-phase microarray technology can detect more than ten pathogens in parallel in one experiment,is especially suitable for the detection of mixed infectious pathogens. |
