Question

rate for answering questions compeletly.

You plan to use a PCR based assay to determine whether 3 mosquitoes that you have collected are infected with West Nile virus. You have available to you three different virus-specific primers. The primers recognize sequences at the following nucleotide positions on the viral genome: primer 1: 155-176 primer 2: 745-720 primer 3: 926-905 The 5' and 3' ends of the genome are indicated on the long line The first nucleotide at the 5' end of the genome is position #1 The 5' ends of the primers are indicated (a) List all the steps you would use in the PCR based assay you develop for the detection of viral-specific sequences. Briefly describe what is occurring at each step. You may use any of the primers listed above. Indicate which primers you choose to use in your diagnostic assay. (b) What size product to do expect to see with the virus-specific primers you used? (c) Another person in the lab tests the same samples using a PCR assay. The PCR products are separated on a 1.5% agarose gel. The three individual mosquitos are labeled A, B, and C. The molecular size markers are indicated in lane M. Please explain the results that your colleague obtained.

Answer 1

a)

sequence:-

1 ) Sample collection.The nasopharyngeal aspirates included in this study were collected for diagnostic testing from children with respiratory illness

2)Ethics statement.In compliance with relevant laws and institutional guidelines, ethical approval was obtained from the institutional review board of the Faculty of Medicine.

3 Sequence-independent next-generation sequencing.

4) Assembly.

5)Metagenome analysis.

6)Diagnostic real-time RT-PCR assays.

7)Phylogenetic analysis.

8)Nucleotide sequence accession numbers.

B)Total nucleic acid was extracted from an aliquot (200 μl) of the 81 nasopharyngeal aspirates using the MagNA Pure LC total nucleic acid isolation kit and the MagNA Pure LC isolation station (Roche) and eluted in 50 μl of elution buffer, according to the manufacturer's instructions. Subsequently, the 81 samples were screened for the presence of human rhinovirus, enterovirus, and human metapneumovirus by a real-time RT-PCR with primers and probes used in the routine molecular viral diagnostics setting of Erasmus Medical Center essentially as described previously (22), except hMPV-probe-2 was not used. For bocaviruses, 4 μl extracted nucleic acid was amplified by a real-time PCR

C)