# Presentation: 782705

## HIGH THROUGHPUT TESTING FOR WEST NILE AND OTHER ARBOVIRUSES: A Case Study

**The Arbovirus Laboratory**
**Wadsworth Center**
**New York State Dept of Health**

## Flow chart for virologic testing of vertebrate and mosquito specimens

**Flow chart for virologic testing of**** ****vertebrate ****and**** ****mosquito**** ****specimens**

## RT-PCR assays on avian tissue, 2001

**Number of tests**

**RT-PCR assays on avian tissue, 2001**

**Number**

**Tests done**

**Birds tested**

## RT-PCR assays on mosquito pools, 2001

**RT-PCR assays on mosquito pools, 2001**

**Tests done**

**Mosq pools tested**

**Week of year**

**Number**

##  Disrupts

-  **Disrupts**
** ****2 x 96 samples ****(1.2 ml) **
** ****or            **
** ****2 x 24 samples ****(2.0 ml)**
** ****in 2-4 min**

**              ****TRITURATION: Qiagen Mixer Mill MM 300**

## High Throughput Testing

- Automated Nucleic Acid Workstation
- Automates sample and reaction preparation for nucleic acid analysis
**increase in productivity **
**cost efficient**
**high quality of product**
**decreased cross-contamination**
**consistency and reproducibility**

## 	

Class II biosafety cabinet enclosure (HEPA filtered)
Sets up dilutions and replicate samples in up to four 96-well output trays for TaqMan analysis
Automatically seals output trays with a full cover optical blanket and holds them at 4C
Completely compatible with TaqMan sequence detection system without additional manipulation
Software synergy 
One-step process from RNA purification  to assay plate
Disadvantage: Chemistry works on cells only

**Class II biosafety cabinet enclosure (****HEPA filtered****)**
**Sets up ****dilutions**** and ****replicate**** samples in up to four 96-well output trays for TaqMan analysis**
**Automatically seals**** output trays with a full cover optical blanket and holds them at 4C**
**Completely ****compatible ****with TaqMan sequence detection system without additional manipulation**
**Software synergy **
**One-step process**** from RNA purification  to assay plate**
**Disadvantage****: Chemistry works on cells only**

**ABI Prism 6700**

## Slide 8

## Slide 9

## Slide 10

## Slide 11

## Slide 12

## Slide 13

##   Comparison of TaqMan Ct values on RNA samples from naturally infected bird kidneys

**  ****Comparison of TaqMan Ct values on RNA samples from naturally infected bird kidneys**

## 	Reproducibility of robotic assays

**	****Reproducibility of robotic assays**
**	****      ****( ****INTRA****-assay variability )**

## Reproducibility of robotic assays

**Reproducibility of robotic assays**
**      ****( ****INTER****-assay variability )**

## Comparison of linearity of assays on avian tissue

## Comparative assays on infected mosquito parts*

**Comparative assays on infected mosquito parts***

- * Added to pool of 50 uninfected mosquitoes

## Qiagen 9604 nucleic acid workstation

**Qiagen 9604 nucleic acid workstation**

## Slide 20

## Slide 21

## Summary of High Throughput  Techniques

- Submission of sample data to laboratory on Excel spreadsheets
- High capacity mixer mill
- Robotic workstation for RNA extraction and real time RT-PCR setup
- Real time RT-PCR

## Arbovirus Laboratory staff

**Arbovirus Laboratory staff**

**2002**