Automated sample preparation for Bluetongue testing

In the past month Bluetongue has hit livestock on these shores for the first time – in order to keep on top of the outbreak it is essential that we can respond rapidly to the first signs of the disease. To do this, automated sample preparation is key writes Dr Claudia Bunzenthal

The Chemical and Veterinary Institute (Chemisches und Veterinäruntersuchungsamt Rhein-Ruhr-Wupper, CVUA-RRW) in Krefeld, Germany, is a government institution that provides a number of veterinary diagnostic tests on animal samples from a variety of sources. The institute’s pathology, serology, bacteriology, virology and molecular biology departments are involved in diagnosing a variety of animal diseases, including virus-based diseases such as bluetongue, avian influenza and classical swine fever. The institute also provides investigations into food and feed quality, and performs pathogen detection in foodstuff.

Bluetongue is an insect-borne disease caused by an orbivirus, it occurs primarily in sheep and cattle and, currently, there is no effective treatment for the disease. The infection is caused by bites from midges of the genus Culicoides. These insects are normally found in warmer, Mediterranean parts of Europe, but bluetongue was discovered in the Netherlands for the first time in August 2006 and this outbreak spread to the North Rhine-Westphalia region. During the past few weeks, an outbreak has occurred for the first time in the UK.

“The disease is potentially devastating to farmers and a country’s economy, so it is critical that it can be strictly monitored using rapid, sensitive and reliable detection methods”

Bluetongue is characterised by several symptoms that differ in sheep and cattle, but generally include tiredness, swelling of the head and neck, and changes to the mucous linings of the mouth and nose, and the coronary band of the foot. The disease is potentially devastating to farmers and a country’s economy, so it is critical that it can be strictly monitored using rapid, sensitive and reliable detection methods. The Chemical and Veterinary Institute (CVUA-RRW) in Germany plays a vital role in monitoring infectious diseases in livestock, and one of the challenges it faces is the unpredictable number and type of samples that can arrive each day, at different times. It is essential that the institute can respond rapidly to sudden outbreaks of diseases and continue to deliver test results on time, regardless of the sample load. The testing methods must also be consistent and reliable so, in order to meet these demands, an automated sample preparation platform has recently been installed at the institute’s molecular biology department, based on a Tecan Freedom EVO 150 liquid handling workstation.

The workstation was chosen for a number of reasons, as Dr Claudia Bunzenthal from the CVUA-RRW’s molecular biology department explained: “Our serology department already had an automated ELISA processor from Tecan that has been very satisfactory, so we were familiar with the company. We chose the Freedom EVO for its flexibility, speed and reliability; it is important that we can adapt the platform for testing a variety of viruses, and also achieve our daily throughput requirements. Previously we used manual procedures to extract viral RNA from our samples, but these can be a great deal more time consuming and laborious. Our colleagues at Germany’s National Reference laboratory, the Friedrich-Loeffler Institute in Insel Riems, use a Freedom EVO workstation for similar assays, so this was another reason for choosing this platform.”

The CVUA-RRW’s Freedom EVO set-up can be easily adapted to use for RNA preparations of other viruses and is fully compatible with a variety of labware, including Eppendorf tubes, deep-well plates and blood tubes; it is currently used to detect avian influenza, bluetongue and classical swine fever viruses.

Samples to be tested for the bluetongue virus usually arrive at the CVUA-RRW in the form of whole blood EDTA samples1. These are labeled with barcodes and entered into the serology department’s laboratory information system (LIS). The serology department carries out ELISAs on all the samples to detect viral antibodies (the bluetongue virus belongs to serotype 8), using an automated ELISA processor from Tecan. Aliquots from each sample are also distributed into deep-well plates and delivered to the molecular biology department for RNA isolation and purification, using the Freedom EVO workstation. Subsequent detection of the specific RNA segment of the virus is tested by reverse transcription and real-time PCR.

Viral RNA is prepared in 96-well plates, allowing 86 samples to be tested together with negative and positive controls. Every eleventh sample serves as an internal negative control to check for cross-contamination and, as long as these remain negative, the process is considered to be sufficiently free of contamination. Processing of the entire 96-well plate with the automated platform takes 2 hours and 15 minutes. RNA preparations are used immediately for qualitative real-time PCR, according to the protocol provided by the Friedrich-Loeffler Institute.

“Automating the nucleic acid extraction steps has greatly benefited our laboratory,” said Dr Claudia Bunzenthal. “In the field of animal disease diagnostics, the number of samples that we need to process each day is very difficult to predict. The Freedom EVO provides us with the flexibility and capacity that we need for handling highly variable workloads, and to be able to cope with sudden disease outbreaks.” 

The automated Freedom EVO workstation for viral RNA preparation for real-time PCR at the CVUA-RRW molecular biology laboratory provides a reliable, consistent and fast platform to monitor the spread of potentially devastating diseases in livestock. Its flexibility makes it easy to switch between different disease tests, allowing the laboratory to respond rapidly to changing sample loads on any given day. The workstation is suitable for integrating with a variety of additional devices, including from third parties, making it easy to adapt for future application requirements.

References

1. The RNA preparation procedure described here is based on a protocol from the Friedrich-Loeffler Institute in Insel Riems, the National Reference Laboratory, which is also equipped with a Tecan liquid handling platform.

By Dr Claudia Bunzenthal. Dr Bunzenthal works in the molecular biology department at the Chemical and Veterinary Institute (Chemisches und Veterinäruntersuchungsamt Rhein-Ruhr-Wupper, CVUA-RRW) in Krefeld, Germany.