February 14, 2008

Gene sequencing discovers and identifies mystery microbes could help find sources of thousands of previously unidentified cases

In the picture, cells infected with the new virus have been stained using antibodies.

After several other identification techniques failed, the new sequencing approach was used to discover a never-before-seen virus that was likely responsible for the deaths of three transplant patients who received organs from the same donor.

As many as 40 percent of cases of central nervous system disease cannot be traced back to a specific culprit. For respiratory illness, the figure is 30 to 60 percent. In the United States alone, 5,000 deaths each year result from unidentified food-borne infections.

It would also be useful in identifying any newly synthesized virus or microbes.

As powerful as 454 sequencing is for discovering new pathogens, it is not fast or cost efficient enough for use in routine screening of transplant tissue. But microbes discovered using this technique could be incorporated into existing screening techniques.

The technique, called unbiased high-throughput pyrosequencing, or 454 sequencing, was developed by 454 Life Sciences, owned by Roche. This is the first time it was used to probe for the cause of an infectious-disease outbreak in humans, and experts say that it could ultimately usher in a new era in discovering and testing for agents of infectious disease.

To find the mystery pathogen responsible for the deaths, Lipkin's team extracted RNA from the tissues of two of the patients and prepared the sample by treating it with an enzyme that removed all traces of human DNA; this enriched the sample for viral sequences. The researchers then amplified the RNA into millions of copies of the corresponding DNA using a reverse transcriptase polymerase chain reaction (PCR). Usually, PCR requires some advance knowledge of the sequence in question because it relies on molecular primers that match the string of code to be amplified. But 454 sequencing avoids that problem by using a large number of random primers.

The resulting strands of DNA were sequenced using pyrosequencing, which determines the sequence of a piece of DNA by adding new complementary nucleotides one by one in a reaction that gives off a burst of light. Pyrosequencing allows for fast, simultaneous analysis of hundreds of thousands of DNA fragments. Although traditional pyrosequencing generally produces relatively short chunks of sequence compared with earlier sequencing techniques, 454 Life Sciences has improved upon the technology such that longer reads are possible.

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