Wednesday, June 1, 2016

Barcoding with microfluidic dynamic arrays

The family of tephritid fruit flies is very large and contains about 5000 described species. Many species are of major economic importance in agriculture. Some have negative effects, some positive. Various species cause extensive damage to fruit and other plant crops. The genus Bactrocera is globally known for its destructive impact on agriculture. The high morphological similarity between many tephritid species makes identification notoriously difficult and species boundaries are hotly debated.

About 350  species are  considered  economically  important and most of them belong  to  five genera:  Anastrepha, Bactrocera, Ceratitis, Dacus, and Rhagoletis.

China  is  a  globally  significant  importer  of  fresh  produce;  hence,  fruit  flies  are  of  particular concern.  The  very  large  trade  of  fresh  commodities  into  China,  the  country’s  range  of climatic conditions  which  make  it  suitable  for  most  exotic  fruit  fly  species,  and  the  economic and  social importance  of  its  domestic  fresh-commodity  production,  means  that  exotic  pest  fruit flies  pose  a continual and high profile threat to the country. Intercept information from Chinese ports in the last ten years show that fruit flies have been intercepted from baggage and cargo on a staggering 19000 occasions, with each interception consisting of one or more individuals.

Due to the increasing number and diversity of intercepted fruit flies, a high-throughput method combined  with  high  accuracy, high speed, and low cost  has  become  a  necessity  not  only  for China,  but  all  major  trading  countries. A group of Chinese researchers developed a promising approach to this challenge. They designed species-specific  qPCR  primer  and  probe  combinations for  27  economically  important  tephritidae species of the six genera mentioned above. The probes were designed using some 1000 DNA barcodes of fruit fly species available on BOLD. Each primer pair was tested through qPCR. In a follow-up step the colleagues developed a standardized reaction system for detecting all target species based on a microfluidic dynamic  array,  and  also  applied  the method  to  identify  unknown  immature  samples  from  port interceptions and field monitoring.

They were quite successful and were able to properly detect all 27 species in a rather short time (7.5h), using  only  0.2μl  of  reaction  system  in  each  reaction  chamber. All intercepted specimens were correctly identified which they confirmed by rearing and morphological identification of adults.

Microfluidic dynamic arrays have been used in a number of novel applications, such as medical diagnosis, gene expression, genotyping, and GMO analysis but it has not been applied for  species detection  in  plant  quarantine  and  invasion  biology,  where  it  offers  great  potential  for high-throughput and species diversity screening. Available chips can perform as many as 9000 reactions in a single run which makes high-throughput screening a realistic prospect.

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