Inhibitor-free DNA for real-time PCR analysis of synovial fluid from horses, cattle and pigs.

This research study explored the effectiveness of five commercial DNA isolation methods in eliminating real-time PCR inhibitors from the synovial fluid of animals such as horses, cattle, and pigs. The results showed that all tested kits can be used to analyze pelleted synovial fluid, but when analyzing non-pelleted synovial fluid, only three kits proved effective.

Study Overview

The researchers investigated five different commercial methods for the isolation of DNA in an attempt to remove substances that could inhibit a process known as Real-Time Polymerase Chain Reaction (PCR) when analysing synovial fluid from horses, pigs, and cattle.

• Real-Time PCR is a lab technique used in molecular biology and genetics to amplify and simultaneously monitor the amplification of a selected DNA molecule.
• Synovial fluid is the clear fluid in the cavities of synovial joints, like the knee or elbow. It reduces friction between the articular cartilage during movement.

DNA Isolation Methods

Out of the five methods on test, four used a silica column-based process to purify the DNA. The fifth kit used a desalting process.

• A silica column-based purification involves passing the sample through a column made of a silica gel. DNA binds to the silica, allowing unwanted material to be washed away.
• A desalting process removes salts and other small contaminants from a solution, again to purify the DNA.

Testing Procedures

The team used an innovative comparator method involving the use of a synthetic target and statistical analysis. Each PCR reaction included an artificial target and the isolated DNA. Control samples were created with water instead of isolated DNA for comparison.

Results

It was found that inhibition and a reduction of endpoint fluorescence (which is an indicator of the presence and quantity of the DNA target) often occurred. In some cases, the quantities of DNA target were underestimated. Regardless, no negative impact from the reduced endpoint fluorescence on the detection limit of the PCR was found.

Conclusion

In conclusion, testing shows that all kits work well with analysing pelleted synovial fluid but only three of the kits, two silica-based and one desalting process based, were successful in providing inhibitor-free DNA for analysis when working with non-pelleted synovial fluid.