Inhibition of PCR by aqueous and vitreous fluids

Abstract

The detection of viral nucleic acids in intraocular fluids and tissues by PCR has become increasingly important in clinical ophthalmology. While much attention has been directed toward minimizing false-positive reactions resulting from specimen contamination or amplicon carryover, relatively little attention has been given to the causes of false-negative PCRs. This report describes a PCR inhibitor in normal aqueous and vitreous fluids that can produce false-negative PCR results. As little as 0.5 l of vitreous fluid and 20 l of aqueous fluid can completely inhibit DNA amplification in a 100-l PCR mixture. This inhibition was not primer specific, nor was it due to chelation of Mg 2؉ ions or DNase activity in the ocular fluid. The inhibitor was completely resistant to boiling for 15 min. However, the inhibitory effects were completely removed by a single chloroform-isoamyl alcohol (24:1) extraction. The extent of PCR inhibition depended upon the type of thermostable DNA polymerase used in the reaction. Taq DNA polymerase was very sensitive to the inhibitor, while thermostable DNA polymerases from Thermus thermophilus HB-8 (Tth) and Thermus flavus (Tfl) were completely resistant. Thus, the inhibitory effects of intraocular fluids on PCRs can be removed by diluting the specimen, by chloroform extraction, or by using Tth or Tfl DNA polymerases.

Sensitive and Rapid Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA by Loop-Mediated Isothermal Amplification

“…Evaluation and comparison of LAMP and PCR for detection of DNA in clinical samples. Previous reports have described clinical samples as containing some PCR inhibitors (8,9,15,21); therefore, DNA preparation is required to obtain sensitive results for PCR assays. In order to clarify whether a DNA preparation procedure was necessary to obtain sensitive results with LAMP, we tested 40 genital and 20 ocular samples, with and without a DNA extraction procedure, by both the LAMP and PCR assays.…”

“…Next, we evaluated the necessity of DNA extraction. Biological fluids, e.g., aqueous and vitreous humor, contain substances that inhibit PCR(8,9,15,21).…”

Comparison of 9 different PCR primers for the rapid detection of severe acute respiratory syndrome coronavirus using 2 RNA extraction methods

“…Although no inhibition was observed with fecal samples spiked with SARS-CoV, it does not mean that inhibition problems associated with clinical samples have been eliminated by using silica-or magnetic-based types of RNA extraction. In Drosten et al's (2004) study, inhibition was seen with one PCR assay but not the other using the same extracted RNA; this might because of the types of enzyme and reagents used in the amplification assay (Wiedbrauk et al, 1995). There was no available clinical sample in this investigation, but both Artus and Roche kits have improved detection of SARS-CoV, and the sensitivity in stool samples have reached 78-87% (Drosten et al, 2004) versus 58-63% reported earlier .…”

Advances in Directly Amplifying Nucleic Acids from Complex Samples

“…DNA collection from swabs is attractive because it is simple, minimally invasive, and even enables self-sampling. However, swab-collected specimens are likely to contain polymerase inhibitors such as secreted minerals, electrolytes, hormones, enzymes, immunoglobulins, and cytokines, as well as topical medications [137,[174][175][176][177]. As a result, many swab tests now on the market remove these inhibitors via extraction methods that are too involved and complex to be suitable for the POC [178].…”

“…Rodriguez et al used a similar approach in detecting clinical levels of H1N1 by passing an in-house elution buffer with the sample of interest through a filter paper-based setup, then amplifying on the filter membrane [205]. Future developments could focus on direct reactions with swabs that integrate extraction, Typically, elution either at room temperature [51, 176,[182][183][184][185][186] or with heating [43,44, [187][188][189][190][191] is sufficient to generate PCR-amplifiable template from swabs in solution. These methods of dilution or heating can save over thirty minutes of processing time, as with Nihonyanagi et al's heating protocol to release MRSA in CellEaseII (Biocosm Inc., Hyogo, Japan) diluents [169].…”