Helminths PCR Kit

BIOSYNEX AMPLIQUICK Helminths is a real-time PCR test for the qualitative detection of ten intestinal helminths in DNA extracts from stool. Microscopy remains the traditional method for these parasites; a PCR panel detects the species that shed few eggs or shed them intermittently.

Targets and layout

Master mix A: Schistosoma mansoni (FAM) and Strongyloides stercoralis (HEX). B: Ascaris lumbricoides and Trichuris trichiura. C: Diphyllobothrium latum and Taenia species. D: Hymenolepis nana and Enterobius vermicularis. E: Ancylostoma duodenale and Necator americanus. The CIEZ internal control is read in the Cy5 channel of master mixes A to D and identifies PCR inhibition; the procedural control in master mix E validates extraction and purification.

Performance

The limit of detection is 0.5 copies per µl for Diphyllobothrium latum and Enterobius vermicularis, 1 copy per µl for Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiura, Hymenolepis nana and Ancylostoma duodenale, and 2.5 copies per µl for Schistosoma mansoni, Taenia species and Necator americanus. The clinical section reports raw counts rather than percentages: 120 true positives, 18 false negatives, 72 samples PCR-positive and microscopy-negative confirmed with another commercial kit, and 16 true negatives.

Sample, instruments and format

Stool pretreated with the BIOSYNEX AMPLIQUICK Fecal Pretreatment kit (REF 3150065). The PCR runs 3 minutes at 95 °C followed by 50 cycles of 5 seconds at 95 °C and 20 seconds at 58 °C. Validated thermocyclers are the Bio-Rad CFX96 Touch and CFX96 Opus, the Bioer QuantGene 9600, the Applied Biosystems QuantStudio 5, the DNA Technologies Dt Lite 48/96 and, for REF 3150066_SEC01 only, the Roche LightCycler 480. Several extraction systems are validated, but the Macherey-Nagel NucleoSpin DNA Stool kit is explicitly not compatible. The kit contains five microplates divisible into strips of 8 wells, one strip per patient, plus positive, negative and procedural controls and a control master mix, and is stored at -20 °C.

1. Lysis Buffer: A solution containing chaotropic salts and detergents that break down the cell walls and membranes of helminth parasites to release genomic DNA into solution.
2. Proteinase K: An enzyme that digests proteins and enzymes, including nucleases and proteases that could degrade the extracted DNA.
3. Beads: Silica-coated magnetic beads that capture and isolate genomic DNA from the lysed helminth samples.
4. Wash Buffer: A solution used to wash the magnetic beads and remove any remaining contaminants.
5. Elution Buffer: A solution used to release the captured genomic DNA from the magnetic beads.
6. DNase/RNase-free Water: Water that has been treated to remove contaminating DNases and RNases, which can degrade the extracted DNA.

The kit also includes instructions for use, as well as a protocol for the extraction of genomic DNA from helminth parasites using a magnetic bead-based method.