16S rRNA qPCR detection of skin mites (Vespertilionidae)

Knowledge graph centered on 16S rRNA qPCR detection of skin mites (Vespertilionidae) with 46 nodes and 139 connections. Top connected: Rocky Mountain Biological Laboratory, Marmota, Yellow-bellied Marmot, Beetles, Pekania pennanti.

Method synopsis

Real-time PCR using universal 16S primers to detect mite DNA in mammalian hair samples, with melting curve analysis for specificity assessment.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Detection, prevalence and phylogenetic relationships of <i>Demodex</i> spp and further skin prostigmata mites (Acari, Arachnida) in wild and domestic mammals (2016), PLoS ONE. Implementations in other papers (listed below) may differ.

1. Real-time qPCR amplification
   Universal 16S primers described by Ferreira et al. were used to amplify an approximately 338-bp fragment of the mitochondrial 16S rRNA gene. Each sample was amplified in duplicate using FastStart Universal SYBR Green Master (Roche Life Science), 0.5 μM of each primer and 5 μL of 1:5 diluted DNA. Thermal cycling qPCR profiles were 95°C for 10 min followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. Real-time qPCRs were performed in a Roche LightCycler 480 Real-Time PCR System with melting curve analysis at the end.
   Quantities: 338-bp target fragment, 0.5 μM of each primer, 5 μL of 1:5 diluted DNA, 15 μL final volumeDuration: 95°C for 10 min, then 45 cycles of 95°C for 15 s and 60°C for 1 minConditions: Laboratory PCR conditions with duplicate samples
   Equipment: Roche LightCycler 480 Real-Time PCR System, FastStart Universal SYBR Green Master, LightCycler 480 Multiwell Plate 384

Typical Equipment

• Roche LightCycler 480 Real-Time PCR System
• FastStart Universal SYBR Green Master

Output Measurements

• mite DNA presence/absence
• melting temperature
• amplification cycles

Papers Using This Protocol (1)

Species Studied With This Protocol

Places Where Used