A short interspersed nuclear element-based quantitative PCR assay for simultaneous human and dog DNA detection and quantification

This study aimed to develop a quantitative PCR assay to simultaneously quantify human and dog DNA in a multispecies mixture to inform downstream analyses in routine forensic casework and scientific research. The human target is the Alu Yb8 element, which has approximately 2000 copies per cell, and the canine target is the SINEC_Cf element, which has approximately 200,000 copies per cell. The internal positive control is a universal exogenous assay consisting of forward and reverse primers, a NED-labeled probe with an MGBNFQ quencher and a 65-bp synthetic template. Results suggest a potentially robust assay with a fast run time and a high degree of sensitivity, precision and species specificity, with direct application to domestic pet samples in veterinary genetics and forensics.

SARS-CoV-2 RNA detection in nasal mid-turbinate swabs

In this protocol, we describe a method to investigate the presence of SARS-CoV-2 RNA in nasal swabs, using a commercially available high-throughput Real-Time Polymerase Chain Reaction (RT-PCR) assay (TaqPath™ Covid-19 kit, ThermoFisher Scientific) in a 384-Well Plate. After Viral RNA extraction with QIAamp Viral RNA Mini kit, reverse transcription and cDNA amplification, all samples are assessed for the presence of three specific SARS-CoV-2 viral genomic regions and an internal positive control (IPC), in one Multiplex RT-PCR reaction.

Mitochondrial haplotype analyses of the mite Varroa destructor (Acari: Varroidae) collected from honeybees Apis mellifera (Hymenoptera, Apidae) in Uruguay

The ectoparasite Varroa destructor is currently the main health problem faced by honeybees Apis mellifera in the world. It has been identified that two haplotypes were able of infecting and reproducing in this species: the “Korean haplotype” (K) and the “Japanese haplotype” (J). Both haplotypes differ in their virulence, being the K haplotype more harmful than the J. V. destructor was detected for the first time in Uruguay in 1978, but its effect on honeybees is very different depending on the region of the country. Although the K haplotype predominates in South America, the presence of J haplotype in Uruguay is possible, and may be the cause of different virulence of the mites. The aims of this study were to identify the haplotype of V. destructor existing throughout Uruguay and, at the same time, to present a new strategy of RFLP which that presents an internal positive control of digestion. Despite the differences in the effect of V. destructor on honeybees in different areas of the country, all the colonies evaluated presented the K haplotype. The presence in the past of haplotype J cannot be ruled out, and could be assessed with independent nuclear markers.

Real-time RT-PCR detection of Citrus bark cracking viroid (CBCVd) in hops including an mRNA-based internal positive control

Citrus bark cracking viroid (CBCVd), formerly known as pathogen in the genus Citrus and first detected in Slovenian hops in 2014, threatens hop production as it leads to important economic losses. Reduction in yield and quality and even death of the infected plants within a few years are typical observations due to CBCVd infections of hops. The viroid is easily transmitted and spreads rapidly. As it cannot be controlled by plant protection measures, avoiding its introduction into hop gardens and eradicating first centres of infection are of utmost importance. An indispensable prerequisite is a reliable detection method suitable for large-scale routine testing. In this study, the development of primers and probe for real-time RT-PCR for sensitive CBCVd detection is described. To exclude “false negative” results, a nad5 mRNA-based internal positive control was included. To our knowledge, this is the first time such a duplex real-time RT-PCR detection method for CBCVd at least in hops is described. In addition, first method validation data are presented.

Development and application of a canine endogenous internal positive control for use in real-time PCR assays

Real-time PCR (rtPCR) tests have become a method of choice in many diagnostic settings, both animal and human. A concern remains, however, regarding rtPCR assay inhibition during nucleic acid extraction and/or rtPCR reaction process that may result in false-negative results. The use of an internal positive control, either endogenous or exogenous, to mitigate this issue has become more commonplace. We identified and standardized an endogenous internal positive control that can be utilized in rtPCR assays targeting canine-specific pathogens in either a singleplex or multiplex format. The target chosen for the endogenous internal positive control (EIPC-K9) was a highly conserved region in canine mitochondrial DNA. Samples from 240 dogs and 11 other species were screened with EIPC-K9; all canine samples were detected, and no cross-amplification with other species tested was observed. Additionally, no inhibition was noted when comparing singleplex to multiplex rtPCR formats.

Validation of Modifications to the ANSR for Listeria Method for Improved Internal Positive Control Performance

A study was conducted to validate a minor reagent formulation change to the ANSR for Listeria method, Performance Tested MethodSM 101202. This change involves increasing the master mix volume prelyophilization by 40% and addition of salmon sperm DNA (nontarget DNA) to the master mix. These changes improve the robustness of the internal positive control response and reduce the possibility of obtaining invalid results due to weak-positive control curves. When three foods (hot dogs, Mexican-style cheese, and cantaloupe) and sponge samples taken from a stainless steel surface were tested, no significant differences in performance between the ANSR and U.S. Food and Drug Administration Bacteriological Analytical Manual or U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedures were observed for any of the matrixes as determined by probability of detection analysis. Inclusivity and exclusivity testing yielded 100% expected results for target and nontarget bacteria. Accelerated stability testing was carried out over a 7 week period and showed no decrease in assay performance over time.