Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

Summary

The present work describes the steps for producing ready-to-use qPCR for T. cruzi DNA detection that can be pre-loaded on the reaction vessel and stored in the refrigerator for several months.

Abstract

Real-time PCR (qPCR) is a remarkably sensitive and precise technique that allows for amplifying minute amounts of nucleic acid targets from a multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8 ^oC. Details of preparing each solution are shown here along with the expected aspect of a gelified reaction designed to detect T. cruzi satellite DNA (satDNA). A similar procedure can be applied to detect other organisms. Starting a gelified qPCR run is as simple as removing the plate from the refrigerator, adding the samples to their respective wells, and starting the run, thus decreasing the setup time of a full-plate reaction to the time it takes to load the samples. Additionally, gelified PCR reactions can be produced and controlled for quality in batches, saving time and avoiding common operator mistakes while running routine PCR reactions.

Introduction

Chagas disease was discovered in the early 20^th century in rural regions of Brazil, where poverty was widespread^1,2. Even today, the disease continues to be connected to social and economic determinants of health in the Americas. Chagas disease is biphasic, comprising an acute and a chronic phase. It is caused by infection by the Trypanosoma cruzi parasite, being transmitted by insect vectors, blood transfusions via congenital route, or oral ingestion of contaminated food^3,4.

The diagnostic of Chagas dise....

Protocol

1. Preparation of stock solutions and gelification mixture

NOTE: Four stock solutions will be prepared (400 mg/mL of melezitose, 400 mg/mL of trehalose, 0.75 mg/mL of lysine, and 200 mg/mL of glycogen) and mixed according to the proportion shown in Table 1 to produce the gelification mixture. Although the protocol describes 10 mL of stock solutions production, it can be adapted for lower or higher volumes.

1. Melezitose solution
   1. Weigh.......

Discussion

Recent years have highlighted the need to find more sensitive and specific technologies to help diagnose tropical and neglected diseases. Although important for epidemiological control, parasitological (optical microscopy) and serological tests have limitations, especially regarding sensitivity and point-of-care applicability. DNA amplification techniques such as PCR, isothermal amplification, and respective variations have long been used in laboratory settings, but technological hurdles preclude it from being used in fi.......

Materials

Name	Company	Catalog Number	Comments
Bentonite clay bags (activated)	Embamat Global Packaging Solutions (Barcelona, Spain)	026157/STD	Not to be confused with silica gel packs
Glycogen	Amersham Bioscience	Cat# US16445
Lysine	Acros Organic	Cat# 365650250
Melezitoze	Sigma-Aldrich	Cat# 63620
Nuclease-free water	preferred vendor
Oligonucleotides	preferred vendor
PCR mastermix	preferred vendor or Instituto de Biologia Molecular do Paraná (IBMP, Curitiba, Brazil)	Chagas NAT kit
PCR thermocycler	preferred vendor
software for vacuum oven	Memmert Gmbh	Celsius v10.0
Trehalose	Sigma-Aldrich	Cat# T9531
Trypanosoma cruzi DNA	from in-house cultivated parasites, or purchased from accredited vendors such as ATCC
Vacuum oven	Memmert Gmbh	VO-400