Microbiology laboratory 5 The technology explained

The innovative molecular biology techniques

Constantly updating and using the latest molecular biology technologies to meet current and future challenges.

What do DNA-based studies involve?

In 1953, James Watson and Francis Crick discovered the double helix structure of deoxyribonucleic acid (DNA). This discovery revolutionized not only the scientific community but also our everyday lives.

DNA is the memory of a living being, serving as a code programming the development of cells and tissues, and provides a method of transmitting genes (reproduction, heredity).

The study of DNA allows:

the identification of a person or a species
the detection of anomalies or mutations

The study of DNA and other genetic information uses molecular biology tools permitting the collection of information about a living being at the molecular level (DNA).

Our growing knowledge on genetics and molecular biology over the last 20 years has permitted huge advances, including:

new therapies in the fight against certain diseases
detection of viruses such as SARS-CoV-2 during the Covid-19 pandemic.

The innovative new generation sequencing (NGS) technology

New generation sequencing (NGS) is a DNA sequencing approach allowing the decoding of genetic information. This new innovative method can handle large amounts of information allowing ultra-high throughput at much higher speeds compared to traditional methods.

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The standard technique based
on quantitative PCR (qPCR)

Also referred to as real-time PCR (RT-PCR), this technique remains the gold standard of all molecular biology methods in the field of diagnostics. It is reliable, sensitive, robust and can be used in high throughput.

The detection and quantification of microorganisms

Detection of bacteria, viruses or fungi in different matrices:

plant
soil
wastewater
cosmetic cream
air samples

Case study

Since 2014, a qPCR method is used in the early detection of the agent responsible for powdery mildew of grapevines (Erysiphe necator). This method enables an early alert on the presence of powdery mildew before the appearance of symptoms. Since 2022 this method has been made easily accessible, in a ready-to-use kit in the idetect^TM range, to laboratories able to perform molecular analyses.

Monitoring resistance to fungicides

Detection and quantification of genetic variants: for example, mutations of genes coding for proteins targeted by and thus offering resistance to fungicides

Case study

Resistance to quinone outside inhibitors (QoI), fungicides acting to block respiration, in agents responsible for downy mildew of grapevines is caused by a mutation in the cytochrome b gene (G143A). The frequency of this mutation is monitored in mixed populations to evaluate the level of resistance by qPCR.

The method of sequencing small fragments, pyrosequencing

Small fragments (<200pb) at a time are sequentially sequenced via enzymatic reactions. Following PCR-based amplification of the region of interest, the amplicons are sequenced by incorporation of labeled nucleotides.

The signal translated as nucleotide sequences is displayed in real time allowing the detection and quantification of genetic variations. With the PyroMark^® 48 from Qiagen, pyrosequencing has received a new make-over and gained in speed, reliability and accessibility.

Case study

What are the limitations of qPCR? Difficulties can be encountered:

When several mutations are present at the same site
With unknown mutations
With deletions or insertions of several nucleotides.

In these cases, pyrosequencing offers a more precise solution:

SARS-CoV-2 in wastewater: detection and quantification of variants. To meet the challenge relating to the emergence of numerous variants of the virus, pyrosequencing ensures their efficient tracking.

Resistance to fungicides: our team has discovered a new mechanism of resistance shown by the agent responsible for grapevine powdery mildew (Plasmopara viticola) to a fungicide called cyazofamid (cellular respiration inhibitor). This resistance comes from a 6 nucleotide insertion-induced modification of the fungicide target. The frequency of genes carrying this insertion can be quantified by pyrosequencing but not by qPCR.