Further advantages of the technique lie in the ability to scan a single sample for many compounds, and the excellent data quality.

Our goal is to develop custom analyses for our cherished customers and collaborators on demand. Our vision is to extend the human lifespan by simplifying the treatment of complex diseases. In the future, cancer, infectious, neurodegenerative and mental diseases should be as easy to treat as a common cold.

At the moment, our highly skilled and trained personnel is implementing a test to accurately quantify a multitude of vitamins from as little as 20µL of blood (Vit Bs, Ds, A, E, K). Feel free to contact us for more information.

Der Beitrag Investment in high precision diagnostics erschien zuerst auf BioVariance - data-driven diagnostics.

qPCR diagnostics in the BioVariance lab

The abbreviation „qPCR“ stands for “quantitative polymerase chain reaction”. SARS-CoV-2 is a so-called RNA virus, that means that its genome consists of RNA and not of DNA like in animals or humans. An organism can be clearly identified by analyzing its genome, which is the main point of the PCR analysis. A pharyngeal sample contains RNA in the form of potential viruses as well as DNA in the form of human mucosal cells. Therefore, the contained RNA is isolated and purified before the actual PCR. This RNA is then converted to DNA using the enzyme reverse transcriptase, because the PCR can only work with DNA. After this process the actual PCR starts: The process being called “amplification”, characteristic segments of the produced DNA are multiplied using the enzyme polymerase. BioVariance targets 3 specific DNA segments, while 2 of them belong to genes found in SARS-CoV-2: The RdRP gene, which exists only in SARS-CoV-2 specifically. And the E gene, which occurs generally in the subgenus Sarbecovirus, whereto also SARS-CoV-2 belongs. The evidence of the RdRP gene alone would be enough to identify a sample as positive, but BioVariance presupposes the detection of both virus genes for the generation of a positive report to avoid wrong-positive results. The third targeted segment, the RNAse P gene, is a human gene, which serves as an internal control factor for checking whether the sample contains enough biological material to avoid wrong-negative results.

Evaluation of the PCR analysis

  But how can we check whether the amplification is successfull? Using a special PCR cycler (see Fig. 1), fluorescence markers are embedded into the multiplied DNA sequences during the PCR. Every target sequence is associated with another fluorescence colour. The more DNA is produced by the polymerase, the stronger is the fluorescence of the sample in the real time measurement. For generating an unequivocal result – no matter if negative or positive – the fluorescence signal of the human RNAse P gene must be sufficient. If there is additional fluorescence of both virus genes, the sample will be marked positive. The Ct value serves as a scale unit for the number of existing genes. It describes the number of DNA multiplication steps being necessary to differentiate the fluorescence signal clearly from the so-called background noise and to generate a reliable test signal. The lower the Ct value is, the higher is the number of existing target genes and thus the number of virus particles. Conversely, a high Ct value shows a low concentration of virus particles. During the PCR, the fluorescence signal arises until it peaks after about 1 hour. After this peak it flattens out because the added components for DNA multiplication are used up. This process creates the typical sigmoidal curve of the fluorescence (see Fig. 2).  

  But even the best procedure is subjected to limitations. In about 1 % of cases the result of the PCR is not unequivocal, because the sample material is insufficient or the fluorescence signal is too low, for example. In these cases, the PCR is repeated to gain a clear result.

Identification of virus variants using qPCR

In the BioVariance lab all positive samples additionally undergo a special variant PCR analysis to identify the respective SARS-CoV-2 variant, targeting several SARS-CoV-2 specific gene segments containing the yet known mutations. These analyses revealed that in the last weeks the former virus, the so-called wildtype, was gradually replaced by the British variant B.1.1.7, today called “Alpha“, in the county of Tirschenreuth. Furthermore, positive samples are also sent to a partner lab of BioVariance for further variant analyses, so that new and yet unknown mutations can be identified.

Screening of schools using pooled gargle samples

In May 2021, BioVariance has introduced a new pooling procedure, with several schools in the county of Tirschenreuth participating in the context of a study. This procedure does not need the pupils to take a nose or pharyngeal sample, but only to gargle with some tap water in the morning at home. At school, these gargle samples are “pooled” per class, that means mixed in one sample cup (see Fig. 3). Thus, only one PCR analysis has to be done for each class. Only if a pool sample is positive, the gargle samples of the referring pupils will be analyzed individually. This process allows great savings of lab resources while maintaining a high quality of results.  

Quality management in the BioVariance lab

BioVariance regularly faces external evaluations to ensure the quality and safety of their PCR analyses. For these so-called ring trials, the lab receives several unknown samples from a central office. Only if all samples are identified correctly as negative or positive, the lab will pass the trial and gain a certificate. In May 2021, BioVariance has passed again the external test from the European Society for External Quality Assessment (ESfEQA). Further, all incoming samples are treated under sterile conditions and conforming with strict hygienic regulations in the BioVariance lab, avoiding the contamination of the workstations and a possible infection of employees (see Fig. 4).  

Speed of PCR diagnostics in the BioVariance lab

In Germany, it usually takes 24-48 hours until a tested person receives the PCR result. In the BioVariance lab, results can be produced in less than 3 hours after the samples arrived in the lab. So 99 % of the tested people receive their result at the same day. Two special components allow BioVariance to reach this speed: On the one hand, BioVariance uses an optimized procedure for the RNA extraction which guarantees high efficiency, precision and safety. On the other hand, the IT specialists of BioVariance developed a purpose-built software called TubeLab enabling the digital monitoring of the lab processes, which clearly accelerated and facilitated the handling of samples. Using TubeLab for a transparent tracing and documentation, the BioVariance lab meets the highest requirements. Besides, the partner lab doing the further sequencing of the positive samples also uses TubeLab for monitoring their PCR analyses since April 2020. BioVariance also increased its personnel and expanded the lab team by 2 technical lab employees and 2 student assistants from the scientific area.

Strategic success in Tirschenreuth

With the help of the administrative district office and the health office, the administrative barriers for the establishment of the diagnostic lab as from 26.02.2021 in Waldsassen could be removed within a very short time. Short ways between the test station and the lab are indispensable for rapid testing. In the best case, both stations are located in the same building like in Waldsassen. And thanks to the BRK, 4 permanent and several mobile test stations could be established. More than 40.000 pharyngeal samples from people living in the county of Tirschenreuth have been analyzed since the commissioning of the lab. Using the highly efficient PCR diagnostics, infected people could be identified and quarantined within a few hours. This fact also had a strong positive influence on the local incidence. In February 2021 when the lab was commissioned, the 7-day-incidence was more than 350 infections per 100.000 inhabitants in the county of Tirschenreuth. Now, 15 weeks later, the region is the first county in Bavaria to show a 7-day-incidence of 0. The effective collaboration of the administrative district office, the health office, the BRK and the BioVariance lab as well as the consistent endurance of the population made it possible to signficantly reduce new infections and to finally conquer the high incidence in the county of Tirschenreuth.

Future orientation of the BioVariance lab

BioVariance aims to expand the application possibilities in the lab and to integrate the diagnostics of cancer diseases into their portfolio, additionally to the existing diagnostics of infectious diseases. The company has always focused on the personalization of cancer diseases by sequencing tumors and using the homemade software OncoVariant. This pioneering step allows the selection of the most suitable therapy for cancer patients individually to achieve the greatest possible effectiveness. Further, BioVariance plans to take advantage of the mass spectrometry, which allows the measurement of metabolic products for example.   Conntact person: Kerstin Hammer

Der Beitrag BioVariance Express-PCR-Lab conquers incidence in the county of Tirschenreuth erschien zuerst auf BioVariance - data-driven diagnostics.

Due to the Black Friday Week, there is another blog post. This time it’s about the craziest offer this week – sequencing at a great price – and why it’s still important – also in regards to personalized medicine.

Black Friday

In the USA Black Friday is the Friday after Thanksgiving, which is always celebrating on the fourth Thursday in November marking the start of a traditional family weekend and the beginning of the Christmas shopping season. The term Black Friday was first used in the 1960s in the United States and may have been resulted from the traffic chaos prevailing during this day and the crowds dragging through the streets like a „black mass“. Another definition could be derived from the black numbers written on this day which were generated by the huge sales every year. There is no connection to the in Germany known „Black Friday“ (known in the US as Black Thursday) , which reminiscent on the day of the New York market crash in 1929.

This custom was brought to Germany in 2006 by Apple and is now celebrated with corresponding special and free offers.

Dante Labs

Probably the most interesting offer discovering this week comes from Dante Labs. They’re represented in Europa as well as in the USA and maintain partnerships in various laboratories worldwide. The company offers sequencings of the whole genome in 8 weeks for just €850 – this Friday on the occasion of Black Friday even for only €169!

Genetics

In order to understand the enormous importance of this offer, there is a brief insight and review into the genetics of man.

Humans have 23 chromosomes – 22 autosomes and one gonosome, the so-called sex chromosome. They contain DNA (= desoxyribonucleic acid), which is called the molecule of life.

The DNA is composed of nucleotides, which in turn consists of a phophate, a sugar and one of the four available bases (adenine, cytosine, guanine and thymine). The structure of the DNA was decrypted in 1953 by Watson & Crick.¹

Thus, what constitutes and distinguishes one individual from another is only determined in the order of these four bases. DNA encodes genes providing all the information needed for the development and appearance of an individal. Humans have about 23,000 genes. That may sound like a lot – even considering that we humans are the most complex organisms – but it isn’t. Caenorhabditis elegans (threadworm) has 19,500 genes and Arabidopsis thaliana (thale cress), a plant of the cruciferous family, has even 27,000 genes and thus more than humans. Since 2012 it is known that even a banana has more genes – namely 36,500!²

The human genome (= the totality of all genes) consists of 3 billion bases. Amazing that the genomes of humans are similar to 99.5%. What then makes the enormous diversity between us? Exactly, the remaining 0.5%. That’s 15,000,000 bases! Also suprising is the fact that our genome is 99.9% similar to that of a chimpanzee and – to return to the previous example – 50 % similar to that of a banane!³

Through a method called sequencing, one can now read the sequence of the bases, also called the human code, and recognize changes, so-called mutations.

Human Genome Project

A revolution in this area represented the Human Genome Project, which was founded in October 1990.⁴ It was an international research project with the objective of fully decrypting a human genome, so to find out the sequence of 3 billion base pairs in the human DNA.⁵ The knowledge gained from this project should serve as the basis for investigation of many biological processes, such as heriditary diseases or molecular mechansims leading to cancer.⁵ The project was first led by James Watson, who has clarified the structure of the DNA. In total, more than 1,000 scientists from 40 countries participated. Since April 2003, the human genome has been officially decoded.⁴ Follow-up projects like the 1000 Genome Project have joined. This has also been regarded as finished since October 2012 – with more than 1,000 decoded genomes.

Sequencing

Since then a lot has happened in this field:⁶

In the 1990s it just took about eight years to sequence a genome, you can do that now in just a few days. The number of sequenced genomes amounts to several thousand in the meantime.

This is important in terms of medicine and research. As a result, the mechanisms of various diseases can be explained and the treatments adopted accordingly. Everyone is different – also in regard to the compatibility and effectiveness of drugs. The information that can be obtained from the DNA allows the personalization and individualisation of therapies.

A very important point – and now return to the introductory subject – are the costs for sequencing:⁶

A few years ago it cost a billion dollar to sequence a genome, now only a few thousand.

Or you wait for the Black Friday – then a sequencing costs only $199 (169€)!!

Contact Person:

Kristina Schraml (kristina.schraml@biovariance.com)

Sources

Der Beitrag Sequencing – a real bargain erschien zuerst auf BioVariance - data-driven diagnostics.