Allelic discrimination are methods to discriminate between two or more sequence changes at a particular gene locus. Typically, one allele (“wild type” DNA sequence) is common, and the other allele (mutation) is rare. In a SNP there are 3 genotypes: – a diploid homozygote (two wild type alleles (PP)), a diploid homozygote (two mutant alleles (pp)) and a heterozygote (one wild type allele and one mutant allele (Pp)).

SNPs (single nucleotide polymorphisms) help predict an individual’s resistance or susceptibility to certain drugs (pharmacogenetics), susceptibility to environmental factors such as toxins, and risk of developing diseases. SNPs can also be used to track the inheritance of disease-associated genetic variants within families.

qPCR (Real-time PCR) enables you to screen known SNPs. The benefits of qPCR are that it is easy, accurate, and can scale to high throughput. The bioinformatic analysis is also less complex than for other technologies, such as sequencing and microarrays. 5′-nuclease (TaqMan) allelic discrimination uses a primer pair to amplify the target area and two allele-specific probes to detect your target SNP alleles and report the genotypes of your samples. Unlike normal qPCR, in genotyping the fluorescence level is only measured at the end of the PCR and the results are displayed on a scatter plot, allowing hundreds of assays to be run and analysed in parallel.

Immunoglobulin G is the major inhibitor of qPCR in blood, as it can bind to single-stranded genomic DNA, leading to increased Ct values and a lower signal. The other two main inhibitors in blood are hemoglobin and lactoferrin that affect the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin can also cause some fluorescence quenching, particularly for FAM. In addition, inhibitors can be found in the anticoagulants used to stabilize blood samples (e.g. EDTA, citrate, or heparin).

Yes, the Lyo-Ready™ Genotyping Direct qPCR Blood can be used as a liquid mix or lyophilized and stored at ambient temperature without needing to change the reaction conditions and lyophilization/storage will not affect the sensitivity of the test.

Blood contains abundant information about the health status of the organism, it can be used to evaluate organ function, detect proteins, nucleic acids, metabolites, infectious diseases and can be used for diagnosis of cancer (liquid biopsy), heart disease, diabetes. DNA and RNA Blood tests are a simple and easily accessible (low-invasive) method to assess and monitor health.

Immunoglobulin G is the major inhibitor of qPCR in blood, as it can bind to single-stranded genomic DNA, leading to increased Ct values in a quantitative PCR blood test. The other two main inhibitors in blood are hemoglobin and lactoferrin that affect the DNA polymerase activity and thus lower the amplification efficiency. Hemoglobin and hematin can also cause some fluorescence quenching, particularly for FAM, although this should not affect the Ct value. In addition, inhibitors can be found in the anticoagulants used to stabilize blood samples (e.g. EDTA, citrate, or heparin).

We have used up to 20% whole blood with our mix, higher concentrations cause to solution to become viscous and difficult to work with.

We use our own proprietary Taq polymerase and a highly optimized buffer system to overcome inhibition.

It is stable for up to 2 years at ambient temperature if correctly stored in sealed pouches.

Yes, the Air-Dryable™ qPCR Blood kits can be used to detect both cfDNA and cfRNA, making it ideal for liquid biopsy.

Yes, it is possible that you may need to pre-treat the blood in order to release the DNA from the gram-positive bacteria. In some cases a heat shock is sufficient, this will depend on the bacteria being examined.

We do not see a performance difference between the two mixes, which is why we call the Air-Dryable Direct RT-qPCR Blood mix – Air-Dryable™ Direct RNA/DNA qPCR Blood mix, as it can be used for both.

It can be stored frozen or following the instructions from the storage buffer manufacturer.

They are compatible with standard cycling conditions and can also be used with very fast cycling conditions, however some optimization may be required for the amount of blood that is needed for qPCR blood tests.

You should elute in water, or TE buffer.

Yes, the Air-Dryable™ Direct DNA qPCR Blood and Air-Dryable™ Direct RNA/DNA qPCR Blood can be used as a liquid mix or air/oven dried and stored for up to 24 months without needing to change the reaction conditions and air drying/ambient temperature storage will not affect the sensitivity of the test.

We advise not using Triton; however, heat treatment is ok.

The advantages of a quantitative PCR urine test are that it is completely non-invasive, and it can be self-sampled in large quantities. Urine testing is used in the diagnosis and management of infectious diseases (including STDs), hormone and metabolic disorders, renal diseases, bladder cancer, urinary tract infections (UTIs) and for monitoring recreational drug use.

The most common infections are urinary tract infections (UTIs), most commonly Escherichia coli, Protus mirabilis, Enterococcus faecalis, Staphylococcus saprophyticus and Klebsiella pneumoniae. Several other infections can be diagnosed by urine DNA and urine RNA tests including community-acquired pneumonia (CAP), legionellosis, tuberculosis, congenital cytomegalovirus (CMV) infection, and arboviruses, such as Chikungunya virus, dengue virus and Zika virus. Parasites can also be diagnosed from urine testing for the detection of eggs from Schistosoma haematobium. Furthermore, urine has been used for screening of different sexually transmitted diseases (STD) such as Neisseria gonorrhoeae and Chlamydia trachomatis.

Cancer cells or their DNA or RNA are too big to pass through the kidneys and will have to come from the bladder or ureters, so bladder cancer is the most obvious urine cancer, however prostate and cervical cancer are also detectable.

We have used up to 25% human urine with our mix.

It is stable for up to 2 years at ambient temperature if correctly stored in sealed pouches.

That will depend on the bacteria, for some bacteria you may not need to do any pre-treatment, however some may require heating to 95°C for 5 min and for some tougher bacteria, a Proteinase K step (Proteinase K 55°C for 15 min and then at 98°C for 5 min) or using a non-ionic detergent (such as Tween-20 to final concentration of 0.2% followed by incubation at room temperature for 10 min) may be necessary.

We do not see a performance difference between the two mixes, which is why we call the Air-Dryable Direct RT-qPCR Urine mix – Air-Dryable™ Direct RNA/DNA qPCR Urine mix, as it can be used for both.

Yes, the Air-Dryable™ Direct DNA qPCR Urine and Air-Dryable™ Direct RNA/DNA qPCR Urine can be used as a liquid mix or air/oven dried and stored for up to 24 months without needing to change the reaction conditions and air drying/ambient temperature storage will not affect the sensitivity of the test.

A quantitative PCR stool test can allow identification of enteric pathogens such as harmful bacteria, fungi, viruses, or parasites down to the genus or species level, fast and accurately in a few hours, rather than the culture-based stool testing that can take several days. It can also be used as a non-invasive method for screening autoimmune conditions and cancer.

The fecal occult blood test (FOBT) has been the most widely used gastrointestinal immunoassay to screen colorectal cancer (CRC) for over three decades, however, recent large-scale studies have shown that the FOBT lacks the sensitivity to detect early stages of CRC. A qPCR cancer stool test is a better diagnostic tool as it offers significant improvement in sensitivity and accuracy and can be used to screen for multiple targets and so speed up diagnosis, therefore improving prognosis.

We have used up to 25% human stool with our mix, this is homogenized, and clarified by centrifugation and some pre-treatment may be required.

There are several buffers on the market that can be used for stool testing, they include phosphate-buffered saline (PBS), Cary-Blair Medium, Stool Transport and Recovery (STAR) buffer and DNA/RNA Shield.

It is stable for up to 2 years at ambient temperature if correctly stored in sealed pouches.

Yes, most of it comes from cells that are sloughed into the lumen after cells die and are replaced, these cells go through apoptosis, releasing the DNA. Tumor cells may be present, but they will normally be less than 1% of the total human DNA fragments present in the overall stool sample.

Pre-treatment will depend on what you are looking at, cfDNA may not need pre-treatment, but you may need to heat to 95°C for 5 min and for some bacteria, a Proteinase K step (Proteinase K 55°C for 15 min and then at 98°C for 5 min) or using a non-ionic detergent (such as Tween-20 to final concentration of 0.2% followed by incubation at room temperature for 10 min) may be necessary.

We do not see a performance difference between the two mixes, which is why we call the Lyo-Ready™ Direct RT-qPCR Stool mix – Lyo-Ready™ Direct RNA/DNA qPCR Stool mix, as it can be used for both.

Yes, the Lyo-Ready™ Direct DNA qPCR Stool and Lyo-Ready™ Direct RNA/DNA qPCR Stool can be used as a liquid mix or lyophilization and stored for up to 24 months without needing to change the reaction conditions and lyophilization/ambient temperature storage will not affect the sensitivity of the test.