Companies: Cepheid
–STD testing: Everylwell (test for HIV and Syphilis, Hepatitis C, HIV, Herpes Type 2, Chlamydia, and Trichomoniasis Vaginalis at the same time; need a blood sample)
It goes without saying that one of the most important aspects of treating any pathogenic disease is the diagnosis of the pathogen. Without knowing what you are treating, it is hard to treat. In general, diagnosis falls into three different categories: Phenotypic methods, immunologic method and genotypic methods:
Phenotypic Method for the diagnosis of infections
Phenotypic methods for the diagnosis of infection includes the combination of results from tests such as gram staining, growth on different media and simple enzymatic tests. Usually a Gram stain is the first test. This will separate bacteria into gram positive and negative. Another phenotypic test is the Mycobacteril growh indicator tube (MGIT) for Mycobacterium tuberculosis. This test monitors oxygen levels in a tube that been inoculated with a patient speciment. The tub contains a medium that encourages the growh of the tuberculosis bacterium (which is every slow growing). There is a silicon chip at the bottom of the tube that is impregnated with a fluorescent substance that is sensitive to oxygen levels. When first inoculated, there will be a lot of free oxygen but as the bacteria grows, they will use the oxygen and the decreased oxygen levels allows the silicon compound to flouresce.
Another important phenotypic test is testing for antimicrobial susceptibility.
Other phenotypic tests include using bacteriphages which involves inoculating a lawn of bacterial cells onto agar into blocks and applying a different phage to each sectioned area of grwoth. Cleared areas correspond to lysed cells indicating senstivity to that phage, and a bacterial identificaiton may be determined from this pattern.
Microbila culturing has two major drawbacks. (1) it takes a minium of 18-24 hours and often longer to culture and (2) many infections may be caused by nonculturable organisms.
Immunoglogic Methods
Immunologic or serologic methods take advantage of the extreme specificity of antibodies for antigens and includes tests like ELISA and Western blot.
Genotypic Methods (Gene Expression Analysis)
The analysis of gene expression is a common experiment conducted in laboratories. Profiling gene expression provides valuable insight into genes involved in normal cell/tissue homeostasis, organism development and information about genes that when their expression is up or down-regulated, lead to disease.
A number of methods are employed to examine gene expression with the most currently employed being Northern blotting, RNase Protection Assay (RPA) and real-time polyemrase chain Reaction (RT-PCR). Each of these methods has advantages and disadvantages relating to sample size, cost, time involved, use of radioactivity, and sensititivy which is estimated to vary from 10k copies of mRNA (Northern blotting) to one copy (RT-PCR).
An assay called the GeneXpert MTR/RIF test is an automated test using nucleica cid amplictionwhich is a cartridge system that indicates both the identity of the bacterium M. tuberculosis and also whether is is susceptible to the antibiotic, rifampin.
Panbacterail qPCR (pan means “universal”) is a real time PCR techniques that uses a primer (16srRNA) that is common to all bacterai. This is very important when assessing a wound or infection that is polymicrobial. It also can detect bacteria even after antibiotic treatment.
Whole genome sequencing has now become a technique of choice, particularly since it has become so cheap.
Capillary electrophoresis with laser induced fluorescence (CE-LIF): is a method for the direct quantitation of gene expression. CE-LIF provides a fast, direct sensitive and non-radioactive means to detect molecules, for example RNA.
Goldsmith (US7939252) discloses a method of measuring an amount of ribo-nculeic aci (RNA) by incubating an RNA sample with a fluorescently labeled RNA probe complementary to a sequence in a target RNA under conditions whereby the probe can hybridize to the target RNA to form an RNA-fluorescently labeled proble hydribd, passing the RNA fluorescenlty labeled proble hybrid through a capillary electrophoresis system (c) etecting and recoridng changes in fluorescence as a peak as the RNA fluorescenlty labeled probe passes through a detection window and determining an area under the peak, whereby the area under the peak indicates the amount of target RNA. In one aspect, the method is used to diagnose a disease in a subject by comparing the level of expression of the gene in the subject to a control level of expression of a gene from a subject without disease. In one embodiment a fluorescently tagged riboporbe is added in excess to a sample of RNA and allowed to undergo hybridization of the labeled riboprobe to target RNA. The sample, containing free probe, unhybridezed RNA and probe-target hybrids is injected into a silica capilary containing a sieving matrix which will separate the various components of the hybridiczation reaciton based upon their size. Using a fluorescence detector, as the various components of the hybridization reaciton pass through the detection window, only those with a fluorescent molecule incorporated (free probe or probe-target hybrids) will be detected. Because the rpobe is much small than the target probe hybrid, it passes trhough the detection window first followed at a later time by the target probe hybride. The method can simulataneously detect multiple, distinct species of RNA. cause each target RNA fluorexcenlty labeled probe hybride will have a distinct mass and conformation, it will need a characteristic maount of time to pass through the cpaillary and pass the detection window. These differences between two unique target RNA fluorescenlty labeled probe hybrids allows for them to be seaprated form one another in the capillary and thus pass by teh detection window at different times and simultaneously detect in the same sample. At the same time, by using a multiple wavelenght detector it is also possible to sue a fluorophore that only one of the lasers on the instrument can detect (e.g., 488 nm vy 620 nm). A target RNA fluorescenlty labeled probe hybride formm fomr the probe will only be detectable by one particular laser on the system and at the same time a different target RNA: fluorescently labeled proble hybride formed using a different fluoreophore will only be detectable using a different laser. Because the lasers oeprate simultaensouly, it is possible to detect as many unique RNA species as there are lasers of different wavelenghts. This can occur regardless of wehther there is any difference at the time it takes the target RNA: fluorescnelty labeled probe hybrids to travel to the detector window.
Tools and approaches of are having a major impact on drug discovery and development including target identification and verification and high throughput screening of potential drugs. For example, can be used to screen for gene products involved in biological processes of pharmaceutical interest and as in situ protein microarrays for development and assessment of lead compounds. Activity-based/ can also be used to covalently modify a set of related enzymes and facilitate their purification and or identification as potential drug targets.
Yeast expression systems: Yeast is a powerful model organisms to identify genes involved in drug responses. A yeast deletion collection which consists of about 21,000 hapliod strains and heterozygous and homozygous diploid strains of S. cerevisiae that each possess a precise deletion (from the start to the stop codon) in one of about 6000 yeast genes has been generated. In any experimental screen this collection of about 6000 genomes can be assayed and all genes required for growth can be identified.
Additional Diagnosing Techniques
Mass Spectrometry: is used alone and in combination with other technologies such as PCR to provide rapid and highly accurate microbail identificaiton within just minutes.