LABORATORY
Direct test This test is designed to assay the change in DNA sequence in a particular gene that leads to a disease and can be done using an EDTA blood sample or from a cheek swab .
Indirect test This test uses DNA markers referred to as microsatellite markers , which are small pieces of DNA sequence that contain repeats of 2-4 nucleotides and are excellent tools for individual identification , parentage analysis and for linkage analysis to a disease gene . The test is indirect since the disease-causing gene is unknown and the status of the disease-causing gene ; mutant or normal , is inferred from the microsatellite markers . There is also a higher error rate associated with them compared to a direct DNA test . Once a linked microsatellite for a particular disease is identified in one species , the equivalent regions in the genomes of other species are located and candidate genes investigated . If there are no candidate genes based on comparison to other species , then the area near the disease gene is narrowed by checking additional nearby microsatellites . The techniques used to identify a gene without candidates can take many years and can be very expensive .
INFECTIOUS DISEASE DIAGNOSTICS
PCR has changed the clinical practice of infectious disease medicine and with advancement of technology may replace traditional culture-based assays for many animal pathogens . PCR has a wide range of clinical applications including pathogen detection , evaluation of emerging novel infections , and surveillance of infectious disease prevalence in a population .
PCR assays are of great value for documentation of infections that are difficult to culture , ( Ehrlichia and Mycoplasma spp .) or cannot be cultured ( Haemoplasmosis ). Specificity can be very high , depending on the primers used in the reaction . Primers can be designed to detect one genus but not others or designed to identify only one species . For example , PCR can detect all Haemoplasmas within a blood sample or just one species such as Mycoplasma haemofelis .
Although PCR can be a highly sensitive test , a positive result does not always prove that the infection is resulting in clinical illness :
• As PCR detects DNA of both live and dead organisms , a positive result may be achieved even if the infection has been controlled .
• When the organism commonly infects the background population of healthy animals , interpretation of results for a single animal can be difficult . For example , Bartonella henselae can infect up to 20 % of healthy cats and so a positive result in a clinically ill cat does not prove a disease association .
• PCR cannot discriminate between vaccine strains and field strains , thus a positive result does not indicate presence of a pathogenic strain .
• Real-time PCR can be used to determine the amount of microbial DNA in a sample and thus possible that the DNA load will correlate to the presence of disease . However , some agents are very host adapted and can have large amounts of DNA present in samples from healthy carrier cats . For example , the number of M . haemominutum copy numbers per μl of blood does not correlate to the haematocrit .
False positives in PCR testing can occur for a variety of reasons . In clinical settings , background contamination is a common cause of false positive reactions . The predominant source of contamination is derived from “ carry over ” products from previous PCR reactions . Unless a great deal of care is exercised , these products can contaminate reagents , tubes , pipettes , laboratory surfaces and even clothing .
False negative results can occur if the concentration of the pathogen is very low in the sample . This can be overcome by using methods to concentrate and / or purify the sample before initiating the PCR reaction .
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Issue 02 | APRIL 2017 | 23