Molecular assays

Yu C-P, Ahuja R, Sayler G, Chu K-H (2005) Quantitative molecular assay for fingerprinting microbial communities of wastewater and estrogen-degrading consortia. Appl Environ Microbiol 71 1433-1444... 

Invader UGTIAI molecular assay for irinotecan toxicity. A genetic test for an increased risk of toxicity from the cancer chemotherapy drug irinotecan (Camptosar). Med Lett Drugs Ther. 48, 39-40. 

The molecular assays in Clk"mAic2As bom fide rhythms with a predominant effect on circadian rhythm amplitude and no more than a modest effect on phase or period. With circadian per and tim enhancers, we observed reduced enhancer activity and a reduced cycling amplitude in a Clk" background, consistent with the role of Clk in regulating these enhancers. Nonetheless, the phase of oscillating bioluminescence is similar to that of wild-type flies. The presence of molecular rhythms contrasts with the absence of detectable behavioural rhythms. We favour the notion that this reflects a level or amplitude reduction below a critical threshold for behavioural rhythmicity. The absence of anticipation of light—dark transitions makes it very unlikely that an effect restricted to the lateral neurons — the absence of the neuropeptide PDF, for example — is primarily responsible for the behavioural phenotypes. This is also because LD behavioural rhythms are largely normal in flies devoid of PDF or the pacemaker lateral neurons (Renn et al 1999). However, we cannot exclude the possibility of selective effects of Clk" on other behaviourally relevant neurons. 

Total protein assays have the advantage of being relatively straightforward compared to molecular-level analyses. Methods with fluorescence-based detection are also highly sensitive, and thus amenable direcdy to DON. Quantitative interpretation for environmental mixtures such as seawater, however, may be problematic for some samples. Most methods react with specific moieties (e.g., coomassie blue binds to lysine and arginine) and thus results obtained can depend on protein composition, size distribution, and even conformation (Sapan et ai, 1999), making the careful choice of calibration standards important. In addition, common components of natural samples, such as humic materials (e.g., Mayer et ai, 1986), carbohydrates (Sapan et ai, 1999), or NH3 may interfere with quantification. Overall, colorimetric methods can be very useful as quick, Hkely semi-quantitative estimates of total protein or peptide. However, potential biases inherent in the mechanism of a specific method should be considered before one is chosen, and appHcation of newer molecular assays (e.g., CBQCA) should be carefully examined in terms of natural sample matrix (Nunn et ai, 2003). 

Available control materials include patient specimens that have been well characterized by a similar method, synthetic controls such as Armored RNA (Ambion Inc., Austin, Texas), or purified nucleic acid, although purified nucleic acid would not be appropriate for use as an extraction control. Armored RNA uses a bacteriophage coat around an RNA target to produce pseudoviral particles. Control material is currently available for a variety of pathogens, including HIV-1, HCV, hepatitis A virus, enterovirus, Norwalk virus, and West Nile virus. These armored RNA preparations are stable for at least 11 months and are compatible with a large number of different platforms commonly used for quantitative molecular assays. Table 42-3 lists other manufacturers of control materials. 

Proficiency testing for molecular diagnostics laboratories remains a challenge because adequate proficiency testing programs are unavailable to cover the wide variety of molecular assays offered by a number of laboratories. The CAP offers the only proficiency program accredited for molecu-... 

As mentioned above, many molecular assays detect rhi-noviruses and most will detect polioviruses. These two factors can lead to unexpected and misleading positive results when testing respiratory or stool specimens. The diagnosis of enterovirus meningitis should be based on testing of CSF specimens, while sepsis syndrome in the neonate is best made by testing serum, plasma, or CSF samples. 

The diagnosis of CMV disease represents a challenge because of the presence of the latent infection. Immunocompromised individuals can have an asymptomatic, chnicaUy insignificant, low-level, persistent infection that must be distinguished from clinically important active CMV disease. The distinction can be challenging when using sensitive molecular assays that can detect small amounts of CMV DNA in clinical specimens. 

Molecular assays have utility for the diagnosis of active GMV disease, because GMV DNA concentrations are higher in patients with active GMV disease than in those with asymptomatic infection. A study in fiver transplant recipients using the Amplicor PGR assay showed that the median peak viral load in patients with asymptomatic infection was 1850 copies/mL compared with 55,000 copies/mL for those with active disease. The viral load cutoff that was most predictive of the development of active disease was between 2000 and 5000 copies/mL of plasma. A similar study in renal transplant recipients using the Hybrid Gapture assay showed that the risk of developing GMV disease increased from 1.5% with a viral load of 10,000 copies/mLof bloodto 73% when the viral load was 1 million copies/mL of blood. It is important to note that the viral load cutoffs differed in the two studies because of the use of different assays and differences in specimen type (plasma versus whole blood). 

A key consideration with molecular assays is controlling for inhibition of amplification. This is particularly critical for the detection of MTb nucleic acid from respiratory specimens, because these specimen types often contain blood or glycoprotein, which can inhibit amplification. The Amplicor assay contains an internal control, and a negative result cannot be reported unless the internal control is detected. The MTD test does not contain an inhibition control, though many laboratories include such a control by adding a positive control material to a second aliquot of the clinical specimen. 

An important role for molecular assays is in the diagnosis of extra-pulmonary MTb infections, such as meningitis,... 

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