Design and use of group-specific primers and probes for real-time quantitative PCR

Real-time quantitative polymerase chain reaction (qPCR) has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil, water, sediments, and sludge. Although qPCR is a very useful technique for nucleic acid quantification, accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used. Many aspects of conducting qPCR assays have become increasingly routine and automated; however, one of the most important aspects, designing and selecting primer and probe sets, is often a somewhat arcane process. In many cases, failed or non-specific amplification can be attributed to improperly designed primer-probe sets. This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays. We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes. qPCR assays using group-specific primers and probes designed with this method, have been used to successfully quantify 16S ribosomal Ribonucleic Acid (16S rRNA) gene copy numbers from target methanogenic and ammonia-oxidizing bacteria in various laboratory- and full-scale biologic processes. Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.     

An experimental study on the airlift pump with air jet nozzle and booster pump

The experiments for high head airlifting performance with vertical tube were examined for wastewater treatment. Comparing with the centrifugal pump and other pumps, the airlift pump has some problems and limited applications. However, an advantage of an airlift pump is in its geometrical simplicity, not having any moving parts, so it is suitable in lifting fluids including tiny pieces of metal or grit. In this study, for the purpose of high lifting head, an air jet nozzle was used. We have performed experimentally according to various characteristics of the airlift pump system such as the change of submerged depth, lifting head of liquid-air mixture (total head) and air flow rate. This work has verified through experiments that airlift pump shows lifting ability for 3 m (S_r = 0.3) in comparison with conventional height, 2 m (S_r = 0.4). Also, we suggested that the new airlift pump system with the air booster pump be used to improve the higher lifting head performance.     

Specimen‐Based Modeling,Stopping Rules,and the Extinction of the Ivory‐Billed Woodpecker

Abstract: Assessing species survival status is an essential component of conservation programs. We devised a new statistical method for estimating the probability of species persistence from the temporal sequence of collection dates of museum specimens. To complement this approach, we developed quantitative stopping rules for terminating the search for missing or allegedly extinct species. These stopping rules are based on survey data for counts of co‐occurring species that are encountered in the search for a target species. We illustrate both these methods with a case study of the Ivory‐billed Woodpecker (Campephilus principalis), long assumed to have become extinct in the United States in the 1950s, but reportedly rediscovered in 2004. We analyzed the temporal pattern of the collection dates of 239 geo‐referenced museum specimens collected throughout the southeastern United States from 1853 to 1932 and estimated the probability of persistence in 2011 as <6.4 × 10^?5, with a probable extinction date no later than 1980. From an analysis of avian census data (counts of individuals) at 4 sites where searches for the woodpecker were conducted since 2004, we estimated that at most 1–3 undetected species may remain in 3 sites (one each in Louisiana, Mississippi, Florida). At a fourth site on the Congaree River (South Carolina), no singletons (species represented by one observation) remained after 15,500 counts of individual birds, indicating that the number of species already recorded (56) is unlikely to increase with additional survey effort. Collectively, these results suggest there is virtually no chance the Ivory‐billed Woodpecker is currently extant within its historical range in the southeastern United States. The results also suggest conservation resources devoted to its rediscovery and recovery could be better allocated to other species. The methods we describe for estimating species extinction dates and the probability of persistence are generally applicable to other species for which sufficient museum collections and field census results are available.     

Design and use of group-specific primers and probes for real-time quantitative PCR

Real-time quantitative polymerase chain reaction (qPCR) has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil, water, sediments, and sludge. Although qPCR is a very useful technique for nucleic acid quantification, accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used. Many aspects of conducting qPCR assays have become increasingly routine and automated; however, one of the most important aspects, designing and selecting primer and probe sets, is often a somewhat arcane process. In many cases, failed or non-specific amplification can be attributed to improperly designed primer-probe sets. This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays. We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes. qPCR assays using group-specific primers and probes designed with this method, have been used to successfully quantify 16S ribosomal Ribonucleic Acid (16S rRNA) gene copy numbers from target methanogenic and ammonia-oxidizing bacteria in various laboratory- and full-scale biologic processes. Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.     

Methanogenic diversity changes in full-scale anaerobic digesters by co-digestion of food waste and sewage sludge

Journal of Material Cycles and Waste Management - This study aims to investigate the methanogenic community of anaerobic mono-digestion of sewage sludge (SL-digester) and co-digestion of sewage...