Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR)

Forootan, Amin; Sjöback, Robert; Björkman, Jens; Sjögreen, Björn; Linz, Lucas B.; Kubista, Mikael

doi:10.1016/j.bdq.2017.04.001

Cited by 416 publications

(368 citation statements)

References 18 publications

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“…We used the quantitative PCR (qPCR) assay and cycling conditions published in Wilcox, Carim, McKelvey, Young, and Schwartz () following their published primer and probe concentrations with TaqMan™ Universal Master Mix (Applied Biosystems, Thermo Fisher Scientific). This assay has a limit of detection (LOD) of nine copies per µL and limit of quantification (LOQ) of 13 copies per µL, following Forootan et al (). A standard curve using a synthetic gene containing the target sequences, ranging from 3,125 to 5 copies/reaction, was run on each plate.…”

Section: Methodsmentioning

confidence: 99%

Improved detection of rare, endangered and invasive trout in using a new large‐volume sampling method for eDNA capture

et al. 2019

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Environmental DNA (eDNA) detection probability increases with volume of water sampled. Common approaches for collecting eDNA samples often require many samples since these approaches usually use fine filters, which restrict the volume of water that can be sampled. An alternative to collecting many, small volume water samples using fine filters may be to collect fewer, large volume water samples using coarse filters that do not clog as rapidly. We used mesocosm experiments and field evaluations to compare coarse filter‐large water volume samples (hereafter large volume filter samples) versus fine filter‐small water volume samples (hereafter small volume filter samples) for detection and quantification of rainbow trout (Oncorhynchus mykiss) and bull trout (Salvelinus confluentus) DNA. We found that large volume filter sampling can be an effective approach for detecting DNA of low‐density target taxa. In mesocosm experiments, large‐volume and small‐volume water samples detected similar quantities of rainbow trout DNA. In the field, large volume samples more frequently detected bull trout DNA, had higher bull trout DNA copy number, and higher total DNA concentrations than small volume samples. However, sampling higher water volumes increased the potential for PCR inhibition so the DNA workflow had to be altered for large volume samples. Combining larger water volume samples with other strategies, like increasing PCR sensitivity and the number of PCR replicates, will improve detection of rare species, which is crucial for advancing conservation and ecological understanding.

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Section: Methodsmentioning

confidence: 99%

Improved detection of rare, endangered and invasive trout in using a new large‐volume sampling method for eDNA capture

et al. 2019

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“…RNAlater preserved tissues were homogenized to a liquid state with glass beads (Fisher Scientific) or a 5 mm steel ball (Qiagen). For tissues, the limit of detection (LOD) varied depending on the weight of tissue sampled with a mean of 3.5 log10 RNA copies/g of tissue, determined according to the calculations described in (49).…”

Section: Isolation and Quantitation Of Viral Rna From Fluids And Tissmentioning

confidence: 99%

A Combination of Two Human Monoclonal Antibodies Limits Fetal Damage by Zika Virus in Macaques

Rompay¹,

Coffey²,

Kapoor

et al. 2020

Preprint

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Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmissionand fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement in vitro (ADE) and extend their half-lives. Here we report on prophylactic co-administration of the Fc-modified antibodies to pregnant rhesus macaques challenged 3 times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV. Significance statement:Zika virus (ZIKV) infection during pregnancy can cause fetal abnormalities. Vaccines against ZIKV are under development, but because of potential safety concerns due to disease enhancing antibodies, and the time required by active immunization to induce protective antibodies, there is a need to explore alternative strategies. Recombinant monoclonal antibodies can be modified to prevent enhancement of infection, and thus could be an efficacious and safe alternative to vaccines to confer rapid protection. We show that prophylactic administration of two engineered antibodies, Z004 and Z021, to pregnant macaques partially protects against fetal neurologic damage and limits vertical transmission of ZIKV.

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“…Perhaps most noteworthy, however, is the assay's LoD. It is known that the detection of low copy numbers of DNA by qPCR often leads to poor reproducibility, as sampling noise tends to dominate (Forootan et al., ). The LoD has generally been defined as “the smallest amount of analyte that can be detected in a sample with stated probability, but cannot necessarily be quantified as an exact value” (C.L.S.I., ).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Accelerated ISAV replication detection by cell culture methods combined with time‐monitoring RT‐qPCR

Arseneau

Gautreau

Boston

et al. 2018

Journal of Fish Diseases

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Infectious salmon anaemia (ISA) is a viral disease that affects farmed Atlantic salmon (Salmo salar L.), often leading to mass mortalities. A quick detection of the ISA virus (ISAV) is crucial for decision‐making and can prevent the occurrence of future outbreaks. Screening done by Canada's National Aquatic Animal Health Laboratory System (NAAHLS) uses quantitative reverse transcription PCR (RT‐qPCR) followed by sequencing of PCR amplicons. As neither technique provides information regarding the infectivity of the virus, suspected virulent strains are subsequently tested using viral isolation. However, this stepwise process can require significant time to deliver results. To speed up this delivery, we have improved on these pre‐existing techniques by combining the use of cell culture with RT‐qPCR to detect replicative virus in as little as 5 days. Preliminary assays enabled the establishment of a minimal shift in Ct values over time, which is representative of viral replication in cultured cells. Subsequent blind panel analyses allowed the establishment of the optimal sampling days, as well as diagnostic sensitivity (DSe) and specificity (DSp) estimates. This method could be adopted not only by laboratories conducting diagnostic analyses for ISAV, but also for other slow‐replicating viral agents that replicate through a budding mechanism.

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Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR)

Cited by 416 publications

References 18 publications

Improved detection of rare, endangered and invasive trout in using a new large‐volume sampling method for eDNA capture

Improved detection of rare, endangered and invasive trout in using a new large‐volume sampling method for eDNA capture

A Combination of Two Human Monoclonal Antibodies Limits Fetal Damage by Zika Virus in Macaques

Accelerated ISAV replication detection by cell culture methods combined with time‐monitoring RT‐qPCR

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