Laboratory Methods Supporting Measles surveillance in Queensland, Australia, 2010 – 2017

ABSTRACTAustralia was officially recognised as having eliminated endemic measles circulation in 2014. Maintaining laboratory support for surveillance of vaccine-preventable diseases such as measles is an essential component of reaching and maintaining circulation-free status. Between 2010 and 2017 over 13,700 specimens were tested in our laboratory by real-time RT-PCR (RT-rPCR). Positive extracts from travellers were sequenced as required. Sequences were uploaded to GenBank and demonstrated the wide genetic variation expected from detection of MeV among virus introductions due to global travel. We describe the laboratory workflow employed in our laboratory between 2010 and 2017 for the sensitive detection of MeV infection, supporting high-quality surveillance.

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Development of a one step real-time RT-PCR method for sensitive detection of human astrovirus

Journal of Virological Methods ◽

10.1016/j.jviromet.2005.10.012 ◽

2006 ◽

Vol 133 (1) ◽

pp. 14-19 ◽

Cited By ~ 15

Author(s):

Enrique Royuela ◽

Ana Negredo ◽

Alicia Sánchez-Fauquier

Keyword(s):

Real Time ◽

Sensitive Detection ◽

Rt Pcr ◽

One Step ◽

Human Astrovirus ◽

Pcr Method

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Efficient capturing and sensitive detection of hepatitis A virus from solid foods (green onion, strawberry, and mussel) using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles and real-time RT-PCR

Food Microbiology ◽

10.1016/j.fm.2021.103921 ◽

2021 ◽

pp. 103921

Author(s):

Ruiqin Wu ◽

Baozhong Meng ◽

Milena Corredig ◽

Mansel W. Griffiths

Keyword(s):

Iron Oxide ◽

Magnetic Nanoparticles ◽

Real Time ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Sensitive Detection ◽

Fe3o4 Magnetic Nanoparticles ◽

Rt Pcr ◽

Solid Foods

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A multimarker real-time RT-PCR for MAGE-A gene expression allows sensitive detection and quantification of the minimal systemic tumor load in patients with localized cancer

Journal of Immunological Methods ◽

10.1016/j.jim.2007.04.006 ◽

2007 ◽

Vol 323 (2) ◽

pp. 180-193 ◽

Cited By ~ 9

Author(s):

Ingo Mecklenburg ◽

Dorothea Weckermann ◽

Alfred Zippelius ◽

Alexandra Schoberth ◽

Stephanie Petersen ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Sensitive Detection ◽

Rt Pcr ◽

Tumor Load ◽

Detection And Quantification

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SARS-CoV-2 variants in Paraguay: Detection and surveillance with a readily modifiable, multiplex real-time RT-PCR

10.1101/2021.09.15.21263618 ◽

2021 ◽

Author(s):

Magaly Martinez ◽

Phuong-Vi Nguyen ◽

Maxwell Su ◽

Fatima Cardozo ◽

Adriana Valenzuela ◽

...

Keyword(s):

Real Time ◽

Sanger Sequencing ◽

Lower Cost ◽

Population Level ◽

Nanopore Sequencing ◽

Receptor Binding Domain ◽

Rt Pcr ◽

High Quality ◽

Rna Detection ◽

Detection Of Mutations

Objectives The objective of the current study was to develop a lower-cost and scalable protocol to identify and monitor SARS-CoV-2 variants in Paraguay by pairing real-time RT-PCR detection of spike mutations with amplicon Sanger sequencing and whole-genome Nanopore sequencing. Methods 201 acute-phase nasopharyngeal samples from SARS-CoV-2-positive individuals were tested with two rRT-PCRs: 1) N2RP assay to confirm SARS-CoV-2 RNA detection (CDC N2 target), and 2) the Spike SNP assay to detect mutations in the spike receptor binding domain. The assay was performed with probes to identify mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Results All samples were positive for SARS-CoV-2 in the N2RP assay (mean Ct, 20.8; SD 5.6); 198/201 (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%) and most consistent with P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%); and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). Results were confirmed by Sanger sequencing in 181/181 samples (100%) with high-quality amplicon sequences, and variant calls were consistent with Nanopore sequencing in 29/29 samples. Conclusions The Spike SNP assay provides accurate detection of mutations associated with SARS-CoV-2 variants. This can be implemented in laboratories performing rRT-PCR to improve population-level surveillance for these mutations and inform the judicious use of scarce sequencing resources.

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Serological and Molecular Assays for Rapid and Sensitive Detection of Iris yellow spot virus Infection of Bulb and Seed Onion Crops

Plant Disease ◽

10.1094/pdis-92-4-0588 ◽

2008 ◽

Vol 92 (4) ◽

pp. 588-594 ◽

Cited By ~ 24

Author(s):

H. R. Pappu ◽

I. M. Rosales ◽

K. L. Druffel

Keyword(s):

Real Time ◽

Rapid Detection ◽

The United States ◽

Iris Yellow Spot Virus ◽

Yellow Spot ◽

Sensitive Detection ◽

Detection Methods ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Spot Virus

Iris yellow spot virus (IYSV) has spread rapidly in the United States and has become an important economic constraint to the production of both bulb and seed onion crops. Symptoms caused by IYSV may be confused with those caused by other fungal and bacterial pathogens and virus-specific, reliable, sensitive, and rapid detection methods would improve the diagnosis. Antiserum was produced to Escherichia coli-expressed nucleocapsid protein of IYSV and an indirect format of the enzyme-linked immunosorbent assay (ELISA) was developed. IYSV could be detected in onion tissue at dilutions of up to 1:1,000. An IYSV-specific primer pair was designed and used in a real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for the rapid detection of IYSV. Compared with standard RT-PCR, real-time RT-PCR was more rapid and sensitive. A commercially available RNA extraction kit and a total nucleic acid extraction method were compared for the quality of the templates obtained for use in real-time RT-PCR and there was no difference in limits of detection. Availability of ELISA- and PCR-based rapid and sensitive detection methods would facilitate accurate virus diagnosis and aid in better understanding of the epidemiology of the disease and in development of management strategies.

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