High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses v2

This process instruction describes the steps for pre-analytical processing of primary settled solids from wastewater treatment plants for downstream nucleic acid purification and quantification. Previous research has demonstrated that enveloped viral particles, such as SARS-CoV-2, associate with the solids in wastewater. Therefore, concentrating the solids in the sample and removing the water concentrates the viral particles as well and increases the sensitivity of the assay. Bovine coronavirus vaccine (BCoV) is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. The dry weight of the sample is determined to account for sample variability between different treatment plants and between samples collected on different days and allows for the final quantification to be normalized to the precise quantity of solids in the sample. A dry weight conversion factor to adjust for the final amount of solids in the quantified sample is determined by measuring the difference in mass between the “wet” dewatered solid and after the sample is dried at 110°C overnight. For long term storage, up to 50mL of the original primary settled solid sample is stored at 4°C and small aliquot of the dewatered solids is stored at -80°C. This process instruction applies to sample dewatering, BCoV control spike in, homogenization, dry weight measurement, aliquoting and storage.

Extraction of bacterial DNA using MagMAX™ CORE Nucleic Acid Purification Kit on KingFisher™ Flex Instrument v1

This procedure is used to extract genome DNA of bacteria isolates using theMagMAX™ CORE Nucleic Acid Purification Kit on the KingFisher Flex Robot. The process consists of Sample processing, Prepare plates for Robot, and Operate on KingFisher Flex machine.

A protocol for the detection of genetic markers in saliva by polymerase chain reaction without a nucleic acid purification step: examples of SARS-CoV-2 and GAPDH markers.

Background: The objective of this study was to develop a protocol for direct use of saliva in tests for genetic markers, without purification of nucleic acids. Currently, diagnostic tests use purified nucleic acids from clinical samples. This purification step adds time, cost, and affects the quality of testing. Multiple attempts to remove the purification step were reported. Results: We report a protocol for the direct detection of genetic markers in saliva. The protocol is based on collection of saliva in a solution containing a detergent and ethanol, and is compatible with isothermal amplification (LAMP), real-time RT-PCR and RT-PCR. SARS-CoV-2 and GAPDH markers were used as reference markers. We observed that mild detergents allow efficient detection of markers (e.g. GAPDH and SARS-CoV-2), while strong detergent, e.g. sodium dodecyl sulfate, inhibited the PCR reaction. Under these conditions, saliva samples can be stored for 24 h at +40C or -180C with preservation of the markers. Storage at room temperature led to deterioration of marker detection. Snap heating of saliva samples at the time of collection, followed by a storage at the room temperature, provided partial protection.Conclusions: The protocol presented in this report describes collection and storage of saliva for direct detection of genetic markers and is compatible with PCR and LAMP tests.

Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification

The COVID-19 pandemic caused by the SARS-CoV-2 virus motivates diverse diagnostic approaches due to the novel causative pathogen, incompletely understood clinical sequelae, and limited availability of testing resources. Given the variability in viral load across and within patients, absolute viral load quantification directly from crude lysate is important for diagnosis and surveillance. Here, we investigate the use of digital droplet PCR (ddPCR) for SARS-CoV-2 viral load measurement directly from crude lysate without nucleic acid purification. We demonstrate ddPCR accurately quantifies SARS-CoV-2 standards from purified RNA and multiple sample matrices, including commonly utilized universal transport medium (UTM). In addition, we find ddPCR functions robustly at low input viral copy numbers on nasopharyngeal swab specimens stored in UTM without upfront RNA extraction. We also show ddPCR, but not qPCR, from crude lysate shows high concordance with viral load measurements from purified RNA. Our data suggest ddPCR offers advantages to qPCR for SARS-CoV-2 detection with higher sensitivity and robustness when using crude lysate rather than purified RNA as input. More broadly, digital droplet assays provide a potential method for nucleic acid measurement and infectious disease diagnosis with limited sample processing, underscoring the utility of such techniques in laboratory medicine.

Comparison of Deoxyribose Nucleic Acid Purification Methods of Mangosteen (Garcinia mangostana L.) and Its Relatives

Mangosteen (Garcinia mangostana L.) and its relatives (Garcinia hombroniana, Garcinia celebica, Garcinia forbesii, Garcinia malaccensis, Garcinia porecta, Garcinia subeliptica, Chalophylum inophylum) contain polyphenol compound. The polyphenol compound makes pure deoxyribose nucleic acid is difficult to reveal. The aim of this research was to find the deoxyribose nucleic acid purification method of mangosteen leaves and its relatives. The research was conducted from January to August 2015 at the Center of Horticultural Tropical Studies Laboratory, Bogor Agricultural University. The mangosteen leaves were isolated based on cetyl trimethyl ammonium bromide (CTAB) buffer extraction added by 2X chloroform isoamyl alcohol (CIAA 24:1), 3X CIAA 24:1, and sliced gel purification using Fermentas kit extraction. The result showed that CTAB added by 2X CIAA was the best treatment for Garcinia mangostana L. and its relatives for purification of deoxyribose nucleic acid. This modified method produced an apparent amplified polymerase chain reaction using PKBT7 inter simple sequence repeat marker. It was applicable to evaluate genetic diversity interspecies.

Optimization of a molecular diagnostic strategy to verify SARS-CoV-2 infections by RT-qPCR

ObjectivesFast and precise detection of SARS-CoV-2 RNA in infected patients is essential for treatment decisions.MethodsA diagnostic strategy by analyzing nasopharyngeal swabs to detect SARS-CoV-2 RNA in individuals was established. The negative impacts of the individual buffer components on RT-qPCR analysis was reviewed and overcome by RNA purification. To investigate the functionality of the improved protocol we compared the novel diagnostic strategy to a Bead-based RNA extraction method using previously positive tested samples.ResultsA method to extract purify RNA molecules from SARS-CoV-2 was established. We examined the significance of nucleic acid purification and the need for an RNase inhibitor. Evaluation of 3,664 samples from March 23rd until May 18th in 2020 showed the incidence of COVID-19 infections in Thuringia, Germany. Additionally, benefits and limits of three RT-qPCR kits were assessed.ConclusionsOur study suggests that virus capsid lysis combined with nucleic acid purification enables a viable alternative for the molecular diagnostics of SARS-CoV-2 infections. Due to the current delivery delays from different companies, this method offers the possibility to continue diagnosis and to handle the large number of samples.

Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification

The COVID-19 pandemic caused by the SARS-CoV-2 virus motivates diverse diagnostic approaches due to the novel causative pathogen, incompletely understood clinical sequelae, and limited availability of testing resources. Given the variability in viral load across and within patients, absolute viral load quantification directly from crude lysate is important for diagnosis and surveillance. Here, we investigate the use of digital droplet PCR (ddPCR) for SARS-CoV-2 viral load measurement directly from crude lysate without nucleic acid purification. We demonstrate ddPCR accurately quantifies SARS-CoV-2 standards from purified RNA and multiple sample matrices, including commonly utilized universal transport medium (UTM). In addition, we find ddPCR functions robustly at low input viral copy numbers on nasopharyngeal swab specimens stored in UTM without upfront RNA extraction. We also show ddPCR, but not qPCR, from crude lysate shows high concordance with viral load measurements from purified RNA. Our data suggest ddPCR offers advantages to qPCR for SARS-CoV-2 detection with higher sensitivity and robustness when using crude lysate rather than purified RNA as input. More broadly, digital droplet assays provide a potential method for nucleic acid measurement and infectious disease diagnosis with limited sample processing, underscoring the utility of such techniques in laboratory medicine.

Improved COVID-19 testing by extraction free SARS-Cov-2 RT-PCR

ABSTRACT The RNA extraction is an important checkpoint for the detection of SARS-CoV-2 in swab samples, but it is a major barrier to available and rapid COVID-19 testing. In this study, we validated the extraction-free RT-qPCR method by heat-treatment as an accurate option to nucleic acid purification in Algerian population.

The implementation of a rapid sample preparation method for the detection of SARS-CoV-2 in a diagnostic laboratory in South Africa

The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.

Deteksi Staphylococcus aureus dan Staphylococcus sp. Secara Langsung Dari Susu Segar Kambing Peranakan Etawa dengan Teknik PCR

Genus Staphylococcus merupakan salah satu patogen bakteri penyebab mastitis yang menyebabkan kerugian ekonomi pada kambing Peranakan Etawa. Diantara Staphylococcus sp. yang dapat tumbuh dengan baik dalam susu segar, diketahui Staphylococcus aureus dapat membahayakan kesehatan manusia yang mengkonsumsi (food borne disease) karena kemampuannya dalam memproduksi enterotoksin yang tahan terhadap enzim pencernaan maupun pemanasan. Tujuan penelitian ini adalah mendeteksi Staphylococcus sp. dan S. aureus secara langsung dari susu kambing peranakan etawa dengan teknik PCR.Metode yang dilakukan adalah mengekstraksi DNA dari 60 sample susu segar dengan prinsip spin column-based nucleic acid purification dan kemudian dilakukan amplifikasi gen spesifik 23S rRNA Staphylococcus sp. dan S. aureus. Hasil PCR diketahui 37 (61%) sampel susu positif mengandung Staphylococcus sp. dan hanya 1 (1,6%) sampel mengandung S. aureus. Metode deteksi dengan PCR dapat digunakan untuk mendeteksi kontaminan Staphylococcus sp. dan S. aureus dengan waktu yang singkat