Detection and quantification of SARS-CoV-2 RNA in wastewater influent in relation to reported COVID-19 incidence in Finland

AbstractWastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21–42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (∼ 60% of the Finnish population). The relative quantity of SARS-CoV-2 RNA fragments in the 24h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and RT-qPCR assay targeted with N2-assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of new and confirmed COVID-19 cases in the sewer network area.Over the 10-month surveillance period, the detection rate of SARS-CoV-2 RNA in WW was 79% (including 6% uncertain results), while only 24% of all samples exhibited gene copy (GC) numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID-19 incidence was 7, 18 and 36 cases within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 GC required significantly more COVID-19 cases: the quantification rate was 50%, 75% and 95% when the 14-day incidence was 110, 152 and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 GC quantified in WW at 15 out of 28 WWTPs (overall R2 = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the GC of SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered.Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.

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Impact of Endogenous Intronic Retroviruses on Major Histocompatibility Complex Class II Diversity and Stability

Journal of Virology ◽

10.1128/jvi.00097-08 ◽

2008 ◽

Vol 82 (13) ◽

pp. 6667-6677 ◽

Cited By ~ 28

Author(s):

Gaby G. M. Doxiadis ◽

Nanine de Groot ◽

Ronald E. Bontrop

Keyword(s):

Major Histocompatibility Complex ◽

Rhesus Macaques ◽

Gene Copy Number ◽

Population Level ◽

Endogenous Retrovirus ◽

Copy Number Variations ◽

Primate Species ◽

Gene Copy ◽

Major Histocompatibility ◽

Histocompatibility Complex

ABSTRACT The major histocompatibility complex (MHC) represents a multigene family that is known to display allelic and gene copy number variations. Primate species such as humans, chimpanzees (Pan troglodytes), and rhesus macaques (Macaca mulatta) show DRB region configuration polymorphism at the population level, meaning that the number and content of DRB loci may vary per haplotype. Introns of primate DRB alleles differ significantly in length due to insertions of transposable elements as long endogenous retrovirus (ERV) and human ERV (HERV) sequences in the DRB2, DRB6, and DRB7 pseudogenes. Although the integration of intronic HERVs resulted sooner or later in the inactivation of the targeted genes, the fixation of these endogenous retroviral segments over long time spans seems to have provided evolutionary advantage. Intronic HERVs may have integrated in a sense or an antisense manner. On the one hand, antisense-oriented retroelements such as HERV-K14I, observed in intron 2 of the DRB7 genes in humans and chimpanzees, seem to promote stability, as configurations/alleles containing these hits have experienced strong conservative selection during primate evolution. On the other hand, the HERVK3I present in intron 1 of all DRB2 and/or DRB6 alleles tested so far integrated in a sense orientation. The data suggest that multigenic regions in particular may benefit from sense introgressions by HERVs, as these elements seem to promote and maintain the generation of diversity, whereas these types of integrations may be lethal in monogenic systems, since they are known to influence transcript regulation negatively.

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Chromosomal defects Detected by SNP-Array-Based Karyotyping Are Independent Predictors of Survival in Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v112.11.431.431 ◽

2008 ◽

Vol 112 (11) ◽

pp. 431-431 ◽

Cited By ~ 1

Author(s):

Ramon V. Tiu ◽

Lukasz P Gondek ◽

Bhavana Bhatnagar ◽

Christine O’Keefe ◽

Mikkael A. Sekeres ◽

...

Keyword(s):

Loss Of Heterozygosity ◽

Detection Rate ◽

Significant Proportion ◽

Snp Array ◽

Uniparental Disomy ◽

Simultaneous Detection ◽

Gene Copy ◽

Newly Diagnosed ◽

Free Survival ◽

Chromosomal Defects

Abstract Cytogenetics is the most important predictor of outcomes in AML. Traditional metaphase cytogenetics (MC), can detect abnormalities in only 40–60% of AML patients. Whole genome scanning by single nucleotide polymorphism arrays (SNP-A) can identify somatic chromosomal changes in hematopoietic malignancies and, due to its superb resolution, may detect previously cryptic unbalanced defects, even in samples deemed “normal” or uninformative using MC. Through simultaneous detection of loss of heterozygosity (LOH) and gene copy number changes, SNP-A also facilitate the identification of somatic segmental uniparental disomy (UPD). Here we tested whether SNP-A analysis could improve the detection rate of chromosomal defects in AML and enhances the prognostic value of MC. Analyses were performed using 250K and/or 6.0 Affymetrix SNP arrays on 140 primary (p) and secondary AML (sAML) patients (newly diagnosed= 107, relapsed=15, remission= 12, persistent=6) and 116 healthy controls. Data on cytogenetic detection rate, complete remission (CR), overall survival [OS], relapse free survival [RFS], remission duration [RD], and event free survival [EFS]) rates were obtained from patients who received induction chemotherapy. We also performed Flt-3 ITD, Flt-3 TKD and NPM-1 mutation analysis and integrated the clinical outcomes with SNP-A results. For patients in whom new defects were detected, germ-line DNA was also analyzed whenever technically possible. The cytogenetic abnormality detection rate in patients with active disease was higher with SNP-A compared to MC (pAML, 75% vs 43% p=<0.0001; sAML, 81% vs 53% p=0.0015). UPD comprised a significant proportion of the SNP-A detected defects (36% in pAML and 40% in sAML) and included chromosomal defects not described in a previous 10K SNP study, such as 1p, 3p, 7q, 11q, 13q, 17q, 20, and 21q. Newly diagnosed AML patients with SNP-A lesions had less favorable outcomes. This was true for all AML patients (OS [5.8 months vs not reached {NR}, p=<0.0001], RFS [6.4 months vs NR, p=0.04] RD [6.9 months vs NR, p=0.04], EFS [2.7 vs 17 months, p=0.0007]); pAML w/normal MC (OS [10.8 months vs NR, p=0.007], RFS [14.2 months vs NR, p=.04], EFS [7.1 months vs NR, p=0.009]); and pAML/sAML with abnormal MC (OS [4.6 months vs 8.5, p=0.04], EFS [2.5 vs 12.4 months,p=0.05]). Of key importance, the presence of copy-neutral loss of heterozygosity (LOH) also translates to worse outcomes (OS [4.2 vs 15.1 months, p=0.0018], EFS [2.6 vs 8.6 months, p=0.007]), a finding comparable to gains or traditional LOH. SNP-A also improved the ability to predict outcomes in both mutant and wild types (WT) based on Flt-3 ITD and NPM-1 mutation status, with inferior survival in patients with new defects detected by SNP-A (Flt-3 ITD mutant [OS: 8 months vs NR, p=0.0011; LFS: 14.2 months vs NR, p=0.04]; Flt-3 ITD WT [8.5 months vs NR, p=0.024]; NPM-1 mutant [OS: 12.1 months vs NR, p=0.03; LFS: 12.3 months vs NR, p=0.05]; and NPM-1 WT [OS: 5.7 months vs 15.1 months, p=0.04]). Multivariate analysis using the Cox proportional hazard method showed that the presence or absence of SNP-A defects is an independent predictive factor for OS (p=0.0076) and EFS (p=0.007). In conclusion, SNP-A improves the cytogenetic detection rate of MC. The detection of new chromosomal lesions, particularly copy-neutral LOH such as UPD, provides additional informative data to MC that is prognostically significant.

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Vulvar cancer in Germany: Increase of incidence and change of tumor-biological characteristics in the period 1974-2013.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e17031 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e17031-e17031

Author(s):

Jana Barinoff ◽

Jalid Sehouli ◽

Bernd Holleczek

Keyword(s):

Squamous Cell ◽

Vulvar Cancer ◽

Advanced Disease ◽

Population Level ◽

Population Based ◽

Tumor Characteristics ◽

Incidence Data ◽

Incidence Trends ◽

The Past ◽

Icd 10

e17031 Background: Incidence of vulvar cancer in Germany is increasing. Gynecological oncologists reported to observe increasing numbers of women presenting with small tumors. The aim of the presented study is to validate this observation on a population level and to extend available incidence data. Methods: Data from the population-based Saarland Cancer Registry (CR) were used and included 1,136 female patients diagnosed with invasive vulvar cancer (ICD-10 code: C51) between 1974 and 2013. Multiple imputation methodology was used to overcome loss of precision and potential bias. Incidence trends were investigated with regard to patient and tumor characteristics. Results: During the study period, the age standardized rate of incidence increased from 1.6 to 7.9 cases per 100,000 women per year (+390%). Since 1989, the incidence of squamous cell carcinoma increased from 1.7 to 7.1 (+320%), whereas no increase was observed in other morphologic types. Simultaneously, the incidence of small vulvar tumors <= 2 cm (T1) increased from 0.8 in 1989-93 to 6.6 in 2009-13 (the proportion of T1 tumors increased from 34% to 79%, respectively). Patients aged >= 75 years suffered from more advanced disease at the time of diagnosis. Conclusions: The detailed analyses revealed that the observed increase in vulvar cancer in the past 15 years mainly resulted from newly incident squamous cell carcinomas. Furthermore, the analyses showed an almost exclusive increase of T1 tumors. An increase in vulvar cancer incidence as shown for Germany could not be observed for any other European country.

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MATHEMATICAL ANALYSIS OF THE ROLE OF DETECTION RATE IN THE DYNAMICAL SPREAD OF HIV-TB CO-INFECTION

JOURNAL OF ADVANCES IN MATHEMATICS ◽

10.24297/jam.v11i10.800 ◽

2016 ◽

Vol 11 (10) ◽

pp. 5715-5741

Author(s):

SUNDAY OLUMUYIWA Adewale ◽

I.A Olopade ◽

I.T Mohammed ◽

S.O Ajao ◽

O.T Oyedemi

Keyword(s):

Detection Rate ◽

Equilibrium Points ◽

Population Level ◽

Transformation Method ◽

Infection Model ◽

Hiv Model ◽

Public Health Burden ◽

Immunodeficiency Virus ◽

Effect Of Treatment ◽

Disease Free

Human Immunodeficiency Virus (HIV) co-existing with Tuberculosis (TB) in individuals remains a major global health challenges, with an estimated 1.4 million patients worldwide. These two diseases are enormous public health burden, and unfortunately, not much has been done in terms of modeling the dynamics of HIV-TB co-infection at a population level. We formulated new fifteen (15) compartmental models to gain more insight into the effect of treatment and detection of infected undetected individuals on the dynamical spread of HIV- TB co-infection. Sub models of HIV and TB only were considered first, followed by the full HIV-TB co-infection model. Existence and uniqueness of HIV and TB only model were analyzed quantitatively, and we shown that HIV model only and TB only model have solutions, moreover, the solutions are unique. Stability of HIV model only, TB model only and full model of HIV-TB co-infection were analyzed for the existence of the disease free and endemic equilibrium points. Basic reproduction number () was analyzed, using next generation matrix method (NGM), and it has been shown that the disease free equilibrium point is locally asymptotically stable whenever and unstable whenever this threshold exceeds unity. i.e., Numerical simulation was carried out by maple software using differential transformation method, to show the effect of treatment and detection of infected undetected individuals on the dynamical spread of HIV-TB co-infection. Significantly, all the results obtained from this research show the importance of treatment and detection of infected undetected individuals on the dynamical spread of HIV-TB co-infection. Detection rate of infected undetected individuals reduce the spread of HIV-TB co-infections.

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Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

10.21203/rs.3.rs-103611/v2 ◽

2021 ◽

Author(s):

Suttipat Srisut ◽

Kanokon Suwannasin ◽

Rungirun Sugaram ◽

Arjen M. Dondorp ◽

Mallika Imwong

Keyword(s):

Drug Resistance ◽

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Antimalarial Drug Resistance ◽

Quantification Limit ◽

Field Samples ◽

The Cost

Abstract Background: Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum pfplasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with antimalarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to antimalarial drugs.Methods: A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay.Results: There were no significant differences between the GCN results obtained from uniplex andmultiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated.Conclusions: The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of antimalarial drug resistance.

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Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

10.21203/rs.3.rs-103611/v1 ◽

2020 ◽

Author(s):

Suttipat Srisut ◽

Kanokon Suwannasin ◽

Rungirun Sugaram ◽

Arjen M. Dondorp ◽

Mallika Imwong

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Antimalarial Drug Resistance ◽

Quantification Limit ◽

Field Samples

Abstract Background: Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum pfplasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with antimalarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to antimalarial drugs.Methods: The ddPCR assays were developed to detect the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay.Results: Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. The accuracy and %RSD value of the duplex ddPCR assay were 94.88% and 3.71, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). Conclusions: The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of antimalarial drug resistance.

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Genotyping Allelic and Copy Number Variation in the Immunoglobulin Heavy Chain Locus

10.1101/042226 ◽

2016 ◽

Cited By ~ 1

Author(s):

Shishi Luo ◽

Jane A Yu ◽

Yun S. Song

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Allelic Variation ◽

Association Studies ◽

Adaptive Immune Response ◽

Gene Copy Number ◽

Population Level ◽

European Ancestry ◽

Gene Copy ◽

Number Variation

The study of genomic regions that contain gene copies and structural variation is a major challenge in modern genomics. Unlike variation involving single nucleotide changes, data on the variation of copy number is difficult to collect and few tools exist for analyzing the variation between individuals. The immunoglobulin heavy variable (IGHV) locus, which plays an integral role in the adaptive immune response, is an example of a genomic region that is known to vary in gene copy number. Lack of standard methods to genotype this region prevents it from being included in association studies and is holding back the growing field of antibody repertoire analysis. Here, we establish a convention of representing the locus in terms of a reference panel of operationally distinguishable segments defined by hierarchical clustering. Using this reference set, we develop a pipeline that identifies copy number and allelic variation in the IGHV locus from whole-genome sequencing reads. Tests on simulated reads demonstrate that our approach is feasible and accurate for detecting the presence and absence of gene segments using reads as short as 70 bp. With reads 100 bp and longer, coverage depth can also be used to determine copy number. When applied to a family of European ancestry, our method finds new copy number variants and confirms existing variants. This study paves the way for analyzing population-level patterns of variation in the IGHV locus in larger diverse datasets and for quantitatively handling regions of copy number variation in other structurally varying and complex loci.

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Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

Malaria Journal ◽

10.1186/s12936-021-03659-5 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Suttipat Srisutham ◽

Kanokon Suwannasin ◽

Rungniran Sugaram ◽

Arjen M. Dondorp ◽

Mallika Imwong

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Quantification Limit ◽

Field Samples ◽

The Cost

Abstract Background Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs. Methods A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay. Results There were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated. Conclusions The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.

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Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

10.1101/414516 ◽

2018 ◽

Cited By ~ 2

Author(s):

Phil Oberacker ◽

Peter Stepper ◽

Donna M Bond ◽

Sven Höhn ◽

Jule Focken ◽

...

Keyword(s):

Open Source ◽

High Throughput ◽

Magnetic Beads ◽

Acid Extraction ◽

Open Platform ◽

Dna And Rna ◽

Rna Fragments ◽

Different Sources ◽

High Throughput Manner ◽

Selection Of

AbstractCurrent molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge-and column-based protocols require specialised equipment, often use toxic reagents and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA and total RNA from different sources, as well as environmental TNA and PCR amplicons. We also provide a bead-based protocol for bisulfite conversion, and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility and extreme cost-effectiveness of using magnetic beads. These open source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

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How much of SARS-CoV-2 Infections is India detecting? A model-based estimation

10.1101/2020.04.09.20059014 ◽

2020 ◽

Cited By ~ 4

Author(s):

Srinivas Goli ◽

K.S. James

Keyword(s):

Detection Rate ◽

Age Distribution ◽

Population Level ◽

Lower Number ◽

Random Testing ◽

High Detection Rate ◽

High Detection ◽

Late Detection ◽

Huge Variation

AbstractBackground and RationaleAmid SARS-CoV-2 outbreak, the low number of infections for a population size of 1.38 billion is widely discussed, but with no definite answers.MethodsWe used the model proposed by Bommer and Vollmer to assess the quality of official case records. The infection fatality rates were taken from Verity et al (2020). Age distribution of the population for India and states are taken from the Census of India (2011). Reported number of deaths and SARS-CoV-2 confirmed cases from https://www.covid19india.org. The reported numbers of samples tests were collected from the reports of the Indian Council for Medical Research (ICMR).ResultsThe findings suggest that India is detecting just 3.6% of the total number of infections with a huge variation across its states. Among 13 states which have more than 100 COVID-19 cases, the detection rate varies from 81.9% (of 410 estimated infections) in Kerala to 0.8% (of 35487 estimated infections) in Madhya Pradesh and 2.4% (of 7431 estimated infections) in Gujarat.ConclusionAs the study reports a lower number of deaths and higher recovery rates in the states with a high detection rate, thus suggest that India must enhance its testing capacity and go for widespread testing. Late detection puts patients in greater need of mechanical ventilation and ICU care, which imposes greater costs on the health system. The country should also adopt population-level random testing to assess the prevalence of the infection.

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