EP4291684A1 - Detection and mutational analysis of an rna virus in an environmental sample - Google Patents

Detection and mutational analysis of an rna virus in an environmental sample

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Publication number
EP4291684A1
EP4291684A1 EP21716968.9A EP21716968A EP4291684A1 EP 4291684 A1 EP4291684 A1 EP 4291684A1 EP 21716968 A EP21716968 A EP 21716968A EP 4291684 A1 EP4291684 A1 EP 4291684A1
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EP
European Patent Office
Prior art keywords
seq
reverse primer
rna
detection
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21716968.9A
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German (de)
English (en)
French (fr)
Inventor
Andreas Scorilas
Margaritis AVGERIS
Nikolaos THOMAIDIS
Nikolaos VOULGARIS
Panagiotis ADAMOPOULOS
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National and Kapodistrian University of Athens
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National and Kapodistrian University of Athens
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Application filed by National and Kapodistrian University of Athens filed Critical National and Kapodistrian University of Athens
Publication of EP4291684A1 publication Critical patent/EP4291684A1/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • the present invention is related to the detection and the mutational analysis of a virus and more specifically of an RNA virus, in an environmental sample.
  • RNA viruses have emerged as one of the most common class of pathogens causing serious disease. This can be attributed to the fact that they exhibit impressive capabilities to adapt to a variety of environments and deal with the challenges they encounter [Moratorio, G., et al. , Attenuation of RNA viruses by redirecting their evolution in sequence space. Nat Microbiol, 2017. 2: p. 17088] This extraordinary ability to cope with the immune system and defense mechanisms of the host cell as well as their durability in antiviral drugs, mainly arises from their exceptionally high mutation rates that are caused by the absence of proofreading mechanisms of their RNA-dependent polymerase, thus leading to error correction skipping and an increased number of genomic mutations [Simon-Loriere, E. and E.C.
  • RNA viruses include the viruses that cause common cold, influenza, SARS, MERS, Dengue Virus, hepatitis C, hepatitis E, West Nile fever, Ebola, rabies, polio, measles as well as the recently emerged SARS-CoV-2.
  • Coronaviruses constitute a family of enveloped positive-strand RNA viruses, which are characterized by a single-stranded, positive-sense RNA genome of 26-32 kilobases (kb) [Su, S., et al., Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses. Trends Microbiol, 2016. 24(6): p. 490-502]
  • a recently emerged coronavirus is SARS-CoV-2, which was the cause of a cluster of severe pneumonia cases of unknown origin, reported by the Chinese Health authorities in late 2019.
  • Sequencing-based analysis of lower respiratory tract samples identified a novel beta-coronovirus sharing >85% sequence similarity with a bat severe acute respiratory syndrome (SARS)-like coronavirus (CoV), provisionally indicated as 2019-nCoV, as the causative pathogen of the coronavirus disease-2019 (COVID-19) [Zhu, N., et al. , A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med, 2020. 382(8): p.
  • SARS severe acute respiratory syndrome
  • CoV coronavirus
  • This seventh member of CoV family that cause disease in humans was further characterized showing sequence homology of approximately 79% and 50% with the SARS-CoV of 2002 outbreak in China and the Middle East respiratory syndrome (MERS)-CoV of 2012 outbreak in the Middle East, respectively [Lu, R., et al., Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet, 2020. 395(10224): p. 565-574]
  • This novel CoV was thereafter named SARS-CoV-2 [Coronaviridae Study Group of the International Committee on Taxonomy of, V., The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol, 2020. 5(4): p. 536-544], and was rapidly spread to most countries worldwide, leading the announcement of COVID-19 pandemic by World Health Organization (WHO) in 12 March 2020.
  • WHO World Health Organization
  • SARS-CoV-2 belongs to the genus beta-coronavirus, having a genome of 29,903 nt. Human-to-human spread of SARS-CoV-2 mainly occurs via either respiratory droplets generated by an infected person sneezing, coughing and talking or direct contact [Li, Q., et al., Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-lnfected Pneumonia. N Engl J Med, 2020. 382(13): p. 1199-1207] However, the detection of SARS-CoV-2 in faeces of patients [Wang, W., et al., Detection of SARS-CoV-2 in Different Types of Clinical Specimens. JAMA, 2020.
  • SARS-CoV-2 RNA virus genomes cased from humans positive for COVID-19 are deposited in GISAID database (https://www.gisaid.org/) and analyzed by Nextstrain (https://nextstrain.org/sars-cov- 2).
  • WBE screening could capture for example asymptomatic carriers of viruses who are less likely to undergo testing and symptomatic patients avoiding testing due to stigmatization and social isolation, as well as to provide real-time and population-wide monitoring of genomic variations/strains of the virus of interest.
  • RNA viruses such as SARS-CoV-2
  • SARS-CoV-2 RNA viruses
  • the sample site features may have an immediate effect on virus detection.
  • industrial effluences, changes in the pH at the site of the sample as well as rain runoffs can affect the quality of the sample, therefore having a tremendous impact in the detection efficiency of the virus.
  • volume of the sample and the sampling method are two important factors in the detection of viruses in environmental samples. Although several protocols have been developed to augment the volume of the sample (for example, bag filtration and composite sampling) in order to increase the chance of detecting the virus, these samples are often difficult to handle in the laboratory [Larsen, D.A. and K.R. Wigginton, Tracking COVID-19 with wastewater. Nat Biotechnol, 2020. 38(10): p. 1151-1153]
  • RNA viruses in environmental samples, such as wastewater, is the reduced stability of the RNA of the virus, which is not observed in human samples.
  • the currently established SARS- CoV-2 detection and screening method in human individuals includes an RNA extraction step from a nasopharyngeal swab followed by one-step reverse transcription quantitative polymerase chain reaction (RT-qPCR) to detect the extracted viral RNA.
  • RT-qPCR reverse transcription quantitative polymerase chain reaction
  • This approach involves the use of virus-specific RT primers, resulting only in the cDNA synthesis of viral mRNA, which is then exploited as template for qPCR.
  • the existing temperature and pH in the area of sampling can induce random degradation of the viral RNA, thus leading to serious limitations in terms of detection with specific one- step RT-qPCR.
  • the present invention provides a method for the detection of an RNA virus in an environmental sample, which involves extraction of the RNA from the sample, reverse transcription of the extracted RNA with random oligonucleotides to give the complementary DNA (cDNA) and employment of nested PCR.
  • the present invention further provides a method for the mutational analysis of an RNA virus in an environmental sample, wherein the method comprises extraction of the RNA from the sample, reverse transcription of the extracted RNA with random oligonucleotides to give the complementary DNA (cDNA), employment of nested PCR, and sequencing of the products of the nested PCR assays using massively parallel sequencing.
  • the method comprises extraction of the RNA from the sample, reverse transcription of the extracted RNA with random oligonucleotides to give the complementary DNA (cDNA), employment of nested PCR, and sequencing of the products of the nested PCR assays using massively parallel sequencing.
  • the present invention further provides a method for the detection of SARS- CoV-2 in an environmental sample which involves extraction of the RNA from the sample, reverse transcription of the extracted RNA with random oligonucleotides to give the complementary DNA (cDNA) and employment of nested PCR.
  • the present invention further provides a method for the mutational analysis of SARS-CoV-2 in an environmental sample, wherein the method comprises extraction of the RNA from the sample, reverse transcription of the extracted RNA with random oligonucleotides to give the complementary DNA (cDNA), employment of nested PCR, and sequencing of the products of the nested PCR assays using massively parallel sequencing.
  • the method comprises extraction of the RNA from the sample, reverse transcription of the extracted RNA with random oligonucleotides to give the complementary DNA (cDNA), employment of nested PCR, and sequencing of the products of the nested PCR assays using massively parallel sequencing.
  • the present invention further provides kits for the detection of the presence of SARS-CoV-2 or for the detection of certain mutations of SARS-CoV-2 in a sample by nested PCR.
  • Figure 1 shows agarose-gel electrophoresis of PCR products from CDC/2019- nCoV_N1 -based assay and from assays of the present invention.
  • Figure 2 shows agarose-gel electrophoresis of PCR products from assays of the present invention.
  • Figure 3 shows standard curves of nested real-time PCR assays of the present invention.
  • Figure 4 shows standard curves of nested real-time PCR assays of the present invention.
  • Figure 5 shows agarose-gel electrophoresis of the PCR products of nested PCR assays of the present invention.
  • RNA virus in an environmental sample exhibits certain challenges, which are not present in a sample taken from a subject, for example from a human subject.
  • the quality of the sample may be affected by environmental or other factors.
  • the stability of the RNA of the virus in the environment is reduced, resulting in various decomposition products. Therefore, a method for the detection or mutational analysis of an RNA virus in an environmental sample must exhibit high specificity and sensitivity.
  • the present invention provides a method for the detection of an RNA virus in an environmental sample, wherein the method comprises the steps of a) extraction of the RNA from the sample, b) reverse transcription of the extracted RNA with random oligonucleotides to give the complementary DNA (cDNA), c) application of a first nested PCR assay which targets a first region of the RNA of the virus, d) detection of the product of the nested PCR assay.
  • RNA virus is a virus that has RNA as its genetic material.
  • the RNA virus is selected from viruses that cause common cold, influenza, SARS, MERS, Dengue Virus, hepatitis C, hepatitis E, West Nile fever, Ebola, rabies, polio and SARS- CoV-2. More preferably, the RNA virus is SARS-CoV-2.
  • the term “environmental sample” refers to a sample obtained from a non- biological source, such as soil, sediment or water.
  • the environmental sample is a water sample is obtained from a natural setting or from an industrial, health-care or residential setting.
  • the environmental sample is a wastewater sample.
  • RNA from the environmental sample may be carried out by using methods well known in the art, such as magnetic beads-based RNA extraction, or silica column-based RNA extraction or acid guanidinium thiocyanate-phenol- chloroform RNA extraction.
  • the environmental sample is a wastewater sample
  • the extraction of the RNA may be preceded, according to an embodiment of the invention, by a concentration of the sample which may be carried out by using methods well known in the art, such as ultrafiltration or polyethylene glycol (PEG) precipitation.
  • PEG polyethylene glycol
  • Random oligonucleotides are synthesized entirely randomly to give a numerous range of sequences that have the potential to anneal at many random regions on a RNA template and act as a primer to commence first strand cDNA synthesis. They are also commonly referred to as random primers.
  • the random oligonucleotides are hexamers.
  • Nested PCR is a modification of PCR well known in the art, which involves two sets of primers used in two successive PCR reactions.
  • the first set of primers also called external primers
  • Amplicons resulting from the first PCR reaction are then used as templates for a second set of primers (also called internal primers) and a second PCR reaction.
  • a nested PCR assay involves the use of two pairs of primers and two PCR reactions.
  • the product of the second PCR reaction is the product of the nested PCR assay.
  • the detection of the product of the nested PCR assay can be carried out qualitatively or quantitatively using methods well known in the art.
  • qualitatively it may be carried out for example, by agarose gel electrophoresis.
  • quantitatively it may be carried out, for example, by using a fluorescent probe, such as a sequence specific probe.
  • the nested PCR assay is designed to target a region of the RNA of the virus. This means that the primers of the nested PCR assay are designed to amplify only cDNA which is complementary to a region of the RNA of the virus.
  • the sample is subjected to a second nested PCR assay which targets a second region of the RNA of the virus. If no cDNA complementary to the RNA of the virus is detected after the second nested PCR assay, the sample is subjected to a third nested PCR assay which targets a third region of the RNA of the virus.
  • the sample is subjected to a fourth nested PCR assay which targets a fourth region of the RNA of the virus.
  • the first, second third and fourth regions of the RNA of the virus are different regions of the RNA of the virus, which means that there is no overlap between the regions.
  • the present invention further provides a method for the detection of a mutation of an RNA virus in an environmental sample, wherein the method comprises the steps of a) extraction of the RNA from the sample, b) reverse transcription of the extracted RNA with random oligonucleotides to give the complementary DNA (cDNA) c) application of a nested PCR assay, wherein the assay targets a region of the RNA of the virus bearing the mutation of interest, d) detection of the product of the PCR assay, e) sequencing of the product of the nested PCR assay using massively parallel sequencing.
  • Massively parallel sequencing is also called next-generation sequencing (NGS) or second-generation sequencing and involves high-throughput approaches to DNA sequencing.
  • NGS next-generation sequencing
  • massively parallel sequencing involves the creation by PCR of DNA sequencing libraries, the sequencing by synthesis of the DNA and the simultaneous sequencing of segregated, amplified DNA templates in a massively parallel fashion without the requirement for a physical separation step.
  • An example of massively parallel sequencing which may be used according to the present invention is the amplicon sequencing or targeted DNA-seq.
  • the RNA virus is SARS-CoV-2.
  • the present invention also provides genomic regions of SARS-CoV-2, which can be used as targets for the nested PCR assays. Namely, the present inventors have found that the regions of the RNA of SARS-CoV-2 consisting of any one of SEQ ID NO: 1 - 70 exhibit higher stability compared to other regions. This means that a region consisting of any one of SEQ ID NO: 1 - 70 is more likely to be present in an environmental sample comprising SARS-CoV-2 and is therefore a better target for a nested PCR assay compared to other regions of the RNA of the virus.
  • the nested PCR assay of the present invention targets a region of the RNA of SARS-CoV-2 consisting of any one of SEQ ID NO: 1 - 70. More preferably, the nested PCR assay of the present invention, or any one nested PCR assay, if more than one assay is employed, targets a region of the RNA of SARS-CoV-2 consisting of any one of SEQ ID NO: 1 - 36.
  • the nested PCR assay of the present invention targets a region of the RNA of SARS-CoV-2 consisting of SEQ ID NO: 1 - 24.
  • the nested PCR assay of the present invention, or any one nested PCR assay if more than one assay is employed targets a region of the RNA of SARS- CoV-2 consisting of any one of SEQ ID NO: 2, 4, 8, 11 , 14, 15, 18, 19.
  • each assay targets a different region of the RNA of the virus.
  • the present invention further provides pairs of primers which can be used in the nested PCR assays for the detection of SARS-CoV-2 in an environmental sample.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 71 and SEQ ID NO: 72 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 73 and SEQ ID NO: 74 respectively.
  • the probe is a FAM-BHQ1 fluorescent probe consisting of SEQ ID NO: 75.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 76 and SEQ ID NO: 77 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 78 and SEQ ID NO: 79 respectively.
  • the probe is a FAM-BHQ1 fluorescent probe consisting of SEQ ID NO: 80.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 81 and SEQ ID NO: 82 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 83 and SEQ ID NO: 84 respectively.
  • the probe is a FAM-BHQ1 fluorescent probe consisting of SEQ ID NO: 85.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 86 and SEQ ID NO: 87 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 88 and SEQ ID NO: 89 respectively.
  • the probe is a FAM-MGB fluorescent probe consisting of SEQ ID NO: 90.
  • the present invention further provides a kit for the detection of the presence of SARS-CoV-2 in a sample, such as an environmental sample, by nested PCR, wherein the kit comprises a) four oligonucleotides consisting of SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 respectively and optionally a FAM-BHQ1 fluorescent probe consisting of SEQ ID NO: 75, and/or b) four oligonucleotides consisting of SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79 respectively and optionally a FAM-BHQ1 fluorescent probe consisting of SEQ ID NO: 80, and/or c) four oligonucleotides consisting of SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 respectively and optionally a FAM-BHQ1 fluorescent probe consisting of
  • the kit comprises at least two of a), b), c) and d), more preferably, the kit comprises at least three of a), b), c) and d) and even more preferably, the kit comprises a), b), c) and d).
  • the present invention further provides pairs of primers which can be used in the nested PCR assays for the detection of certain mutations of SARS-CoV-2 in an environmental sample according to the present invention.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 91 and SEQ ID NO: 92 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 93 and SEQ ID NO: 94 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 95 and SEQ ID NO: 96 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 97 and SEQ ID NO: 98 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 99 and SEQ ID NO: 100 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 101 and SEQ ID NO: 102 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 103 and SEQ ID NO: 104 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 105 and SEQ ID NO: 106 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 107 and SEQ ID NO: 108 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 109 and SEQ ID NO: 110 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 111 and SEQ ID NO: 112 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 113 and SEQ ID NO: 114 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 115 and SEQ ID NO: 116 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 117 and SEQ ID NO: 118 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 119 and SEQ ID NO: 120 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 121 and SEQ ID NO: 122 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 123 and SEQ ID NO: 124 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 125 and SEQ ID NO: 126 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 127 and SEQ ID NO: 128 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 129 and SEQ ID NO: 130 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 131 and SEQ ID NO: 132 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 133 and SEQ ID NO: 134 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 135 and SEQ ID NO: 136 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 137 and SEQ ID NO: 138 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 139 and SEQ ID NO: 140 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 141 and SEQ ID NO: 142 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 143 and SEQ ID NO: 144 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 145 and SEQ ID NO: 146 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 147 and SEQ ID NO: 148 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 149 and SEQ ID NO: 150 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 151 and SEQ ID NO: 152 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 153 and SEQ ID NO: 154 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 155 and SEQ ID NO: 156 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 157 and SEQ ID NO: 158 respectively.
  • the forward and reverse primer of the first reaction (external set) of the nested PCR assay consist of SEQ ID NO: 159 and SEQ ID NO: 160 respectively
  • the forward and reverse primer of the second reaction (internal set) of the nested PCR assay consist of SEQ ID NO: 161 and SEQ ID NO: 162 respectively.
  • the present invention further provides a kit for the detection of a mutation of SARS-CoV-2 in a sample, such as an environmental sample, by nested PCR, wherein the kit comprises four oligonucleotides consisting of SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID
  • SEQ ID NO: 109 SEQ ID NO: 110 respectively, or four oligonucleotides consisting of SEQ ID NO: 111 , SEQ ID NO: 112, SEQ
  • SEQ ID NO: 113 SEQ ID NO: 114 respectively, or four oligonucleotides consisting of SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118 respectively, or four oligonucleotides consisting of SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 respectively, or four oligonucleotides consisting of SEQ ID NO: 123, SEQ ID NO: 124, SEQ
  • SEQ ID NO: 125 SEQ ID NO: 126 respectively, or four oligonucleotides consisting of SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 respectively, or four oligonucleotides consisting of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134 respectively, or four oligonucleotides consisting of SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138 respectively, or four oligonucleotides consisting of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142 respectively, or four oligonucleotides consisting of SEQ ID NO: 143, SEQ ID NO: 144, SEQ
  • RNA virus such as SARS-Coupled virus
  • SEQ ID NO: 145 SEQ ID NO: 146 respectively, or four oligonucleotides consisting of SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150 respectively, or four oligonucleotides consisting of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154 respectively, or four oligonucleotides consisting of SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 respectively, or four oligonucleotides consisting of SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162 respectively.
  • the present invention enables the detection of an RNA virus, such as SARS-
  • the present invention enables the mutational and genomic profiling of an RNA virus in an environmental sample, which represents an efficient and cost-effective approach towards the establishment of an early-warning system for the monitoring of the genomic epidemiology of an RNA virus at community/population level.
  • MFE minimum free energy
  • a DQ° z-score is calculated for each sequence. ScanFold uses each predicted MFE for the native sequence (MFEnative) and compares it with MFE values calculated for 100 shuffled version of the sequence with the same nucleotide composition (MFErandom), using an approach adapted from Clote et. Al [Clote, P., et al., Structural RNA has lower folding energy than random RNA of the same dinucleotide frequency. RNA, 2005. 11(5): p.
  • the obtained p-values correspond to the number of MFErandom values, which were more stable (more negative) than the MFEnative.
  • analysis with ScanFold enables the characterization of the potential structural diversity of the native sequence, by calculating the ensemble diversity (ED) and the centroid structure.
  • the centroid structure depicts the base pairs that were “most common” (i.e. , had the minimal base pair distance) between all the Boltzmann- ensemble conformations predicted for the native sequence.
  • the ED attempts to quantify the variety of different structures, which were present in the ensemble. In specific, higher ED numbers indicate multiple structures unique from the predicted MFE, while low ED numbers indicate the presence of a dominant MFE structure highly represented in the ensemble.
  • VfoldCPX Server Xu, X. and S.J. Chen, VfoldCPX Server: Predicting RNA-RNA Complex Structure and Stability. PLoS One, 2016. 11(9): p. e0163454].
  • parameters such as loop-loop kissing interactions and the use of physical loop entropy were taken into consideration for identifying the most stable regions of SARS-Cov-2.
  • Extensive analysis with VfoldCPX led to a set of energetically stable structures, ranked by their stabilities, thus providing detailed insights about the most stable SARS-Cov-2 genomic regions.
  • the 24-hour composite influent wastewater samples were collected from the Wastewater Treatment Plant (WWTP) of Athens, which is designed to serve a population equivalent of 5,200,000.
  • the WWTP of Athens is designed with primary sedimentation, activated sludge process with biological nitrogen and phosphorus removal and secondary sedimentation.
  • the pH range (7.5-8.0) and the temperature range (17-20 °C) for the collected samples were provided by the WWTP of Athens. All the samples are flow-proportional.
  • Influent wastewater samples were collected in pre-cleaned high-density polyethylene (HDPE) bottles, transported on ice to the laboratory and stored at 4°C. All the collected samples were analyzed immediately after the arrival at the laboratory. Sampling personnel followed the appropriate regulations and guidelines and wore face standard personal protective equipment (PPE).
  • HDPE high-density polyethylene
  • the collected samples were concentrated immediately after arrival using Polyethylene glycol 8000 (PEG 8000; Promega Corporation, Madison, Wl, USA) precipitation.
  • PEG 8000 Polyethylene glycol 8000
  • 50 ml_ of an influent wastewater were centrifuged at 4,750 g for 30 min at 4 °C to remove debris, bacteria and large particles.
  • the supernatant was transferred in a clean centrifuge tube, containing 3.5 g PEG and 0.8 g NaCI, mixed at ambient temperature until completely dissolved, and centrifuged at 10,050 g for 2 h at 4°C. The most of the supernatant was discarded without disturb the viral pellet and the tube was centrifuged at 10,050 g for 5 min at 4 °C, and finally the viral pellet was reconstituted by 500 pl_ nuclease-free water.
  • RNA extraction was performed, by 200 mI_ concentrate using the Water DNA/RNA Magnetic Bead kit (IDEXX Laboratories Inc., Westbrook, Maine, USA) according to manufacturer’s instruction, immediately following concentration.
  • RNA template from wastewater samples was reverse transcribed in a 20 mI reaction containing 5.0 mI RNA, 1.0 mI of 10mM dNTPs mix (Jena Bioscience GmbH, Jena, Germany), 100 U Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), 50 U RNaseOUT recombinant ribonuclease inhibitor (Invitrogen) and 1.0 mI of 50 mM random hexamers (Invitrogen).
  • the mixture of total RNA, dNTPs and random hexamers was incubated at 65°C for 5 min, while the reverse transcription took place at 25°C for 10 min followed by 50°C for 50 min. Enzyme inactivation was performed at 70°C for 15 min.
  • the AMPLIRUN SARS-CoV-2 RNA control (Vircell S.L., Granada, Spain) was used as SARS-CoV-2 complete genome control.
  • the Veriti 96 well fast thermal cycler (Applied Biosystems, Carlsbad, CA) was used for the nested PCR assays.
  • the 25 mI of the reaction consisted of 5.0 mI cDNA template (1st PCR) or 2.0 mI PCR product (2nd PCR), 1.0 mI of 10mM dNTPs mix (Jena Bioscience GmbH), 500 nM of each forward/reverse primer and 1 U of Kapa Taq polymerase (Kapa Biosystems, Inc., Woburn, MA).
  • the thermal protocol consisted of polymerase activation step at 95°C for 3 min, followed by 15 cycles (1st PCR) or 40 cycles (2nd PCR) of denaturation at 95°C for 30 sec, primer annealing at 60°C for 30 sec and extension at 72°C for 1 min, followed by a final extension step at 72°C for 5 min.
  • 10 mI of PCR product were electrophoresed on 1.5% w/v agarose gel, visualized with ethidium bromide staining, and photographed under UV light.
  • the probe fluorescent-based real-time PCR assays were performed in 7500 Fast Real-Time PCR System (Applied Biosystems).
  • the PCR product of the 1st conventional PCR - as described above - were used as template for the real-time PCR assay (2nd reaction).
  • the 20 pi reaction consisted of 2.0 mI PCR product, 10 mI Kapa Probe Fast Universal 2X qPCR Master Mix (Kapa Biosystems), 500 nM of each forward/reverse primer and 125 nM of fluorescent probe.
  • the thermal protocol included an initial polymerase activation step at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 15 sec and finally the primer/probe annealing and extension step at 60°C for 1 min.
  • FP Forward primer
  • RP Reverse primer
  • Pr qPCR probe
  • serial dilutions of SARS-CoV-2 complete genome RNA control covering 9 order of magnitude (from 1000 to 2.5 RNA copies/reverse transcription reaction) were analyzed by: a. nested PCR/real-time PCR assays, and b. assay using the CDC proposed “2019-nCoV_N1” set of primers and probe (CDC/2019-nCoV_N1 -based assay) (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html).
  • Figure 3A shows the standard curves of the nested real-time PCR N assay.
  • Figure 3B shows the standard curves of the nested real-time PCR ORF3a assay.
  • Figure 4A shows the standard curves of the nested real-time PCR Helicase assay.
  • Figure 4B shows the standard curves of the nested real-time PCR NSP3 assay.
  • SARS-CoV-2 detection in wastewater is improved by amplification of multiple targets
  • the CT values of the positive samples per assay are presented in Table 2, while the agarose gels are included in Figures 5A-D. Table 2.
  • SARS-CoV-2 was detected, by at least one assay, in 17 samples (17/30; 56.7%), while 13 samples were negative (13/30; 43.3%).
  • CDC/2019-nCoV_N1 -based assay detected 5 samples (sensitivity: 29.4%).
  • the in house nested real-time PCR assays resulted to the detection of SARS-CoV-2 in: a. N assay - 10 samples (sensitivity: 58.8%), b. NSP3 assay - 9 samples (sensitivity: 52.9%), c. Helicase assay - 7 samples (sensitivity: 41.2%) and d. ORF3a assay - 5 samples (sensitivity: 29.4%).
  • the Ion Xpress Plus Fragment Library Kit (Ion Torrent, Thermo Fisher Scientific Inc.) was employed for the construction of the DNA-seq library, using 1 pg of purified PCR product mix as input. Adapter ligation, nick-repair and purification of the ligated DNA were carried out based on the manufacturer’s guidelines. The adapter-ligated library was quantified using the Ion Library TaqMan Quantitation Kit (Ion Torrent) in an ABI 7500 Fast Real-Time PCR System (Applied Biosystems).
  • the sequencing template was generated with emulsion PCR on an Ion OneTouch 2 System (Ion Torrent), using the Ion PGM Hi-Q View OT2 kit (Ion Torrent), strictly based on the instructions of the manufacturer.
  • Ion OneTouch ES instrument Ion Torrent
  • semi-conductor sequencing methodology was carried out in the Ion Torrent PGM system for the sequencing of the amplicons bearing the potential variations/mutations of the virus.
  • Genomic variation profiling (list of existing SNVs or indels) of SARS-CoV-2 that was derived from the developed high-throughput sequencing approach is summarized in Tables 4 and 5 below. Table 4. Genomic variation profile (SNVs and indels) of SARS-CoV-2 in samples from September 2020, as obtained from targeted DNA-seq. Table 5. Genomic variation profile (SNVs and indels) of SARS-CoV-2 in samples from September 2020.
  • H625R a previously unknown point mutation within S gene, H625R (23436A>G), was observed in frequency of 5.7% in September samples. More precisely, the 23436A>G missense substitution results to the change of Histidine-to-Arginine at position 625 of the spike protein.
  • H625R mutation involves the substitution of two amino acid residues with positively charged polar side chains. Even though two similar amino acids are substituted, based on the in-silico protein structure analysis, the occurrence of H625R leads to subtle alterations in the spike protein folding.
  • the H625R-mutant spike protein may exhibit differential biochemical properties, which should be further investigated, since they may have a severe impact on the functionality of the protein, making the virus more transmissible or infectious.
  • a novel point substitution, A54V (255530T) was detected in percentage -9% of September samples, resulting to the change of Alanine-to-Valine at position 54 of the ORF3a polypeptide. Both amino acids represent aliphatic, nonpolar neutral residues, and thus it is not expected to induce crucial alterations in the ORF3a functionality.
  • S194L missense mutation S194L (28854C>T), in line with a similar trend observed worldwide (13% of September to 21% of November).
  • S194L mutation is also located on the Ser/Arg (SR)- rich motif of the nucleocapsid protein and involves the substitution of the hydroxylic neutral Serine (S) with the aliphatic neutral Leucine (L). Since this region regulates the N protein oligomerization upon phosphorylation, the mutation-induced absence of the S194 could have a significant impact on the function of nucleocapsid, which also comes in accordance with the dramatic changes in the predicted protein structure and therefore merits further study.
  • SR Ser/Arg

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