METHODS TO QUANTIFY VIRUS FROM WASTEWATER
The present disclosure relates to a method of detecting and quantifying a virus-of-interest from human viral shed in a community, including: concentrating viral material from a wastewater sample to form a viral nucleic acid material; measuring an amount of a virus-of-interest nucleic acid in the viral nucleic acid material to obtain a first amount; measuring an amount of a marker or bacteriophage nucleic acid in the viral nucleic acid material to obtain a second amount; and forming a ratio of the first amount to the second amount to estimate a level of virus-of-interest from human viral shed within a community. In embodiments, methods for quantifying, monitoring, and/or identifying virus-of-interest such as SARS-CoV-2 are also disclosed.
1 . A method of detecting and quantifying a virus-of-interest from human viral shed in a community, comprising:
concentrating viral material from a wastewater sample to form a viral nucleic acid material;
measuring an amount of a virus-of-interest nucleic acid in the viral nucleic acid material to obtain a first amount;
measuring an amount of a bacteriophage nucleic acid in the viral nucleic acid material to obtain a second amount; and
forming a ratio of the first amount to the second amount to estimate a level of virus-of-interest from human viral shed within a community.
2 . The method of claim 1 , wherein the virus-of-interest is an orthomyxovirus, influenza virus, influenza A, influenza B, an RNA virus, or a DNA virus.
3 . The method of claim 2 , wherein the RNA virus is characterized as one of Reoviridae, Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, Retroviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Coronaviridae, Astroviridae, or Bornaviridae.
4 . The method of claim 2 , wherein the DNA virus is characterized as Adenoviridae, Papovaviridae, Parvoviridae, Herpesviridae, Poxviridae, or Hepadnaviridae.
5 . The method of claim 1 , wherein the virus-of-interest is SARS-CoV-2 or a SARS-CoV-2 variant.
6 . The method of claim 1 , wherein the bacteriophage nucleic acid is cross-assembly phage nucleic acid.
7 . The method of claim 1 , wherein concentrating viral material further comprises centrifuging the viral material through a sugar cushion in an ultracentrifuge.
8 . The method of claim 1 , wherein concentrating viral material further comprises centrifuging the viral material through a sugar gradient in an ultracentrifuge.
9 . The method of claim 7 , wherein centrifuging the viral material through a sugar cushion in an ultracentrifuge further comprises forming a solution comprising a buffer and sugar.
10 . The method of claim 9 , wherein the solution comprises a sugar in the amount of 40 to 60 percent weight of the total solution.
11 . The method of claim 10 , wherein the sugar comprises sucrose in an amount of about 50 percent weight of the total solution.
12 . The method of claim 1 , wherein the viral material comprises inactivated or fragmented virus.
13 . The method of claim 1 , wherein concentrating viral material from a wastewater sample to form a viral nucleic acid material is performed under conditions to purify the viral material.
14 . The method of claim 1 , wherein measuring the amount of virus-of-interest nucleic acid and bacteriophage nucleic acid is performed by qPCR analysis comprising one or more fluorescent materials.
15 . A method of monitoring a virus-of-interest human transmission in a community, comprising:
(a) concentrating viral material from a wastewater sample to form a viral nucleic acid material;
(b) measuring an amount of virus-of-interest nucleic acid in the viral nucleic acid material to obtain a first amount;
(c) measuring an amount of bacteriophage nucleic acid in the viral nucleic acid material to obtain a second amount;
(d) forming a ratio of the first amount to the second amount to estimate a level of virus-of-interest human viral shed within a community; and
(e) repeating (a)-(d) in an amount sufficient to monitor virus-of-interest transmissions within a community.
16 . The method of claim 15 , wherein concentrating viral material further comprises centrifuging the viral material through a sugar cushion in an ultracentrifuge.
17 . The method of claim 16 , wherein centrifuging the viral material through a sugar cushion in an ultracentrifuge further comprises forming a solution comprising a buffer and sugar.
18 . The method of claim 17 , wherein the solution comprises a sugar in the amount of 40 to 60 percent weight of the total gradient-forming solution.
19 . The method of claim 18 , wherein the sugar comprises sucrose in an amount of about 50 percent weight of the total solution.
20 . The method of claim 15 , wherein the virus-of-interest is SARS-CoV-2.
21 . A method of detecting and/or quantifying a virus-of-interest in wastewater, comprising:
concentrating viral material from a wastewater sample to form a viral nucleic acid material, wherein concentrating comprises centrifuging the viral material through a sugar cushion in an ultracentrifuge;
measuring an amount of virus-of-interest nucleic acid in the viral nucleic acid material to detect and/or quantify the virus-of-interest.
22 . The method of claim 21 , wherein centrifuging the viral material through a sugar cushion in an ultracentrifuge further comprises forming a solution comprising a buffer and sugar.
23 . The method of claim 22 , wherein the solution comprises a sugar in the amount of 40 to 60 percent weight of the total solution.
24 . The method of claim 23 , wherein the sugar comprises sucrose in an amount of about 50 percent weight of the total solution.
25 . The method of claim 21 , wherein the wastewater is characterized as an aliquot of less than 50 mL of raw wastewater.
26 . The method of claim 21 , wherein concentrating viral material comprises purifying the nucleic acid of the viral material.
27 . The method of claim 21 , wherein the virus-of-interest is SARS-CoV-2 or a SARS-CoV-2 variant.