Detection of infectious agents from environmental air dust
Embodiments of the present disclosure are directed to systems and methods for collection and analysis of environmental air dust (EAD) within an individually ventilated cage rack (IVR) environment for detecting pathogens. The method includes collection of an EAD sample by a collection media, isolation of a plurality of nucleic acids (e.g., RNA and/or DNA) representative of one or more infectious agents from the EAD sample, optional reverse transcription of RNA to cDNA if the isolated nucleic acids contain RNA, amplification of the cDNA and/or DNA (e.g., by polymerase chain reaction (PCR)), and assay interpretation. Optionally, the EAD sample may be analyzed with one or more other sample types (e.g., fecal pellets, oral swabs, body swabs, tissue, etc.) to improve detection of low-copy organisms.
1 . A method for detecting at least one pathogen, comprising:
receiving a test sample comprising environmental air dust released by agitation of soiled bedding, the soiled bedding positioned within an animal enclosure of an individually ventilated cage rack (IVR), the test sample captured from airflow passing through the animal enclosure of the IVR by a collection media, wherein the collection media is disposed on an interior wall of the animal enclosure and not in direct contact with an exhaust for the animal enclosure to capture the environmental air dust within the airflow circulating through an interior volume of the animal enclosure, wherein the test sample is received while the environmental air dust is still retained on the collection media;
isolating a plurality of nucleic acids from the test sample; and
analyzing the isolated plurality of nucleic acids to identify the presence or absence of at least one pathogen from the interior volume of the animal enclosure.
2 . The method of claim 1 , wherein the environmental air dust is released by animal agitation of the soiled bedding within the animal enclosure.
3 . The method of claim 1 , further comprising agitating the soiled bedding within the animal enclosure to release the environmental air dust into the airflow passing through the animal enclosure.
4 . The method of claim 1 , further comprising amplifying at least one of the plurality of nucleic acids.
5 . The method of claim 4 , wherein amplifying at least one of the plurality of nucleic acids comprises at least one of loop-mediated isothermic amplification and polymerase chain reaction (PCR).
6 . The method of claim 5 , wherein amplifying at least one of the plurality of nucleic acids comprises real-time PCR.
7 . The method of claim 5 , wherein:
isolating the plurality of nucleic acids comprises extracting an RNA sample from the test sample and reverse transcribing the extracted RNA sample into a cDNA sample;
amplifying at least one of the plurality of nucleic acids comprises amplifying the cDNA sample by real-time PCR; and
analyzing the amplified nucleic acids comprises measuring a Ct value of the amplified cDNA sample.
8 . The method of claim 5 , wherein:
isolating the plurality of nucleic acids comprises extracting a DNA sample from the test sample;
amplifying at least one of the plurality of nucleic acids comprises amplifying the DNA sample by real-time PCR; and
analyzing the amplified nucleic acids comprises measuring a Ct value of the amplified DNA sample.
9 . The method of claim 5 , wherein amplifying at least one of the plurality of nucleic acids comprises PCR and wherein analyzing the amplified nucleic acids comprises time-of-flight analysis of PCR products.
10 . The method of claim 1 , wherein the test sample comprises environmental air dust captured from the airflow over a time period of at least two weeks.
11 . The method of claim 1 , wherein the further comprises at least one cage for housing a test animal.
12 . The method of claim 1 , wherein the at least one pathogen is selected from the group consisting of: Staphylococcus spp., Pasteurella spp., Proteus spp., Klebsiella spp., Giardia spp., Cryptosporidium spp., Entamoeba spp., Spironucleus spp., Murine norovirus, Pseudomonas spp., and beta-hemolytic Streptococcus spp.
13 . The method of claim 11 , wherein the collection media is disposed on an interior wall of the at least one cage.
14 . The method of claim 1 , wherein isolating the plurality of nucleic acids comprises at least one of magnetic isolation, column-based nucleic acid isolation, organic extraction methods, and alkaline lysis.
15 . The method of claim 1 , wherein the IVR does not comprise a cage housing a sentinel animal.
16 . The method of claim 1 , wherein the collection media comprises a filter selected from the group consisting of mechanical filters, chemical filters, electrostatic filters, and wet scrubbers.
17 . The method of claim 1 , wherein the collection media is a graded filter.
18 . A method for detecting one or more pathogens attached to environmental air dust, comprising:
receiving a test sample comprising environmental air dust released by agitation of soiled bedding, the soiled bedding positioned within an animal enclosure of an individually ventilated cage rack (IVR), the test sample captured from airflow passing through the animal enclosure by air filtering collection media, the animal enclosure comprising a chamber containing an animal cage in fluid communication with the airflow, wherein the test sample is received while the environmental air dust is still retained on the air filtering collection media,
wherein the air filtering collection media is disposed on an interior wall of the animal enclosure and not in direct contact with an exhaust for the animal enclosure to capture the environmental air dust while the environmental air dust is circulating through an interior volume of the animal enclosure;
isolating a plurality of nucleic acids from the test sample; and
analyzing the isolated nucleic acids to identify the presence or absence of a pathogen from the interior volume of the animal enclosure.
19 . The method of claim 18 , wherein the environmental air dust is released by animal agitation of the soiled bedding within the animal enclosure.
20 . The method of claim 18 , further comprising agitating the soiled bedding within the animal cage to release the environmental air dust into the airflow passing through the animal enclosure.
21 . The method of claim 18 , further comprising amplifying at least one of the plurality of nucleic acids.
22 . The method of claim 21 , wherein amplifying at least one of the plurality of nucleic acids comprises at least one of loop-mediated isothermic amplification and polymerase chain reaction (PCR).
23 . The method of claim 22 , wherein amplifying at least one of the plurality of nucleic acids comprises real-time PCR.
24 . The method of claim 22 , wherein:
isolating the plurality of nucleic acids comprises extracting an RNA sample from the test sample and reverse transcribing the extracted RNA sample into a cDNA sample;
amplifying at least one of the plurality of nucleic acids comprises amplifying the cDNA sample by real-time PCR; and
analyzing the amplified nucleic acids comprises measuring a Ct value of the amplified cDNA sample.
25 . The method of claim 22 , wherein:
isolating the plurality of nucleic acids comprises extracting a DNA sample from the test sample;
amplifying at least one of the plurality of nucleic acids comprises amplifying the DNA sample by real-time PCR; and
analyzing the amplified nucleic acids comprises measuring a Ct value of the amplified DNA sample.
26 . The method of claim 22 , wherein amplifying at least one of the plurality of nucleic acids comprises PCR and wherein analyzing the amplified nucleic acids comprises time-of-flight analysis of PCR products.
27 . The method of claim 18 , wherein the test sample comprises environmental air dust captured from the airflow over a time period of at least two weeks.
28 . The method of claim 18 , wherein the one or more pathogens is selected from the group consisting of: Staphylococcus spp., Pasteurella spp., Proteus spp., Klebsiella spp., Giardia spp., Cryptosporidium spp., Entamoeba spp., Spironucleus spp., Murine norovirus, Pseudomonas spp., and beta-hemolytic Streptococcus spp.
29 . The method of claim 18 , wherein isolating the plurality of nucleic acids comprises at least one of magnetic isolation, column-based nucleic acid isolation, organic extraction methods, and alkaline lysis.
30 . The method of claim 18 , wherein the air filtering collection media comprises a filter selected from the group consisting of mechanical filters, chemical filters, electrostatic filters, and wet scrubbers.
31 . The method of claim 18 , wherein the air filtering collection media is a graded filter.
32 . The method of claim 18 , wherein the air filtering collection media is disposed on an interior wall of the animal cage.
33 . The method of claim 5 , wherein amplifying at least one of the plurality of nucleic acids comprises endpoint PCR.
34 . The method of claim 22 , wherein amplifying at least one of the plurality of nucleic acids comprises endpoint PCR.
35 . The method of claim 1 , further including the steps of:
placing the collection media having the environmental air dust collected thereon in a tube;
pipetting a solution directly onto the collection media;
subjecting the tube to a vortex to thoroughly wash the collection media with the solution; and
adding a nucleic acid recovery control to the tube.
36 . The method of claim 18 , further including the steps of:
placing the air filtering collection media having the environmental air dust collected thereon in a tube;
pipetting a solution directly onto the air filtering collection media;
subjecting the tube to a vortex to thoroughly wash the air filtering collection media with the solution; and
adding a nucleic acid recovery control to the tube.
37 . A method for detecting at least one pathogen, comprising:
receiving a test sample comprising environmental air dust released by agitation of soiled bedding, the soiled bedding positioned within an animal enclosure of an individually ventilated cage rack (IVR), the test sample captured from airflow passing through the animal enclosure of the IVR by a collection media, wherein the collection media is disposed on an interior wall of the animal enclosure and not in direct contact with an exhaust for the animal enclosure to capture the environmental air dust within the airflow circulating through an interior volume of the animal enclosure, wherein the test sample is received while the environmental air dust is still retained on the collection media;
placing the collection media having the environmental air dust collected thereon in a tube;
pipetting a solution directly onto the collection media;
subjecting the tube to a vortex to thoroughly wash the collection media with the solution;
adding a nucleic acid recovery control to the tube;
isolating a plurality of nucleic acids from the test sample; and
analyzing the isolated plurality of nucleic acids to identify the presence or absence of at least one pathogen from the interior volume of the animal enclosure.
38 . The method of claim 37 , wherein the environmental air dust is released by animal agitation of the soiled bedding within the animal enclosure.
39 . The method of claim 38 , further comprising amplifying at least one of the plurality of nucleic acids.
40 . The method of claim 39 , wherein amplifying at least one of the plurality of nucleic acids comprises at least one of loop-mediated isothermic amplification and polymerase chain reaction (PCR).
41 . The method of claim 40 , wherein:
isolating the plurality of nucleic acids comprises extracting an RNA sample from the test sample and reverse transcribing the extracted RNA sample into a cDNA sample;
amplifying at least one of the plurality of nucleic acids comprises amplifying the cDNA sample by real-time PCR; and
analyzing the amplified nucleic acids comprises measuring a Ct value of the amplified cDNA sample.
42 . The method of claim 41 , wherein:
isolating the plurality of nucleic acids comprises extracting a DNA sample from the test sample;
amplifying at least one of the plurality of nucleic acids comprises amplifying the DNA sample by real-time PCR; and
analyzing the amplified nucleic acids comprises measuring a Ct value of the amplified DNA sample.
43 . The method of claim 42 , wherein the IVR further comprises at least one cage for housing a test animal, and wherein the collection media is disposed on an interior wall of the at least one cage.
44 . The method of claim 43 , wherein the test sample comprises environmental air dust captured from the airflow over a time period of at least two weeks.
45 . The method of claim 44 , wherein the test sample comprises environmental air dust captured from the airflow over a time period of at least three to four months.