DIRECT NUCLEIC ACID ANALYSIS OF ENVIRONMENTAL AND BIOLOGICAL SAMPLES
Methods and apparatuses are described for nucleic acid analysis of environmental water samples and biological samples without the need for purification.
1 . A method comprising steps of:
obtaining an environmental sample comprising a microorganism, wherein the microorganism comprises a nucleic acid;
concentrating the environmental sample to produce a concentrated sample, wherein the microorganism is concentrated about 2-fold to about 125-fold in the concentrated sample as compared to the environmental sample;
contacting the concentrated sample with a nucleic acid amplification reagent in a reaction vessel, wherein the concentrated sample is directly contacted with the nucleic acid amplification reagent without any intervening steps; and
performing a nucleic acid amplification reaction on the nucleic acid from the microorganism in the concentrated sample.
2 . The method of claim 1 , wherein the environmental sample is a water sample selected from the group consisting of industrial cooling tower water, untreated fresh water, waste water, stagnant water, wash water, grey water and water obtained from a lavatory, shower, bathtub, toilet, sink.
3 . The method of any one of claims 1 and 2 , wherein the microorganism is a bacteria, cyanobacteria, virus, protozoa, fungus or rotifer.
4 . The method of claim 3 , wherein the bacteria is selected from the group consisting of Alicyclobacillus, Aeromonas, Bacteroides, Bifidobacterium, Campylobacter, Citrobacter, Clostridia, Enterobacter, Enteroccocus, Escherichia, Eubacterium, Klebsiella, Lactobacillus, Legionella, Listeria, Mycobacterium, Pseudomonas, Raoultella, Salmonella, Shigella, Streptococcus, Vibrio and combinations thereof.
5 . The method of claim 3 or 4 , wherein the bacteria is selected from the group consisting of Legionella pneumophila, Legionella longbeachae, Legionella bozemannii, Legionella micdadei, Legionella feeleii, Legionella dumoffii, Legionella wasdworthii, Legionella anisa and combinations thereof.
6 . The method of claim 3 or 4 , wherein the bacteria is Escherichia coli.
7 . The method of any one of claims 1 - 6 , wherein the environmental sample is concentrated to produce the concentrated sample by filtration, evaporation and/or centrifugation.
8 . The method of any one of claims 1 - 7 , wherein the environmental sample is concentrated to produce the concentrated sample by filtration.
9 . The method of claim 8 , wherein the filtration step comprises washing a retentate and/or eluting the concentrated sample from the filter.
10 . The method of claim 8 or 9 , wherein the filtration is performed using a hydrophilic filter membrane.
11 . The method of any one of claims 8 - 10 , wherein the filtration is performed using a hydrophilic polyethersulfone (PES) filter membrane.
12 . The method of any one of claims 1 - 11 , wherein the nucleic acid amplification reaction comprises a DNA polymerase at a concentration of at least 1.0 U/reaction and a primer at a concentration of at least 0.2 μM.
13 . The method of any one of claims 1 - 12 , wherein the nucleic acid amplification reaction comprises a probe at a concentration ranging from at least 1.0 μM to about 14 μM.
14 . The method of claim 12 , wherein the DNA polymerase is at a concentration ranging from at least 3.4 U/reaction to about 45 U/reaction.
15 . The method of claim 12 , wherein the primer is at a concentration ranging from at least 1.3 μM to about 15 μM.
16 . The method of any one of claims 1 - 15 , wherein the nucleic acid amplification reagent does not comprise a reagent which is designed to resist DNA polymerase inhibitors.
17 . The method of any one of claims 1 - 16 , wherein the method does not include a step of lysing the microorganism.
18 . The method of any one of claims 1 - 17 , wherein the method does not further include a step of purifying the nucleic acid from the microorganism.
19 . The method of claim 12 , wherein the nucleic acid amplification reaction comprises a DNA polymerase at a concentration ranging from at least 12 U/reaction to about 21 U/reaction, a primer at a concentration ranging from at least 4.0 μM to about 7.0 μM and a probe at a concentration ranging from at least 3.5 μM to about 7.0 μM.
20 . The method of any one of claims 1 - 19 , further comprising a step of determining whether an amplification product was produced as a result of the nucleic acid amplification reaction.
21 . A method comprising steps of:
obtaining a sample comprising a nucleic acid;
contacting the sample with a nucleic acid amplification reagent in a reaction vessel,
wherein the sample is directly contacted with the nucleic acid amplification reagent without any intervening steps and
wherein the nucleic acid amplification reagent comprises a DNA polymerase at a concentration ranging from at least 6 U/reaction to about 42 U/reaction, a primer at a concentration ranging from at least 2.0 μM to about 14 μM and a probe at a concentration ranging from at least 1.9 μM to about 14 μM; and
performing a nucleic acid amplification reaction on the nucleic acid from the sample.
22 . The method of claim 21 , wherein the sample is selected from the group consisting of an environmental sample and a biological sample.
23 . The method of claim 22 , wherein the environmental sample is a concentrated sample.
24 . The method of claim 22 , wherein the environmental sample is a water sample selected from the group consisting of industrial cooling tower water, untreated fresh water, waste water, stagnant water, wash water, grey water and water obtained from a lavatory, shower, bathtub, toilet, sink.
25 . The method of claim 24 , wherein the environmental sample comprises a microorganism and wherein the microorganism comprises a nucleic acid.
26 . The method of claim 25 , wherein the microorganism is a bacteria, cyanobacteria, virus, protozoa, fungus or rotifer.
27 . The method of claim 26 , wherein the bacteria is selected from the group consisting of Alicyclobacillus, Aeromonas, Bacteroides, Bifidobacterium, Campylobacter, Citrobacter, Clostridia, Enterobacter, Enteroccocus, Escherichia, Eubacterium, Klebsiella, Lactobacillus, Legionella, Listeria, Mycobacterium, Pseudomonas, Raoultella, Salmonella, Shigella, Streptococcus, Vibrio and combinations thereof.
28 . The method of claim 26 or 27 , wherein the bacteria is selected from the group consisting of Legionella pneumophila, Legionella longbeachae, Legionella bozemannii, Legionella micdadei, Legionella feeleii, Legionella dumoffii, Legionella wasdworthii, Legionella anisa and combinations thereof.
29 . The method of claim 27 , wherein the bacteria is Escherichia coli.
30 . The method of claim 22 , wherein the biological sample is selected from the group consisting of a cell sample, a body fluid sample and a swab sample.
31 . The method of claim 22 , wherein the biological sample is collected from a foodstuff or a mammal.
32 . The method of claim 31 , wherein the mammal is a human.
33 . The method of claim 21 , further comprising a step of determining whether an amplification product was produced as a result of the nucleic acid amplification reaction.
34 . The method of claim 21 , wherein the step of obtaining comprises collecting the swab sample.
35 . A method comprising steps of:
obtaining an environmental sample from a source, wherein the environmental sample comprises a microorganism and the microorganism comprises a nucleic acid;
optionally concentrating the environmental sample to produce a concentrated sample, wherein the microorganism is concentrated about 2-fold to about 125-fold in the concentrated sample as compared to the environmental sample;
contacting the environmental sample or concentrated sample with a nucleic acid amplification reagent in a reaction vessel, wherein the environmental sample or concentrated sample is directly contacted with the nucleic acid amplification reagent without any intervening steps; and
performing a nucleic acid amplification reaction on the nucleic acid from the microorganism in the environmental sample or concentrated sample, wherein the nucleic acid amplification reaction is completed within less than 1 day from when the environmental sample was originally collected from the source.
36 . The method of claim 35 , wherein the amplification reaction is completed within less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 45 minutes, less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, or less than 1 minute from when the environmental sample was originally collected from the source.
37 . A method comprising steps of:
(a) obtaining a first environmental sample from a source, wherein the environmental sample comprises a microorganism and the microorganism comprises a nucleic acid;
(b) optionally concentrating the environmental sample to produce a concentrated sample, wherein the microorganism is concentrated about 2-fold to about 125-fold in the concentrated sample as compared to the environmental sample;
(c) contacting the environmental sample or concentrated sample with a nucleic acid amplification reagent in a reaction vessel, wherein the environmental sample or concentrated sample is directly contacted with the nucleic acid amplification reagent without any intervening steps;
(d) performing a nucleic acid amplification reaction on the nucleic acid from the microorganism in the environmental sample or concentrated sample, wherein the nucleic acid amplification reaction is optionally completed within less than 1 day from when the environmental sample was originally collected from the source; and
repeating steps (a), (c), (d) and optionally (b) on a second environmental sample from the same source within less than one month.
38 . The method of claim 37 , wherein steps (a), (c), (d) and optionally (b) are repeated on a new environmental sample from the same source on a monthly basis.
39 . The method of claim 37 , wherein steps (a), (c), (d) and optionally (b) are repeated on a second environmental sample from the same source within less than one week.
40 . The method of claim 39 , wherein steps (a), (c), (d) and optionally (b) are repeated on a new environmental sample from the same source on a weekly basis.