Noninterfering Multipurpose Compositions for Collecting, Transporting and Storing Biological Samples
The invention is directed to compositions and methods for collecting, transporting, and storing microorganisms obtained from samples of biological, clinical, forensic, and environmental origin. Compositions preserve the viability of the collected organisms and permit the long-term storage of samples. Compositions are compatible with subsequent manipulation of the sample, including propagation and culture of the collected microorganisms, or isolation, purification, detection, and characterization of proteins, nucleic acids, and all macromolecules. When the compositions containing microorganisms and any polynucleotides therein are further processed, such as by nucleic acid testing, there is an increased ability to detect, isolate, purify and/or characterize select microbes and their components, such as nucleic acids, when compared to conventional microbial transport media that contain interfering substance(s). In particular formulations, the compositions disclosed herein allow biological samples to be collected, transported, and even stored for extended periods, and are compatible with nucleic acid extraction, identification, quantitation, PCR amplification, and genomic analysis methodologies.
1 . A method of detecting a sequence in a biological sample containing nucleic acids comprising:
contacting the biological sample with an effective amount of a transport medium containing a stabilizing agent that functions to maintain the integrity of nucleic acid sequences contained within the biological sample without interfering with subsequent nucleic acid amplification of the sample, wherein the stabilizing agent is a gelatin obtained or derived from fish or fish products;
amplifying nucleic acids of the biological sample; and
detecting the sequence from the amplified nucleic acids.
2 . The method of claim 1 , wherein the biological sample comprises cells suspected of being infected with a pathogen.
3 . The method of claim 2 , wherein the pathogen is a viral, a bacterial, a parasitic or a fungal infection.
4 . The method of claim 2 , wherein the pathogen is Chlamydia, Mycoplasma, Ureaplasma, Adenovirus, Herpes Simplex Virus, Paramyxovirus, or Influenza virus.
5 . The method of claim 4 , wherein the Influenza virus is one or more of Influenza A, Influenza B, or Influenza C, or Influenza serotypes that contain H1, H3, H5, N1, H5N1, or H1N1.
6 . The method of claim 1 , wherein the transport medium is a collection, transport and storage medium and provided in at least a volume equivalent to about the volume of the biological sample.
7 . The method of claim 1 , wherein the transport medium is a collection, transport and storage medium and provided in at least a volume equivalent to about three times the volume of the biological sample.
8 . The method of claim 1 , wherein the transport medium contains from about 1 to 10 g/L of a buffer, from about 10 to 50 g/L of trehalose, from about 1 to 10 g/L of a carbohydrate comprising at least one of glucose, sucrose, mannose, altrose, allose, idose, talose, fructose, methyl-α-D-glucoside, galactose, ribose, deoxyribose, xylose, lactose, maltose, glycogen, amylase, cellulose, 6-deoxy-α-d-gluco-heptopyranosyl 6-deoxy-α-d-gluco-heptopyranoside, or (6-deoxy-α-d-gluco-heptopyranosyluronic acid) 6-deoxy-α-d-gluco-heptopyranosiduronic acid, or any combination thereof, and up to about 2 g/L of the gelatin.
9 . The method of claim 8 , wherein the transport medium contains from about 30 to about 40 g/L of trehalose, from about 5 to about 10 g/L of each of fructose and glucose; and from about 1 to about 1.5 g/L of the gelatin.
10 . The method of claim 8 , wherein the buffer comprises N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (HEPPS), 2-morpholinoethanesulfonic acid monohydrate (MES), 3-morpholinopropanesulfonic acid (MOPS), piperazine-N,N′-bis(2-ethane sulfonic acid) (PIPES), N-tris(hydroxymethyl)methyl glycine (Tricine), or a combination thereof.
11 . The method of claim 1 , wherein the transport medium further comprises at least one antibiotic which comprises vancomycin, polymyxin B, gentamycin, colistin, trimethoprim, amphotericin B, or a combination thereof.
12 . The method of claim 1 , wherein the transport medium further comprises a salt which comprises calcium chloride, potassium chloride, magnesium chloride, sodium chloride, magnesium sulfate, or a combination thereof.
13 . The method of claim 1 , wherein the transport medium further comprises a pH indicator which comprises neutral red, phenol red, or a combination thereof.
14 . The method of claim 1 , wherein the transport medium has a pH of about 7.1 to about 7.5.
15 . The method of claim 1 , wherein the transport medium further comprising at least one amino acid which comprises glutamic acid, aspartic acid, or a combination thereof.
16 . The method of claim 1 , wherein the transport medium further comprises another stabilizing agent selected from the group consisting of a low molecular weight gelatin or serum albumin, agar, carrageenan, a fish-derived gelatin, pectin, a sugar polymer, a galactose polymer, a polysaccharide, a heteropolysaccharide, a linear-sulfated polysaccharide, a protein, a collagen or a hydrolyzed product of collagen, and combinations thereof.
17 . The method of claim 1 , wherein the amplifying comprises a polymerase chain reaction (PCR), amplified fragment length polymorphism, restriction fragment length polymorphism, allele-specific oligonucleotide analysis, polymerase cycling assembly, real time PCR, reverse transcription PCR, quantitative PCR, nested PCR, multiplex PCR, overlap extension PCR, asymmetric PCR, or a combination thereof.
18 . The method of claim 17 , wherein the amplifying by PCR comprises a polymerase chain reaction cycle threshold (C T ) value that equates to about 10 to 100-fold lower than that obtained when the biological sample is maintain in a medium containing bovine serum albumin.
19 . The method of claim 1 , wherein 80 percent or more of the nucleic acid sequences present in the biological sample are detectable.
20 . The method of claim 1 , wherein upon contact with the transport medium, the nucleic acid sequences of the contacted biological sample remain detectable for at least 30 days prior to amplification.
21 . The method of claim 1 , wherein contacting is performed at ambient temperature.
22 . The method of claim 1 , wherein amplification of the nucleic acids is greater than or equal to about 1-log to the base 10 (about 3.3 C T ) when the sample is contacted by a conventional medium including at least one interfering substance.
23 . The method of claim 1 , wherein the transport medium comprises:
about 0.001 mM to about 650 mM of trehalose, about 0.001 mM to about 600 mM of a carbohydrate comprising allose, altrose, arabinose, deoxyribose, erythrose, erythrulose, fructose, galactose, glucose, idose, lyxose, mannose, psicose, ribose, ribulose, sorbose, tagatose, talose, threose, xylose, xylulose, cellobiose, isomaltose, lactose, lactulose, maltose, maltulose, mannobiose, melibiose, sucrose, turanose, xylobiose, 6-deoxy-α-d-gluco-heptopyranosyluronic acid, 6-deoxy-α-d-gluco-heptopyranosiduronic acid, 6-deoxy-α-d-gluco-heptopyranosylic acid, 6-deoxy-α-d-gluco-heptopyranoside, cellulose, dextran, galactan, glycogen, levan, maltodextrin, maltotriose, mannan, melezitose, methyl-α-D-glucoside, raffinose, rhamnose, starch, or a combination thereof;
about 1 μM to about 1 M of a buffer comprising BES, TES, HEPES, HEPPS, MES, MOPS, PIPES, Tricine, or a combination thereof;
up to about 2 g/L of the gelatin;
about 1 μM to about 100 mM of a salt comprising calcium chloride, potassium chloride, magnesium chloride, sodium chloride, magnesium sulfate, or a combination thereof;
about 0.1 μM to about 500 mM of an amino acid comprising glutamic acid, aspartic acid, or a combination thereof;
about 1 μM to about 50 mM of a pH indicator comprising neutral red, phenol red, or a combination thereof; and
about 0.1 μM to about 1 mM of an antibiotic comprising vancomycin, polymyoxin B, gentamycin, colistin, trimethoprim, amphotericin B, or a combination thereof.
24 . The method of claim 1 , wherein the biological sample is maintained in the transport medium at a temperature of from about minus 80° C. to about plus 37° C., for a period of from about one hour to about six months.
25 . The method of claim 1 , wherein the biological sample is maintained in the transport medium at a temperature of from about 0° C. to about 30° C., for a period of from about one day to about 90 days.
26 . The method of claim 1 , wherein contacting is performed at ambient temperature.
27 . A method for detecting a target sequence of interest from a biological sample containing nucleic acids comprising:
contacting the biological sample with an effective amount of an aqueous transport medium to form a mixture, wherein the aqueous transport medium contains up to about 2 g/L of a fish-derived gelatin that does not interfere with quantitative detection of nucleic acid sequences by a polymerase chain reaction (PCR);
storing the mixture for a period of a period of time at a temperature of from about minus 10° C. to about 35° C. such that such at least 80 percent of the nucleic acid of the contacted biological sample remain detectable;
amplifying the target sequence of interest with the PCR; and
detecting the amplified target sequence of interest.
28 . The method of claim 27 , wherein the period of time is about 30 days or more.
29 . The method of claim 27 , wherein the PCR is real time PCR, reverse transcription PCR, quantitative PCR, nested PCR, multiplex PCR, overlap extension PCR, asymmetric PCR, or a combination thereof.
30 . The method of claim 27 , wherein detecting the amplified target sequence is determined by measuring a polymerase chain reaction cycle threshold (C T ) of the sequence.
31 . The method of claim 30 , wherein the polymerase chain reaction cycle threshold (C T ) value equates to about 10 to 100-fold lower than that obtained when a substantial amount of an interfering substance is present in the composition.
32 . The method of claim 27 , wherein the aqueous transport medium contains from about 1 to 10 g/L of a buffer, from about 10 to 50 g/L of trehalose, from about 1 to 10 g/L of a carbohydrate comprising at least one of glucose, sucrose, mannose, altrose, allose, idose, talose, fructose, methyl-α-D-glucoside, galactose, ribose, deoxyribose, xylose, lactose, maltose, glycogen, amylase, cellulose, 6-deoxy-α-d-gluco-heptopyranosyl 6-deoxy-α-d-gluco-heptopyranoside, or (6-deoxy-α-d-gluco-heptopyranosyluronic acid) 6-deoxy-α-d-gluco-heptopyranosiduronic acid, or any combination thereof.
33 . The method of claim 27 , wherein the biological sample comprises cells suspected of being infected with a pathogen and amplification of the target sequence is indicative of the presence of the pathogen.
34 . The method of claim 33 , wherein the pathogen is a viral, a bacterial, a parasitic or a fungal infection.
35 . The method of claim 33 , wherein the pathogen is Chlamydia, Mycoplasma, Ureaplasma, Adenovirus, Herpes Simplex Virus, Paramyxovirus, or Influenza virus.
36 . The method of claim 27 , wherein the aqueous transport medium comprises a chaotrope, an anionic detergent, a reducing agent, a chelator and a buffer in nuclease-free water, and the transport medium kills microbes that may be present in the sample.
37 . A method of detecting Mycobacteria in a biological sample comprising:
contacting the biological sample with an effective amount of an aqueous transport medium containing a stabilizing agent that functions to maintain the integrity of nucleic acid sequences contained within the biological sample without interfering with subsequent nucleic acid amplification of the sample, wherein the stabilizing agent is a gelatin obtained or derived from fish or fish products;
amplifying nucleic acids of the biological sample by PCR; and
detecting the presence of Mycobacteria from the amplified nucleic acids.
38 . The method of claim 37 , wherein the aqueous transport medium comprises a chaotrope, an anionic detergent, a reducing agent, a chelator and a buffer in nuclease-free water, and the transport medium kills Mycobacteria that may be present in the sample.
39 . The method of claim 37 , wherein amplification of the nucleic acids is greater than or equal to about 1-log to the base 10 (about 3.3 C T ) when the sample is contacted by a conventional medium including at least one interfering substance.
40 . The method of claim 37 , wherein the aqueous transport medium contains from about 1 to 10 g/L of a buffer, from about 10 to 50 g/L of trehalose, from about 1 to 10 g/L of a carbohydrate comprising at least one of glucose, sucrose, mannose, altrose, allose, idose, talose, fructose, methyl-α-D-glucoside, galactose, ribose, deoxyribose, xylose, lactose, maltose, glycogen, amylase, cellulose, 6-deoxy-α-d-gluco-heptopyranosyl 6-deoxy-α-d-gluco-heptopyranoside, or (6-deoxy-α-d-gluco-heptopyranosyluronic acid) 6-deoxy-α-d-gluco-heptopyranosiduronic acid, or any combination thereof, and up to about 2 g/L of the gelatin.