Capturing, concentrating, and detecting microbes in a sample using magnetic ionic liquids and recombinase polymerase amplification
In this disclosure, a method or kit for using the method of extracting, concentrating, and detecting microbes from a sample is disclosed. The method disclosed herein use transition or rare earth metal-based magnetic ionic liquids (MILs) to extract viable microbes from a sample and to detect them via an amplification-based method and/or a non-amplification-based methods. The method and kit can be used in-field and on-site for detection of viable microbes in a sample within about an hour, without using any powered heat source or powered tool.
1. A method of extracting and detecting microbes in a food sample, comprising:
contacting the food sample with a magnetic ionic liquid (MIL) for the period of a contacting time;
extracting the microbes or their diagnostically useful cellular components from the magnetic ionic liquid using an aqueous extracting medium to generate an extracted microbe sample;
detecting the microbes; wherein the detecting is performed by an amplification-based detection method comprising using Recombinase Polymerase Amplification (RPA) on the extracted microbe sample; wherein the RPA is carried out with a power-free heat source comprising a sodium acetate heat pack;
wherein the food sample comprises a liquid food or an aqueous suspension of a solid food, wherein the food sample comprises viable microbes, and wherein the viable microbes comprise gram-negative bacteria, gram-positive bacteria, viruses, or a mixture thereof.
2. The method according to claim 1 , wherein the microbes are gram-negative bacteria selected from the group consisting of Cronobacter sakazakii, E. coli, Klebsiella aerogenes, Pantoea eucalypti, Pantoea stewartii, Pectobacterium carotovorum, Salmonella bongori, Salmonella enterica, Serratia marcescens, Yersinia enterocolitica , and mixtures thereof.
3. The method according to claim 1 , wherein the food sample comprises milk or egg.
4. The method according to claim 1 , wherein the extracting medium is a Luria-Bertani-derived nutrient broth comprising more than 10 g/L of tryptone, more than 5 g/L of yeast extract, more than 10 g/L of NaCl, or combination thereof.
5. The method according to claim 1 , wherein a volume ratio between the magnetic ionic liquid and the extracting medium is from about 1:5 to 1:15.
6. The method according to claim 1 , wherein the RPA on the extracted microbe sample amplifies DNA or RNA of the microbes; and wherein RPA is applied to the extracted microbe sample for amplifying characteristic DNA of the microbes at a temperature of from about 20° C. to about 50° C.
7. The method according to claim 6 , wherein the microbes are Salmonella and the RPA comprises using a dienelactone hydrolase primer.
8. The method according to claim 6 , wherein the RPA comprises using a reverse transcriptase (RT) enzyme for characteristic RNA of the microbes.
9. The method according to claim 1 , wherein results of the RPA are visualized using nucleic acid lateral flow immunoassay (NALFIA) or gel electrophoresis.
10. The method according to claim 1 , wherein the magnetic ionic liquid comprises a paramagnetic anionic component and a cationic component,
wherein the cationic component has a general formula (I), (IV), or (V)
[(PR 1 R 2 R 3 R 4 ) + ] (I)
wherein each of the R 1 , R 2 , R 3 , and R 4 is independently an unsubstituted or substituted alkyl;
wherein the paramagnetic anionic component has the following general formula (II),
[M(Y) x − ] (II)
wherein M is transition metal or rare earth metal ion; and
Y is a chelating agent having the general formula (III),
each of the R 10 and R 11 are independently a substituted or unsubstituted methyl, phenyl, thiophenyl, napthyl, alkyl, or aryl group; and x is 3 or 4
wherein R is one or more of the following:
and wherein Ti comprises Co, Ni, Mn or a combination thereof and Ln comprises Dy, Gd, Ho, or a combination thereof.
11. The method according to claim 10 , wherein the MIL has the anionic component of [Co(hfacac) 3 − ], [Ni(hfacac) 3 − ], ([Mn(hfacac) 3 − ]), ([Dy(hfacac) 4 − ]), ([Gd(hfacac) 4 − ]), ([Nd(hfacac) 4 − ]), or combination thereof; wherein hfacac is
and has the cationic component of [(PR 1 R 2 R 3 R 4 ) + ], wherein each of the R 1 , R 2 , R 3 , and R 4 is independently a C 2 -C 20 unsubstituted alkyl.
12. The method according to claim 1 , wherein the magnetic ionic liquid comprises a paramagnetic cationic component and an anionic component, wherein the anionic component has a general formula (VI) or (VII)
wherein R is one or more of the following:
and wherein Ti comprises Co, Ni, Mn or a combination thereof and Ln comprises Dy, Gd, Ho, or a combination thereof.