DEVELOPMENT OF A NUCLEIC ACID BASED LATERAL FLOW IMMUNOASSAY FOR BK VIRUS DETECTION FROM ESRD URINES AND CONTAMINATED SEWAGE SAMPLES

Abstract

A nucleic acid probe set is disclosed. The nucleic acid probe set comprises a detection probe and a capture probe, and the detection probe and the capture probe both include a nucleotide sequence that is extracted from a conserved region of a genome sequence belong to a BK virus. A nucleic acid lateral flow immunoassay for using in detection of BK virus is also disclosed. The nucleic acid lateral flow immunoassay comprises: the forgoing nucleic acid probe set, a test strip, and a streptavidin (SA) solution. Experimental data have proved that, the nucleic acid lateral flow immunoassay can be adopted for conducting a BK virus detection on a sample that is collected from environmental water, sewage water, drinking water, urine, or serum.

Claims

1. A nucleic acid probe set, comprising: a capture probe, comprising a first primer that comprises at least 19 bases and a combiner connected to one terminal base of the first primer; and a detection probe, comprising a second primer that comprises at least 19 bases and a labeling connected to one terminal base of the second primer; wherein the first primer and the second primer are both a nucleotide sequence that is extracted from a conserved region of a genome sequence belong to a BK virus.

2. The nucleic acid probe set of claim 1, wherein the combiner is a biotin that is used to combine with a streptavidin (SA), and the labeling being made of a fluorescent material that is selected from a group consisting of gold nanoparticles, silver nanoparticles, carbon nanoparticles, quantum dots (QDs), colloidal gold, colloidal silver, and colloidal QDs.

3. The nucleic acid probe set of claim 1, wherein the terminal base of the first primer connected with the combiner is a front-terminal base or a rear-terminal base, the terminal base of the second primer connected with the labeling is a front-terminal base or a rear-terminal base.

4. The nucleic acid probe set of claim 1, wherein the nucleotide sequence is selected from a group consisting of 5′-GAAAGGAAGGTAAGTTGTTAAG-3′ and 5′-TATGTATGAATAGAGTCTTAGGT-3′.

5. The nucleic acid probe set of claim 4, wherein the capture probe further comprises a spacer that is connected between the terminal base and the combiner, and the spacer comprising 10 adenines.

5. The nucleic acid probe set of claim 4, wherein the detection probe further comprises a spacer connected to the terminal base and a thiol group connected between the spacer and the labeling, and the spacer comprising 10 adenines.

7. The nucleic acid probe set of claim 1, wherein the labeling is made of gold nanoparticles having a particle size in a range between 25 nm and 65 nm.

8. A nucleic acid lateral flow immunoassay, comprising: a capture probe, comprising a first primer that comprises at least 19 bases and a combiner connected to one terminal base of the first primer; a detection probe, comprising a second primer that comprises at least 19 bases and a labeling connected to one terminal base of the second primer; wherein the first primer and the second primer are both a nucleotide sequence that is extracted from a conserved region of a genome sequence belong to a BK virus; a lateral flow strip, comprising a membrane substrate that is formed with a test line made of a first capture antibody and a control line made of a second capture antibody thereon; and a tetrameric protein solution; wherein when adopting the nucleic acid lateral flow immunoassay to carry out a BK virus detection, a sample having a DNA of the BK virus being heated firstly, and then the capture probe and the detection probe are mixed into the sample so as to obtain a test sample; subsequently, the test sample being added into the tetrameric protein solution so as to obtain a test solution; consequently, the lateral flow strip being disposed into the test solution, and an optical reader being operated to read out a T/C ratio value from a first colored line showing up in the test line and a second colored line showing up in the control line.

9. The nucleic acid lateral flow immunoassay of claim 8, wherein the sample is selected from a group consisting of sample collected from an environmental water, sample collected from a sewage water, sample collected from a drinking water, sample collected from a urine, and sample collected from a serum.

10. The nucleic acid lateral flow immunoassay of claim 8, wherein the tetrameric protein solution comprising a buffer liquid and a tetrameric protein dissolved or dispersed in the buffer liquid.

11. The nucleic acid lateral flow immunoassay of claim 8, wherein the first capture antibody is an anti-streptavidin antibody, and the second capture antibody is an anti-BSA antibody.

12. The nucleic acid lateral flow immunoassay of claim 10, wherein the tetrameric protein is a streptavidin (SA), and the buffer liquid is a phosphate buffer solution.

13. The nucleic acid lateral flow immunoassay of claim 8, wherein the lateral flow strip further comprises: a supporting substrate, wherein the membrane substrate is disposed on the supporting substrate; and an absorption pad, being formed on the supporting substrate, and being located at a rear-end side of the supporting substrate.

14. The nucleic acid lateral flow immunoassay of claim 13, wherein the membrane substrate is made of a material that is selected from a group consisting of nitrocellulose (NC), polyvinylidene difluoride (PVDF) and nylon.

15. The nucleic acid lateral flow immunoassay of claim 8, wherein the combiner is a biotin, and the labeling is made of a fluorescent material that is selected from a group consisting of gold nanoparticles, silver nanoparticles, carbon nanoparticles, quantum dots (QDs), colloidal gold, colloidal silver, and colloidal QDs.

16. The nucleic acid lateral flow immunoassay of claim 8, wherein the nucleotide sequence is selected from a group consisting of 5′-GAAAGGAAGGTAAGTTGTTAAG-3′ and 5′-TATGTATGAATAGAGTCTTAGGT-3′.

17. The nucleic acid lateral flow immunoassay of claim 16, wherein the terminal base of the first primer connected with the combiner is a front-terminal base or a rear-terminal base, the terminal base of the second primer connected with the labeling is a front-terminal base or a rear-terminal base.

18. The nucleic acid lateral flow immunoassay of claim 16, wherein the capture probe further comprises a spacer that is connected between the terminal base and the combiner, and the spacer comprising adenines.

19. The nucleic acid lateral flow immunoassay of claim 16, wherein the detection probe further comprises a spacer connected to the terminal base and a thiol group connected between the spacer and the labeling, and the spacer comprising adenines.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The invention as well as a preferred mode of use and advantages thereof will be best understood by referring to the following detailed description of an illustrative embodiment in conjunction with the accompanying drawings, wherein:

[0043] FIG. 1 shows a schematic diagram for describing a nucleic acid probe set according to the present invention;

[0044] FIG. 2 shows a first schematic stereo diagram of a lateral flow strip of a nucleic acid lateral flow immunoassay according to the present invention;

[0045] FIG. 3A to FIG. 3E show schematic diagrams for describing a flow of adopting the nucleic acid lateral flow immunoassay to carry out a BK virus detection;

[0046] FIG. 4 shows a second schematic stereo diagram of the lateral flow strip;

[0047] FIG. 5 shows nine real images of the lateral flow strip;

[0048] FIG. 6 shows a bar chart of target DNA levels (nM) versus T/C ratio values by relative intensity;

[0049] FIG. 7 shows four real images of the lateral flow strip;

[0050] FIG. 8 shows a bar chart of different virus versus T/C ratio values by relative intensity;

[0051] FIG. 9 shows six real images of the lateral flow strip; and

[0052] FIG. 10 shows a bar chart of different virus versus T/C ratio values by relative intensity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] To more clearly describe a nucleic acid probe set and a nucleic acid lateral flow immunoassay comprising the nucleic acid probe set according to the present invention, embodiments of the present invention will be described in detail with reference to the attached drawings hereinafter.

Nucleic Acid Probe Set

[0054] With reference to FIG. 1, there is shown a schematic diagram for describing a nucleic acid probe set according to the present invention. As FIG. 1 shows, the nucleic acid probe set 1 comprises a capture probe 11 and a detection probe 12. The capture probe 11 comprises a first primer 111 that comprises at least 19 bases and a combiner 112 connected to one terminal base of the first primer 111, and the detection probe 12 comprises a second primer 121 that comprises at least 19 bases and a labeling 122 connected to one terminal base of the second primer 121. According to the present invention, the first primer 121 and the second primer 121 are both a nucleotide sequence that is extracted from a conserved region of a genome sequence belong to a BK virus. Moreover, in a specific embodiment, melting temperature (T.sub.m) of the second primer 121 is greater than that of the first primer 111.

[0055] As explained in more detail below, the combiner 112 is a biotin that is used to combine with a streptavidin (SA), and the labeling 122 is made of a fluorescent material that is selected from a group consisting of gold nanoparticles, silver nanoparticles, carbon nanoparticles, quantum dots (QDs), colloidal gold, colloidal silver, and colloidal QDs.

Manufacture of the Nucleic Acid Probe Set

[0056] In one exemplary embodiment, method for extracting the aforesaid nucleotide sequence from a conserved region of a BK virus's genome sequence comprises following operation steps: [0057] (a) collecting 232 genome sequences (i.e., a complete nucleotide sequence) of BKV, 464 genome sequences of JCV and 34 genome sequences of SV40 from DNA database of National Center for Biotechnology Information (NCBI); [0058] (b) using DNAS TAR Lasergene 8.0 to carry out a multiple sequence alignment between the 730 genome sequences, thereby finding out a consensus sequence; [0059] (c) applying a conserved region identifying process to the 232 genome sequences of BKV, thereby finding out four conserved regions, including 987-1100 (114 bp), 1102-1210 (109 bp), 1809-1876 (68 bp), and 3306-3389 (84 bp); [0060] (d) using Vector NTI Advance 11.0 to complete a primer design based the four conserved regions, therefore obtaining eight nucleotide sequences that are listed in following Table (1); and [0061] (f) synthesizing a plurality of capture probes 11 and a plurality of detection probes 12 by using the obtained eight nucleotide sequences.

TABLE-US-00001 TABLE (1) Sequence BK4 5′-TCACATAGACTCCGAAGAC-3′ p0001 BK4 5′-CCGACTTTAACGACGACCC-3′ p0002 BK5 5′-CGCATTGAGGAGTTTGTATA-3′ p001 BK5 5′-CGACATTAACGACCACGAG-3′ p002 BK7 5′-TCTTTTTTGATAACGGGGTC-3′ p01 BK7 5′-ATTAGTTTCTTGACGAGGAG-3′ p02 BK12 5′-TATGTATGAATAGAGTCTTAGGT-3′ P60011 BK12 5′-GAAAGGAAGGTAAGTTGTTAAG-3′ P60012

[0062] It needs to further explain that, the first primer 111 of the capture probe 11 can be any one of the nucleotide sequences listed in Table (1). Therefore, it is able to know that, the first primer 111 includes at least 19 bases. According to the present invention, the capture probe 11 further comprises a spacer that is connected between the terminal base and the combiner 112, wherein the forgoing terminal base is a front-terminal base or a rear-terminal base of the nucleotide sequence. Moreover, the spacer comprising 10 adenines, and has a notation of A10. On the other hand, the second primer 121 of the detection probe 12 can be any one of the nucleotide sequences listed in Table (1). Therefore, it is able to know that, the second primer 121 also includes at least 19 bases. According to the present invention, melting temperature (T.sub.m) of the second primer 121 is greater than that of the first primer 111. In addition, the detection probe 12 further comprises a spacer (i.e., A10) connected to the terminal base and a thiol group connected between the spacer and the labeling 122.

[0063] After specificity tests of the synthesized capture probes 11 and the synthesized detection probes 12 are all completed, test result reveals that, the capture probe 11 containing the first primer 111 including nucleotide sequence of P60011 or P60012 exhibits outstanding specificity. Similarly, the detection probe 12 containing the second primer 121 including nucleotide sequence of P60011 or P60012 also shows outstanding specificity. As a result, there are two kinds of capture probes 11 and two kinds of detection probes 12 synthesized and listed in following Table (2).

TABLE-US-00002 TABLE (2) Sequence (5′.fwdarw.3′) detection HS-A10-TATGTATGAATAGAGTCTTAGGT probe detection TATGTATGAATAGAGTCTTAGGT-A10-SH probe capture Biotin-A10-GAAAGGAAGGTAAGTTGTTAAG probe capture GAAAGGAAGGTAAGTTGTTAAG-A10-Biotin probe

[0064] Therefore, it is understood that, “A10” represents the spacer comprising 10 adenines, “HS” represents the thiol group, and “Biotin is the combiner 112 that is used to combine with a streptavidin (SA). Herein, it needs to particularly explain that, the labeling 122 is not shown in the two kinds of detection probes 12. In one exemplary embodiment, the labeling 122 can be made of gold nanoparticles having a particle size in a range between 25 nm and 65 nm.

Nucleic Acid Lateral Flow Immunoassay

[0065] According to the present invention, a nucleic acid lateral flow immunoassay comprising a nucleic acid probe set 1 as shown in FIG. 1 and a lateral flow strip is also disclosed. FIG. 2 shows a first schematic stereo diagram of the lateral flow strip of the nucleic acid lateral flow immunoassay according to the present invention. In one exemplary embodiment, as FIG. 2 shows, the lateral flow strip 2 comprises a supporting substrate 20, a membrane substrate 21 and an absorption pad 23. In which, the membrane substrate 21 can made of nitrocellulos (NC), polyvinylidene difluoride (PVDF) or nylon, and there are a test line 21T and a control line 21C formed on the membrane substrate 21. The test line 21T is formed by spraying a first solution containing a first capture antibody onto the membrane substrate 21, and the control line 21C is formed by spraying a second solution containing a second capture antibody onto the membrane substrate 21. In one embodiment, the first solution is made by dissolving or dispersing an anti-streptavidin antibody (i.e., the first capture antibody) in a PBS buffer, and the second solution is made by dissolving or dispersing an anti-BSA antibody (i.e., the second capture antibody) in a PBS buffer.

[0066] Adopting the nucleic acid lateral flow immunoassay to carry out a BK virus detection

[0067] FIG. 3A to FIG. 3E show schematic diagrams for describing a flow of adopting the nucleic acid lateral flow immunoassay to carry out a BK virus detection. A flow of using the nucleic acid lateral flow immunoassay to carry out a BK virus detection comprising following operation steps:

[0068] (1) As FIG. 3A shows, a sample containing BKV DNA is heated under a process temperature of 95° C. for 2 minutes, thereby separating BKV'S double-stranded DNA into two single-stranded DNAs;

[0069] (2) As FIG. 3B shows, the capture probe 11 is mixed into the sample, so as to make the first primer 111 be connected to the single-stranded DNA;

[0070] (3) As FIG. 3C shows, the detection 12 is mixed into the sample, thereby making the second primer 121 be connected to the same single-stranded DNA;

[0071] (4) As FIG. 3D shows, producing a tetrameric protein solution by dissolving or dispersing a streptavidin (i.e., tetrameric protein) in a PBS buffer, and then adding the tetrameric protein solution into the sample for making the combiner 112 of the capture probe 11 combine with the streptavidin 13, thereby obtaining a test sample; and

[0072] (5) As FIG. 3E shows, disposing lateral flow strip 2 into the test solution 14, and then using an optical reader to read out a T/C ratio value from a first colored line showing up in the test line 21T and a second colored line showing up in the control line 21C.

[0073] FIG. 4 shows a second schematic stereo diagram of the lateral flow strip. As FIG. 4 shows, the absorption pad 23 provides a driving force for making the test sample laterally move toward the test line 21T. When the test sample reaches the test line 21T, hybrid complex constituted by the capture probe 11, the detection probe 12 and the single-stranded DNA is fixed at the test line 21T because the biotin (i.e., the combiner 112) is captured by the anti—streptavidin antibody (i.e., the first capture antibody). Moreover, after there are more and more hybrid complex accumulated on the test line 21T, a first colored line (red band) shows up in the region of the test line 21T. Furthermore, the test sample continuously moves toward the control line 21C. When the test sample reaches the control line 21C, hybrid complex is fixed at the control line 21C because the detection probe 12 is captured by the anti-BSA antibody (i.e., the second capture antibody). Moreover, after there are more and more hybrid complex accumulated on the control line 21C, a second colored line (red band) shows up in the region of the control line 21C.

Sensitivity of BKV Detection of the Nucleic Acid Lateral Flow Immunoassay

[0074] FIG. 5 shows nine real images of the lateral flow strip. According to the nine real images, it is known that, in case of the BKV DNA level contained in the test sample reaching 50 nM, the red band showing up in the test line 21T and the red band showing up in the control line 21C are both recognizable by human eyes. Of course, in case of the BKV DNA level being below 50 nM, an optical reader (e.g., a smartphone installed with a lateral flow assay reader App) can be used to read out a T/C ratio value from the test line 21T and the control line 21C. FIG. 6 shows a statistical bar chart of target (i.e., BKV) DNA levels (nM) versus T/C ratio values (labeled by relative intensity) that are measured by using the optical reader. Therefore, according to the data of FIG. 6, it is understood that the nucleic acid lateral flow immunoassay of the present invention exhibits a linear range of detection for BKV, and the linear range of detection is in a range between 5 nM and 500 nM.

Specificity of BKV Detection of the Nucleic Acid Lateral Flow Immunoassay

[0075] FIG. 7 shows four real images of the lateral flow strip, and FIG. 8 shows a statistical bar chart of different virus (BKV, JCV, and SV40) versus T/C ratio values (labeled by relative intensity) that are measured by using the optical reader. From experimental data of FIG. 7 and FIG. 8, it is clear that the nucleic acid lateral flow immunoassay shows specificity on BKV detection. Therefore, experimental data have proved that, the nucleic acid lateral flow immunoassay of the present invention can be adopted for conducting BK virus detection on a sample that is collected from environmental water, sewage water, drinking water, urine, or serum.

Adopting the Nucleic Acid Lateral Flow Immunoassay to Detect Plasmid DNA of BK Virus

[0076] FIG. 9 shows six real images of the lateral flow strip. According to the six real images, it is known that, in case of the plasmid DNA level contained in the test sample reaching 10.sup.8 copies/mL, the red band showing up in the test line 21T and the red band showing up in the control line 21C are both recognizable by human eyes. Of course, in case of the plasmid DNA being below 10.sup.8 copies/mL, an optical reader (e.g., a smartphone installed with a lateral flow assay reader App) can be used to read out a T/C ratio value from the test line 21T and the control line 21C. FIG. 10 shows a statistical bar chart of different virus versus T/C ratio values (labeled by relative intensity) that are measured by using the optical reader. Therefore, according to the data of FIG. 9 and FIG. 10, it is understood that the nucleic acid lateral flow immunoassay of the present invention exhibits a detection range of plasmid DNA of BK virus, and the detection range is in a range between 10.sup.7 copies/mL and 10.sup.10 copies/mL. As a result, experimental data have proved that the nucleic acid lateral flow immunoassay of the present invention shows a clinical significance on BK virus detection.

[0077] Nowadays, BK virus is found to exist in natural water and home sewage water. For this reason, BK virus has become one of a pathogenic indicator of sewage water and natural water defined in the United Nations World Water Development Report (UN WWDR). It is worth mentioning that, experimental data have proved that, the nucleic acid lateral flow immunoassay can be adopted for conducting a BK virus detection on a sample that is collected from environmental water, sewage water, drinking water, urine, or serum.

[0078] Moreover, when adopting the nucleic acid lateral flow immunoassay of the present invention to carry out a BKV detection, a sample for the BKV detection is not needed to be applied with a PCR process and/or a RPA process. Moreover, there is no RNA transcription process conducted during the operation of the BKV detection. Therefore, the nucleic acid lateral flow immunoassay disclosed by the present invention including advantages of low cost, achieving rapid BKV detection, and able to be conducted by common people, able to be used in BKV detection without needing using any professional machine.

[0079] Therefore, through above descriptions, all embodiments and their constituting elements of the nucleic acid probe set and the nucleic acid lateral flow immunoassay using the same according to the present invention have been introduced completely and clearly. The above description is made on embodiments of the present invention. However, the embodiments are not intended to limit scope of the present invention, and all equivalent implementations or alterations within the spirit of the present invention still fall within the scope of the present invention.