EP2291541A1

EP2291541A1 - Multiplexed pcr-coupled pna labeled beads flow cytometric assay for simultaneous detection of multiple biological agents

Info

Publication number: EP2291541A1
Application number: EP09716939A
Authority: EP
Inventors: Wan Wen Su; Jeffry Golden
Original assignee: Clean Earth Technologies LLC
Current assignee: Clean Earth Technologies LLC
Priority date: 2008-03-05
Filing date: 2009-03-05
Publication date: 2011-03-09
Also published as: WO2009111072A1; EP2291541A4

Abstract

A method for detection of multiple biological entities, such as biological warfare and terrorism agents, and public health threat pathogens, is accomplished by multiplexing PCR to amplify different DNA targets and to produce dsDNA products, then mixing the multiple PCR dsDNA products with beads of specific sizes that are labeled with specific complementary PNA labels and fluorescent staining dye so that the PNA-labeled beads bind with specific dsDNA by complementary sequences and fluorescent dsDNA dye combines with dsDNA, and then performing either flow cytometry to count beads by size and determine the intensity of fluorescence by size or performing size separation of two or more bead size fractions and detection by measuring the fluorescent signals of two or more bead size fractions.

Description

Multiplexed PCR-Coupled PNA Labeled Beads Flow Cytometric Assay for Simultaneous Detection of Multiple Biological Agents

FIELD OF THE INVENTION

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional

Application No. 61/033952 filed on March 5, 2008 and the disclosure is incorporated herein by reference.

[0002] This invention relates to a series of DNA amplification and hybridization for the detection of multiple biological entities.

BACKGROUND OF THE INVENTION

[0003] The advent of bioterrorism, large scale pathogenic contamination of food supply commodities leading to significant outbreaks of food- borne illness, and emerging public health threats has highlighted the need for rapid, simple and robust diagnostic assays to detect biological entities, which may include biological warfare or terrorism agents, pathogens that cause pandemic illness, and microbial contamination of food, water, and other commodity items. Mortality from biological agents or pathogens that cause outbreaks of disease may be greatly reduced by prompt treatment if the specific biological entity (agent) can be identified early. Rapid and multiplex methods are useful to identify agents of concern for prompt treatment in the event of an unknown biothreat agent release or microbial threat to public health. Both nucleic acid-based and immunological methods have been used to simultaneously detect multiple biological agents with different degrees of sensitivity in addition to physical or physicochemical methods. However, immunological methods generally lack sensitivity and require approximately 10⁴ to 10⁵ organisms to be detected.

[0004] Currently used nucleic acid technologies include multiplexed real-time Polymerase Chain Reaction (PCR) using molecular beacons or TaqMan® assay [Wittwer, 2001] and multiplexed PCR coupled with microarray [Nosarabadi, US 7,083951] aiming at many targets in a single reaction. Normal multiplexed PCR has high sensitivity. This varies from 20 - 2000 organisms for each amplicon type. The limitations in these methods are as follows. The multiplex real-time PCR assay has a 5-dye limit because of current maximum instrumentation capacity. It can simultaneously detect only five deoxyribonucleic acid (DNA) markers. Further, microarrays are time consuming, require denaturing amplified DNA to hybridize to the probes, and require expensive dye-labeled primers. In addition, the probe oligonucleotides have to compete with the template complementary single-strand DNA (ssDNA) to bind dye-labeled ssDNA fragments, thus decreasing the overall assay efficiency. Recently, Lawrence Livermore National Laboratory (LLNL) developed a multiplexed PCR-coupled liquid bead array for the simultaneous detection of four biothreat agents [Wilson, 2005] for rapid screening of environmental samples. The liquid array employs beads embedded with different ratios of red and infrared fluorescent dyes and each PCR product sequence to be detected is assigned one set of uniquely dyed beads. The beads are conjugated with their assigned probe, a reverse complementary internal PCR product sequence. The biotin-labeled PCR products from a multiplexed PCR assay are allowed to hybridize to the bead set and are then labeled with a second reporter dye complex, Streptavidin-phycoerythrin (SAPE), for detection using an automated flow cytometer. This method is better, as it is less time-consuming and less costly.

[0005] Multiplexed PCR for simultaneous amplification of multiple targets has been applied in many areas of DNA testing since 1988. The crucial part is post-PCR analysis of amplicons. Amplification of multiple DNA targets can be achieved with carefully designed primers and reaction conditions. Some of the methods for post-PCR analysis are: 1) Gel electrophoresis [Pusey, US 7,291,459]; 2) Real-time PCR with molecular beacon or TaqMan® assay [Wittwer, 2001]; 3) Microarray [Nosarabadi, US 7,083,951], and 4) Flow cytometry. Separation of amplicons by gel electrophoresis requires the PCR products of different lengths and in certain size ranges, which may be a limiting constraint in addition to the fact that gel electrophoresis analysis of amplicons is time consuming. Amplification of multiple targets in a single reaction being the objective of multiplexed PCR, the amplification products should have a large range of DNA fragment sizes that will make a specific PCR reaction difficult. For real-time PCR with molecular beacon or TaqMan® assay, there is a 5-dye limit for maximum instrumentation capacity. Analysis by microarray requires expensive dye-labeled primers and a large amount of probes to compete and bind ssDNA targets with the original complementary ssDNA. Flow cytometry, a common method, uses specific oligonucleotides covalently bonded to beads and labeled primers to amplify DNA. For conventional cytometry methods, DNA must be denatured and hybridized with coated-beads. It is also expensive and time-consuming. [0006] An alternative approach, not found in the prior art, for multiplexed flow cytometric assay exploits peptide nucleic acid (PNA), which is a nucleic acid analog containing a neutral, chiral backbone of repeating N-(2- aminoethyl) glycine units linked by amide bonds, with purine and pyrimidine bases attached by methylene carbonyl linkages. Common practice is to denature double- strand DNA (dsDNA) resulting from PCR, but contrary to common practice, use of dsDNA provides an opportunity for exploitation with PNA complement labeling for the simulataneous detection of organism specific DNA that is associated with target organisms in a sample with multiple organisms possibly present. The PNA peptide backbone is more flexible than the natural ribose-phosphate backbone present in DNA. PNA is more stable and resistant to degradation by nucleases and proteinases. It not only binds to complementary ssDNA, but it also binds specifically to dsDNA. This provides unique characteristics to detect complementary dsDNA.

[0007] Flow cytometry is an analytical method that allows both the rapid measurement of scattered light for particle size determination and measurement of fluorescence emission produced by suitably illuminated particles. The particles are suspended in liquid and produce signals when they pass through a beam of light individually. Because measurements of each particle are made separately, the results are a correlated set of each individual particle's characteristics. An important analytical feature of flow cytometry is its ability to measure multiple particle parameters such as scattered light and fluorescence emission. Scattered light collected in the same direction as the incident light reflects cell size where as the fluorescence is dependent upon the presence of fluorochromes on particles. Thus, a combination of light scattering and fluorescence is a powerful approach to detect multiple targets in one sample without the need for a separation step.

[0008] In the commonly used detection scheme, a probe that attaches to ssDNA is used to link to a fluorescent dye, e.g., a biotin-based probe with cy5 or cy3 dye, or PCR primer is used to link to cy5 dye. In contrast, dsDNA can directly bind to a fluorescent dye.

SUMMARY OF THE INVENTION

[0009] The invention is a method for detection of multiple biological agents such as bacteria, viruses, spores, molds, and mycoplasma. The method comprises the three steps of (1) multiplexing PCR to amplify different DNA targets and to produce dsDNA products, (2) mixing the multiple PCR dsDNA products with beads of various sizes wherein each size bead has a specific complementary PNA label and a fluorescent staining dye, and whereby the mixing causes the PNA-labeled beads to bind specific dsDNA by complementary sequences, and the fluorescent dsDNA dye to combine with dsDNA, and (3) flow cytometry is then performed to count the number of beads of each size and to measure the intensity of fluorescence of the beads of each size. In an alternative embodiment of the instant invention, in the third step, instead of performing flow cytometric detection, size separation of the labeled beads with bound dsDNA is performed and then the fluorescent signal of each size-separated fraction is measured.

BRIEF DESCRIPTION OF THE DRAWINGS [00010] The instant invention will become more fully understood from the detailed description and the accompanying drawings, wherein:

[00011] Fig. 1. is a schematic diagram showing the three steps of the method of the instant invention.

DETAILED DESCRIPTION OF THE INVENTION

[00012] The present invention comprises a method for detection and analysis of multiple biological agents. Figure 1 shows the three steps of the multiplexed PCR-coupled PNA-labeled-beads flow cytometric assay.

[00013] In the first step, multiplexed PCR using oligonucleotide primers is preformed to obtain amplification of different DNA targets and to produce dsDNA products. In the second step, the multiple PCR dsDNA products are mixed with the PNA labeled beads and a single fluorescent staining dye, wherein beads of various sized have been prepared with various PNA labels so that a specific PNA label is applied to all beads of a specific size, and the beads with a specific PNA-label will bind to specific dsDNA by complementary sequences, and the fluorescent dsDNA dye will combine with dsDNA. In the third step, flow cytometry is performed to count and record the size and number of beads of each size and to measure and record the fluorescence intensity of the beads of a given size. Although PNA can bind ssRNA that may be present in the PCR sample, it will be in a very small amount compared to the concentration of dsDNA amplicons. The method offers high specificity and sensitivity, and it provides reductions in time and cost. Table 1 presents a comparison of differences and advantages between the instant invention and the LLNL method. Table 1. Com arison of the instant invention and the LLNL method.

Multiplex PCR:

[00014] For the method of the instant invention, the length of multiplex

PCR products can be from 80 bp to 1000 bp. In a preferred embodiment, the best range is 100 bp to 150 bp, which is very suitable for efficient capture by PNA-beads and is conducive to the generation of ample fluorescent signal.

PNA labeled Beads:

[00015] For this method, it is important that the PNA efficiently capture specific PCR product according to complementary DNA sequences. The efficiency of capturing dsDNA by PNA depends on the length of PNA. The length of PNA can. range from 15 bp to 100 bp. In a preferred embodiment, the range is 18 bp to 25 bp, which is sufficient to capture complementary ds DNA fragments.

[00016] The beads may be made of any of a variety of materials. There are many types of suitable, commercially available beads. Exemplary materials are plastic, glass, silica gel, silica, latex, and ceramic. In a preferred embodiment, the beads are made of polystyrene, polycarbonate, silica gel, latex or glass. The beads may be modified by the addition of a carboxyl group to the bead material. The beads may also be coated with a coating having desired chemical or physical properties such as anti-agglomeration, hydrophobicity, hydrophillicity, hygroscopicity, opacity, reflectance, and color. The size of the beads, i.e., the diameters of the various size beads, can be the range from 0.1 μm to 100 μm. In a preferred embodiment, the range is 1 μm to 30 μm. For preparation of the bead population prior to labeling for use in the method of the instant invention, nearly monodisperse bead size fractions can be obtained by separation techniques using graded screen sieves or by flow cytometric separation methods.

[00017] The population of beads has a distribution of sizes. Ideally, this distribution is partitioned into set of discrete size ranges (size "bins") so that the beads can be sorted by size and in the flow cytometric determination of size, beads can be accurately identified as being within a specific size "bin". Flow cytometers have size resolution that typically is < 0.5 μm, so discrimination of bead bins with width greater than 1 μm is readily achieved. Further, if the bead sizes are selected so that the bins are separated by a gap of about 0.25 to 1 μm according to the resolution of the instrument, then identification of bead size is nearly error free. The distribution function of bead size may be relatively "flat", i.e., a constant as a function of bead size, or it may be selected so that smaller or larger beads are present in greater number to improve detection or to adjust the selectivity of the method in the case where some agents or their multiplex PCR product may be more or less abundant. If it is anticipated or suspected that an agent and its corresponding multiplex PCR product are in relatively small abundance, i.e., comprise a rare class, then weighting the bead size distribution to have a greater number of smaller beads can yield a disproportionately larger signal for the rare class. Similarly, if an agent class is expected to be greatly abundant, then fewer beads associated with the abundant class so that its signal does not overwhelm the less abundant or rare classes.

[00018] For labeling the beads, an NH3 group of PNA can be covalently linked (bonded) with a COOH group of the bead material or coating. This link is very strong and will remain unbroken during the performance of the assay. dsDNA dye:

[00019] Fluorescent DNA dyes are essential for flow cytometric detection and measurements. There are many dsDNA dyes used for flow cytometry and other detection methods [e.g., see Glazer, US 5,312,921]. Useful dsDNA dyes include SYBR green 1, ethidium bromide, thiazole orange (TO) and its derivatives, and propidium iodide (PI) [see Cosa, et al. and also, Fei, et al., and also, Nygren et al.]. Many of these are commercially available. Preferred dsDNA dyes are SYBR green I, ethidium bromide, thiazole orange (TO), and propidium iodide (PI) which can bind dsDNA and generate a large signal. Flow Cytometric Detection: [00020] For this method, flow cytometric detection comprises the determination of bead size, the counting of beads by size, and the measurement of fluorescent intensity by size of the beads. Each size of bead represents a specific targeted sequence that is associated with a corresponding biological agent because beads of a specific size were coated by the specific PNA, which specifically hybridizes with the specific PCR product generated in the multiplex PCR step. For improved signal-to-noise, thresholds may be set. If the fluorescent intensity for an individual bead is greater than a threshold value, then, this bead represents a positive count for the detection of the specific agent. Thresholds can be set for upper and lower limits of bead size for each size "bin", so that discrimination of bead size is improved and incorrect assignment of a bead to a size "bin" can be minimized or avoided. Accumulation of the statistics of counting and fluorescent intensity for each bead size provides detection and can be used for the determination of the relative abundance of the detected agents. Size Separation and Fluorescent Measurement Detection

[00021] In an alternative embodiment of the instant invention, in the third step, instead of performing flow cytometric detection, size separation of the labeled beads with bound dsDNA is performed and then the fluorescent signal of each size-separated fraction is measured. Physical size separation is readily accomplished by a set of sized or graded screens or sieves so that each successive screen captures a smaller size fraction than the preceding screen. Backwashing or other commonly used means can be used to remove the captured size fraction prior to measurement of the fluorescent signal of that size fraction, or the fluorescent signal may be measured in place if the captured beads comprise an approximately single bead thick layer on the screen. In flow cytometry, signals from individual beads can be measured. In the method using physical size separation instead of flow cytometry, resolution of individual beads is more difficult, but measurement of the collective signal of a given size fraction of the bead distribution can be made more easily, more quickly, and without expensive flow cytometric equipment. Moreover, the size separation version of the method can be made for high throughput, which is in contrast to flow cytometry. Thus, size separation and fluorescent measurement detection offers a more economical means of detection than flow cytometry. Detection with two or more dyes

[00022] Using multiple dsDNA dyes with specific dyes assigned to specific size fractions enables improved discrimination and simultaneous monitoring. Two or more aliquots (the initial set of aliquots) of the multiplexed PCR dsDNA products can be mixed with a selected single size fraction or multiple selected size fractions of the bead size distribution for which specific PNA labels have been applied to selected size fractions. Then, selected dye can be applied to a given aliquot, and then, the size-selected PNA labeled beads with bound dsDNA can be separated from the unbound PCR dsDNA products. The selected dyes are chosen so that their emission wavelength or excitation wavelength differ and are sufficiently separated from each other so that detection at two or more emission wavelengths can be performed simultaneously or detection at a given wavelength with two different and sequentially pulsed excitation wavelengths is performed either when flow cytometric detection or size separation/fluorescent detection is performed on the separated beads or on a mixture of the previously separated beads of the initial the set of aliquots. An example of two suitable dyes are SYBR green I, which, typically, is excited by light with wavelength of approximately 450-520 nm (peak ~ 488 nm) and emits with wavelengths in the vicinity of 490-640 nm (peak ~ 522 nm), and ethidium bromide, which, typically, is excited by ultraviolet light (280-330 nm, peak ~ 300 nm) and emits orange light (560-720 nm, peak ~ 600). So, using two detectors with spectral filters, e.g., one with cutoff below 640 nm and one with cutoff above 550 nm, simultaneous detection of fluorescence of the ethidium bromide and the SYBR green I can be performed. The use of two or more dyes in this manner enables the use of wavelength discrimination as an additional detection parameter. This can be used to improve the signal-to- noise ratio of the fluorescence detection, and it can be used to reduce false positive detection results.

Claims

WHAT IS CLAIMED:

1. A method for detection of multiple biological entities such as bacteria, viruses, spores, molds, and mycoplasma, the method comprising the step of performing multiplexing PCR to amplify different DNA targets and to produce dsDNA products; and the following steps, mixing the multiple PCR dsDNA products with beads of various sizes wherein each bead of a specific size or being in a specific size range has a specific complementary PNA label and a fluorescent staining dye, and by the said mixing causes the PNA-Iabeled beads to bind specific dsDNA by complementary sequences and the fluorescent dsDNA dye to combine with dsDNA, and the additional step of performing flow cytometry whereby the number of beads of each size is counted and to the intensity of fluorescence of the beads of each size is determined.

2. The method of claim 1 wherein said DNA dye comprises either SYBR green I or ethidium bromide; said flow cytometric detection includes setting a threshold for fluorescent intensity and upper and lower thresholds for each bead size bin.

3. The method of claim 1 wherein said the length of PCR products is range from 80 bp to 1000 bp; said beads comprise material principally consisting of one of the following polystyrene, polycarbonate, latex, silica gel, silica, ceramic, and glass; said beads is in the range from 0.1 μm to 100 μm; said the length of PNA is in the range from 15 bp to 100 bp; and said bead labeling has a covalent bond link.

4. The method of claim 1 wherein said the length of PCR products is in the range from 100 bp to 150 bp; said beads comprises material selected from the following: polystyrene, latex and glass; said beads is in the range from 1 μm to 30 μm; said the length of PNA is in the range from 18 bp to 25 bp; and said beads labeling has a covalent bond link.

5. The method of claim 1 wherein said beads are coated with a material having a functional group for bonding to a PNA label.

6. The method of claim 1 wherein said beads are coated with a material to obtain a specific chemical or physical property.

7. The method of claim 1 wherein the biological entities are biological warfare or terrorism agents or pathogenic threats to public health.

8. The method of claim 1 wherein the distribution of bead sizes is selected so that a greater number of beads of a given size are provided with a PNA label that is specific to PCR dsDNA product associated with a less likely biological entity.

9. The method of claim 1 wherein the PCR dsDNA products are divided into two or more aliquots that are then each subsequently mixed with selected size fractions of PNA-labeled beads and different selected dye is applied and mixed with the different aliquots so that wavelength discrimination can be used in the detection step of the method.

10. The method of claim 9 wherein two selected dyes are SYBR green 1 and ethidium bromide.

1 1. A method for detection of multiple biological entities such as bacteria, viruses, spores, and mycoplasma, the method comprising the step of performing multiplexing PCR to amplify different DNA targets and to produce dsDNA products; and the following steps, mixing the multiple PCR dsDNA products with beads of various sizes wherein each bead of a specific size or being in a specific size range has a specific complementary PNA label and a fluorescent staining dye, and by the said mixing causes the PNA-labeled beads to bind specific dsDNA by complementary sequences and the fluorescent dsDNA dye to combine with dsDNA, and the additional step of performing size separation of the PNA-labeled beads with bound specific dsDNA and dye to obtain two or more desired size fractions of the bead size distribution, and detection by the measurement of fluorescence of two or more size fractions of said PNA-labeled beads with bound specific dsDNA and dye.

12. The method of claim 11 wherein the distribution of bead sizes is selected so that a greater number of beads of a given size are provided with a PNA label that is specific to PCR dsDNA product associated with a less likely biological entity.

13. The method of claim 1 1 wherein the PCR dsDNA products are divided into two or more aliquots that are then each subsequently mixed with selected size fractions of PNA-labeled beads and different selected dye is applied and mixed with the different aliquots so that wavelength discrimination can be used in the detection step of the method.

14. The method of claim 13 wherein two selected dyes are SYBR green 1 and ethidium bromide.