WO2020174495A1

WO2020174495A1 - Endpoint fluorescence detection system for amplified nucleic acid

Info

Publication number: WO2020174495A1
Application number: PCT/IN2020/050177
Authority: WO
Inventors: V Suri; Aseem SURI; Yogesh Patil; Anant SURI
Original assignee: V Suri
Priority date: 2019-02-27
Filing date: 2020-02-27
Publication date: 2020-09-03

Abstract

Disclosed is an that invention relates to a portable system and method for endpoint fluorescence detection of amplified nucleic acid that can be coupled with conventional thermal cyclers (PCR) for amplification of nucleic acids wherein the system and method is low-cost, robust, hand-held, portable, battery operated and easy-to-use; enabling rapid and accurate detection of targeted amplified nucleic acids. The system is especially useful for diagnosis of infectious diseases at a rural setup.

Description

ENDPOINT FLUORESCENCE DETECTION SYSTEM FOR AMPLIFIED NUCLEIC ACID

Field of the Invention

The present invention relates to a portable system and method for endpoint fluorescence detection of amplified nucleic acid that can be coupled with conventional thermal cyclers (PCR) for amplification of nucleic acids and more particularly to a portable endpoint fluorescence detection of amplified nucleic acid that can be operated at rural setup for diagnosis of infectious diseases.

Background of the Invention

Rapid detection of any biological agents such as pathogens, is important for accurate diagnosis of infectious diseases, particularly in remote areas where access to clinical laboratories is not available. Molecular biology based methods play an important role in early diagnosis of infectious diseases in less time that enables an early therapeutic intervention and strongly supports an efficient and successful patient recovery. Polymerase chain reaction (PCR) is one of the major common tools used to identify and diagnose the presence or absence of biological agents, because it is highly specific and accurate tool as compared to other techniques. Conventionally PCR-amplified nucleotide sequences are detected by expensive and labor intensive techniques, such as Gel electrophoresis, Real time PCR machines like the recently developed GeneXpert Systems. Further these techniques require expensive charge coupled cameras and detectors that add to the cost of instrumentation and cost of the test per sample. Other known conventional methods for detection of fluorescent labelled DNA are well established and have their own advantages and limitations.

One such method for detection of targeted DNA molecules is the GeneXpert assay, a real-time PCR based assay. Such instrumentation is not easily deployable in remote areas or rural settings. Real-time PCR system is one of the most commonly utilized assays for detection of infectious diseases. This known system mainly amplifies extracted nucleic acids and detect them simultaneously. These systems also require special operating conditions and also heavy power supplies, which limits their use at only tertiary healthcare centers. Also these known systems utilize various optoelectronic components for detection of nucleic acids on real time basis which adds on the overall cost of the equipment and make them bulky as well. The GeneXpert assay utilizes integrated mechanisms of extraction of nucleic acids, amplification and detection of targeted nucleic acids. This system is expensive and has limited applications for rural areas due to its high cost and requirement of power backup and special environmental conditions. The machine is recommended by WHO for diagnosis of well-known diseases such as TB and HIV that are difficult to diagnose by conventional microbiological methods.

The machine utilizes various pneumatic and mechanical systems for automation of nucleic acid extraction, amplification and uses expensive detection systems for detection of amplified nucleic acids which leads to increase overall cost of the system. On the other hand conventional PCR methods uses Gel Electrophoresis technique for endpoint detection of amplified nucleic acids which is time consuming, labour intensive, and requires detection technologies viz. Gel documentation systems which are very expensive and bulky.

Thus, there is a need for low-cost, robust, portable, battery operated, easy-to-use method and system enabling rapid and accurate endpoint detection of PCR-amplified nucleic acids that can be coupled with conventional thermal cyclers for diagnosis of infectious diseases at a rural setup.

Summary of the invention

In a general aspect, the present invention relates to a portable system for detection of amplified nucleic acid. In a preferred embodiment, the present invention relates to a portable system for endpoint fluorescence detection of polymerase chain reaction (PCR) amplified nucleic acid that can be coupled with conventional thermal cyclers (PCR) for amplification of nucleic acids. The portable system for endpoint fluorescence detection of a amplified nucleic acid, comprises a hand held box comprising a bio-fluidic sample holder for placing a target sample therein. An excitation source is configured to emit a pulsating monochromatic light of a first wavelength on the target sample.

A light sensor is configured to detect fluorescence light of a second wavelength emitted by the target sample upon absorption of the pulsating monochromatic light, and generate an electrical signal corresponding to the second wavelength of the light. A signal processing unit in communication with the light sensor is configured to process said electrical signal to detect the presence or absence of the amplified nucleic acid. A display unit in communication with the signal processing unit indicate the presence or absence of the targeted amplified nucleic acid.

In this embodiment, the portable system further comprises a fluorescence optical filter disposed in an emission path of the fluorescence light to filter the light beam prior to detection thereof by the photo detector. The excitation source is selected from a light emitting diode (LED) or a laser diode. The first wavelength of the monochromatic light is about 480 nm and the second wavelength i.e. the wavelength of the emitted fluorescence light when the sample is excited, is about 520 nm. The light sensor is selected from a light detecting resistor (LDR) or an avalanche photodiode (APD).

In an embodiment, the signal processing unit is configured to generate numerical values through cables, indicative of strength of the detected nucleic acid. In an embodiment, the display unit includes a multi-colour lamp configured to emit a first colour (Red) indicating the presence of the amplified nucleic acid and a second colour indicating the absence of the amplified nucleic acid (Green). A display panel is configured to display the numerical values.

In another embodiment the present invention relates to a method for endpoint detection of an amplified nucleic acid. The method comprises transferring, to a bio-fluidic sample holder, a target sample, emitting by an excitation source, a pulsating monochromatic light of a first wavelength on the target sample. Detecting, by a light sensor, a fluorescence light of a second wavelength emitted by the target sample upon absorption of the monochromatic light and generating an electrical signal corresponding to the second wavelength of the fluorescence light.

Processing is achieved by a signal processing unit, said electrical signal and detecting the presence or absence of the amplified nucleic acid. The display unit displays the presence or absence of the targeted amplified nucleic acid. In this embodiment, the fluorescent light beam generated by the target sample passes through a fluorescence optical filter that is specific to 510-530 nm wavelengths, prior to detection by the light sensor. In another embodiment, the LDR sensor is placed at a 90° to the laser beam to avoid noise signal.

In this embodiment, before transferring to the bio-fluidic sample holder, the target sample is amplified using fluorescence probes labelled with 6-carboxyfluorescein (6- FAM) molecules. In another embodiment, the signals are converted in to numerical values through the cable wires in terms of multi-colour lamp showing a red and a green signal and further the numerical values are displayed on the display panel in this embodiment, 450 units is the threshold value for predicting whether the sample is positive or negative for the amplified nucleic acid.

In this embodiment, sensor reading below the threshold value of 450 indicated the sample is negative for targeted nucleic acid and sensor will indicate green light and on the other hand if sensor value is above threshold value of 450 it will indicate sample is positive for targeted nucleic acid and the sensor will give signal in the form of red light. The positive sample emits fluorescence signal proportional to tagged nucleic acid molecules. The negative sample also emits fluorescence corresponding to noise signal due to random tagged molecules. By prolonged stimulation of test sample by first wavelength, 15-20 seconds in general and 17 seconds in specific case, a high signal to noise ratio is achieved. On a calibrated scale, the positive sample reads more than 450 units, whereas the negative sample reads less than 450 units. In this embodiment, the system only shows fluorescence if and only if particular targeted nucleic acid is present in the sample.

Brief description of the drawings

FIG. 1 is the schematic representation of the detector system, in accordance with the embodiment of the present invention.

Description of the invention

The present invention relates to a portable endpoint detection of target specific amplified nucleic acids which are labelled with fluorescence tag, particularly PCR (Polymerase chain reaction) products with specific fluorescence dyes based on the principle of excitation of fluorescent dye attached with target molecules with the help of either LED or laser diode (light source) and then detection of the emitted & filtered fluorescent light from the sample by using photo detectors basically LDR/ (Light Detecting Resistor) or Avalanche Photodiodes (APDs). The present invention relates to a portable system and a method for endpoint fluorescence detection of polymerase chain reaction (PCR) amplified nucleic acids. In a preferred embodiment, the present invention relates to a portable system and a method for endpoint detection of amplified nucleic acids. In an embodiment, a portable system (100) for endpoint detection of a nucleic acid is discussed in Figure 1. Referring to Figure 1, the portable system (100) for endpoint detection of an amplified nucleic acid, comprises a hand held box (102) comprising: a bio-fluidic sample holder (104) for placing a target sample therein; an excitation source (106) configured to emit a pulsating monochromatic light (108) of a first wavelength on the target sample; a light sensor (114) configured to detect a fluorescence light (110) of a second wavelength emitted by the target sample upon absorption of the pulsating monochromatic light (108), and generate an electrical signal corresponding to the second wavelength of the fluorescent light (110); a signal processing unit (116) in communication with the light sensor (114) and configured to process said electrical signal to detect the presence or absence of the amplified nucleic acid; and a display unit in communication with the signal processing unit (116) to indicate the presence or absence of the nucleic acid.

In an embodiment, the portable system (100) further comprises a fluorescence optical filter (112) disposed in an emission path of the fluorescence light (110) to filter the light (110) prior to detection thereof by the light sensor (114). In an embodiment, the excitation source (106) is selected from a light emitting diode (LED) or a laser diode.

In an embodiment, the first wavelength i.e. wavelength of the monochromatic beam of light (108) used for excitation is about 480 nm and the second wavelength i.e. the wavelength of the emitted fluorescence light (110) when the sample is excited, is about 520 nm.

In an embodiment, the light sensor (114) is selected from a light detecting resistor (LDR) or an avalanche photodiode (APD). In an embodiment, the signal processing unit (116) is configured to generate numerical values through cables, indicative of strength of the detected amplified nucleic acid. In another embodiment, the display unit includes:

a multi-colour lamp (120) configured to emit a first colour indicating the presence of the amplified nucleic acid (Red) and a second colour indicating the absence of the amplified nucleic acid (Green); and a display panel (122) configured to display the numerical values.

In this embodiment, the red signal is assigned to DNA positive samples indicating the presence of the amplified nucleic acid and green signal is assigned to negative samples indicating the absence of the amplified nucleic acid.

In another embodiment, referring to the method of the present invention as shown in figure 1 , method for endpoint detection of amplified nucleic acid is discussed.

The method for endpoint detection of an amplified nucleic acid comprising:

transferring, to a bio-fluidic sample holder (104), a target sample;

emitting, by an excitation source (106), a pulsating monochromatic light (108) of a first wavelength on the target sample after receiving a command from detector (not shown);

detecting, by a light sensor (114), a fluorescence light (110) of a second wavelength emitted by the target sample upon absorption of the monochromatic light and generating an electrical signal corresponding to the second wavelength of the fluorescence light (110);

processing, by a signal processing unit (116), said electrical signal and detecting the presence or absence of the amplified nucleic acid and

displaying, by a display unit, the presence or absence of the targeted amplified nucleic acid.

In an embodiment, the fluorescent light beam generated by the target sample passes through a fluorescence optical filter (112) that is specific to 510-530 nm wavelength, prior to detection by the light sensor. In another embodiment, the LDR sensor is placed at a 90° to the laser beam to avoid noise signal. In a further embodiment, before transferring to the bio-fluidic sample holder (104), the target sample is amplified on conventional thermal cyclers using fluorescence probes selected from 6-carboxyfluorescein (6-FAM).

In another embodiment, the signals are converted in to numerical values through the cable wires (118) in terms of multi-colour lamp (120) showing a red and a green signal and further the numerical values Eire displayed on the display panel (122). in this embodiment, 450 units is the threshold value for predicting whether the sample is positive or negative for the amplified nucleic acid. in this embodiment, sensor reading below the threshold value of 450 indicated the sample is negative for targeted nucleic acid and sensor will indicate green light and on the other hand if sensor value is above threshold value of 450 it will indicate sample is positive for targeted nucleic acid and the sensor will give signal in the form of red light. The positive sample emits fluorescence signal proportional to tagged nucleic acid molecules. The negative sample also emits fluorescence corresponding to noise signal due to random tagged molecules. By prolonged stimulation of test sample by first wavelength, 15-20 seconds in general and 17 seconds in specific case, a high signal to noise ratio is achieved. On a calibrated scale, the positive sample reads more than 450 units, whereas the negative sample reads less than 450 units.

In this embodiment, the system only shows fluorescence if and only if particular targeted nucleic acid is present in the sample.

In context of the present invention, the detection system is used for detection of infectious diseases of bacterial, viral and fungal origin, for example MTB, HIV, Dengue etc.; detection of food and water borne pathogens or of plant pathogens; detection of biomarkers specific for cancer etc.

In context of the present invention, the detection system can be clubbed with conventional amplification systems available in the market for endpoint detection of amplified nucleic acids like DNA & RNA. The system of the present invention is low cost, rapid and sensitive for endpoint detection of amplified nucleic acids labelled with fluorescence molecules which emits excitation light of 520 nm. The device of the present invention is portable, handy and can be operated by battery. No environmental control is needed for operation and can be operated at normal room temperature. Operation is simple and paramedics can use it for diagnosis in rural settings. The system of the present invention is maintenance free wherein low cost components like LDR and Laser diode is used and uses only one fluorescence filter. The system of the present invention is robust wherein the design through optonics concepts is used, and it is devoid of expensive precision optics, components viz. lenses, prisms, eyepiece etc. and their assembly and electronic components such as Lamps etc. No custom built bio-chip is used in the present invention, instead conventionally available eppendorf tubes are used for sample processing, and hence the present invention is universal as well as low cost sample processing system.

LDR is not able to differentiate the fluorescence signal emitted from positive and negative samples moreover it gets saturated when sample is excited by continuous exposure of laser source on the sample directly. The use of pulsating laser as excitation source overcomes this problem and helps the LDR sensor to clearly differentiate the fluorescent signal emitted from positive and negative sample of targeted DNA. If the laser source and sensor are in same line, due to reflected light etc, signal gets saturated after a few seconds and there is no differentiation between positive and negative signal. By placing the LDR sensor at 90° from the laser beam, the aforesaid problem is solved and noise signal is avoided.

The system of the present invention can be clubbed with any known conventional nucleic acid amplification systems (Conventional PCR Systems) for amplification of targeted nucleic acids by attaching fluorescence molecules to sequence specific probes. The amplified nucleic acids are detected on the detection system of the present invention within a minute. Thus the present invention’s detection system utilizes only detection part of the process of nucleic acid detection. The existing known technologies are expensive and unaffordable by the poor strata of the society in developing countries like India. In order to screen the infectious diseases at rural setup the developed portable DNA detection system of the present invention can be operated at rural setup. EXAMPLES

Examples and implementations are provided herein below for the illustration of the invention. Variations, modifications and enhancements to the described examples and implementations and other implementations can be made based on what is disclosed. Examples are set forth herein below and are illustrative of different amounts and types of reactants and reaction conditions that can be utilized in practicing the disclosure. It will be apparent, however, that the disclosure can be practiced with other amounts and types of reactants and reaction conditions than those used in the examples, and the resulting devices various different properties and uses in accordance with the disclosure above and as pointed out hereinafter.

Example 1: Detection of TB

TB DNA Extraction: The DNA of the sample is extracted after decontamination of the sputum sample by using conventionally available kit in the market.

Amplification of the samples: Extracted DNA from the samples are amplified for sequences mainly mpt64 gene specific for Mycobacterium Tuberculosis by using Mycobacterium Tuberculosis real time Probe based PCR kit (Cat No. MBPCR 108) on Hi media Wee- 16™ Thermal Cycler reference number LA 1059 by the standard PCR cycles mentioned in the kit manual as Initial denaturation for 95 °C for 15 minutes and 40 cycles of Denaturation at 95 °C for 15 seconds and annealing at 60°C for 1 minute(SignaI Detection). The reaction mixture was prepared by mixing 10 pL of 2X PCR Taq Mixture (MBT061), 2 pL of Primer Mix for M. tuberculosis (DS0132A), 0.5 pL MTB Probe (FAM Labelled) (DS0253) and 5 pL of Extracted Template DNA, the volume of reaction mixture is then balanced by adding 2.5 pL of Molecular Biology Grade Water for PCR (ML065).

Detection of the results on detector system: The amplified samples are then directly placed inside the detection system and the results are then analyzed by observing the reading and signal given by the detector system for particular samples. Sensor reading below 450 indicates that the sample is negative for TB and is indicated by green light on the display and sensor reading above 450 indicates that the sample is positive for TB and is indicated by red light on the display. Experimental Data: The readings of some of the representative experiments are as shown in the following Table 1: For TB Detection total of 12 samples were amplified which includes a negative control (Sample Number 12). The sample number 4, 5, 7 were found positive by the present invention detection system as well as by GeneXpert and Real Time-PCR (RT-PCR) assay. The sample number 1,2,3,6,8,9,10,11,12 were found negative by all the three methods viz. present invention detection system, Real Time PCR assay and GeneXpert assay.

Vote: Sample Number 12 is a negative control sample that includes water in place of a DNA sample that confirms nonspecific amplifications have not occurred during the process.

The results of the present invention detector system are in same lines with Real Time PCR Experiment as well as with GeneXpert assay which means that the present invention has the sensitivity and specificity equivalent to both of the known assays. Example 2: Detection of HIV RNA Extraction: Viral nucleic acid extraction was performed by using conventional RNA extraction kits in the market and extracted nucleic were then subjected to RNA amplification process specific for HIV.

Amplification of the Viral RNA: The extracted RNA samples are subjected for amplification by using Ozofind™ Quantitative PCR Kit. The reaction mixture was formulated by mixing 7.5 pi of PCR Mix, 7.5 mΐ of Nuclease free water, HIV Detection Mix 1.2 mΐ and extracted RNA samples of 12 mΐ. The reaction mixture is then subjected to amplification cycles inside Himedia Wee- 16™ Thermal Cycler reference number LA1059 by using following amplification cycle as mentioned below; PCR Cycles:

Reverse transcription: 50°C for 15 minutes

RT Incubation: 95°C for 20 seconds

Amplification: 45 cycles

95 °C for 5 seconds

60°C for 30 seconds

Detection of the results on detector system: The amplified samples are then directly placed inside the detection system and the results are then analyzed by observing the reading and signal given by the detector system for particular samples. Sensor reading below 450 indicates that the sample is negative for HIV and is indicated by green light on the display and sensor reading above 450 indicates that the sample is positive for HIV and is indicated by red light on the display.

The readings of some of the representative experiments are as shown in the following Table 2:

Total of 9 Samples were tested with negative control (Sample Number 9) and it was found that Sample number 1 , 4, 6 were positive by using present invention detection system as well as GeneXpert and Real Time PCR assay and other samples viz. 2, 3, 5,7, 8, 9 were found negative by Real time PCR, GeneXpert as well as by present invention detection system.

Note: Sample Number 9 is a negative control sample that includes water as a sample which confirms that the nonspecific amplifications have not occured during the process of assay. The results given by the present invention detection system for detection of HIV by kit method are consistent with Real Time PCR assay and GeneXpert assay which confirms that the sensitivity and specificity of the present invention detection system is equivalent to GeneXpert and PCR assay.

The test sample is stimulated by pulsating monochromatic light (108) on it. The emitted fluorescence signal energy increases with time for both positive and negative samples. However, for negative sample the fluorescence signal saturates up to 400 units, whereas the positive sample crosses the threshold value of 450 units. It is observed that the signal saturates at 17 seconds of exposure to pulsating monochromatic light (108) and its value is proportional to the quantity of the target nucleic acid present in the sample.

The foregoing description of specific embodiments of the present invention has been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.

The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others, skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the spirit or scope of the present invention.

Claims

1. A portable system (100) for endpoint fluorescence detection of an amplified nucleic acid, the system comprising:

a bio-fluidic sample holder (104) for placing a target sample therein;

an excitation source (106) configured to emit a pulsating monochromatic light (108) of a first wavelength on the target sample;

a light sensor (114) configured to detect a fluorescence light (110) of a second wavelength emitted by the target sample upon absorption of the monochromatic light (108), and generate a signal corresponding to the second wavelength of the fluorescence light (110);

a signal processing unit (116) in communication with the light sensor (114) and configured to process said electrical signal to detect the presence or absence of the nucleic acid based on the second wavelength of the fluorescence light (110); and

a display unit in communication with the signal processing unit (116) to indicate the presence or absence of the amplified nucleic acid.

2. The portable system (100) as claimed in claim 1, wherein the system (100) further comprises a fluorescence optical filter (112) disposed in an emission path of the fluorescence light (110) to filter the light (110) prior to detection thereof by the light sensor (114).

3. The portable system (100) as claimed in claim 1, wherein the excitation source (106) is selected from a light emitting diode (LED) or a laser diode.

4. The portable system (100) as claimed in claim 1, wherein the first wavelength is about 480 nm and the second wavelength is about 520 nm.

5. The portable system (100) as claimed in claim 1, wherein the light sensor (114) is selected from a light detecting resistor (LDR) or an avalanche photodiode (APD).

6. The portable system (100) as claimed in claim 1, wherein the light sensor is placed at an angle of 90° from the laser beam.

7. The portable system (100) as claimed in claim 1, wherein the signal processing unit (116) is configured to,

detect the presence of the amplified nucleic acid in the event that the sensor reading is above a threshold value, and

detect the absence of the amplified nucleic acid in the event that the sensor reading is below the threshold value.

8. The portable system (100) as claimed in claim 6, wherein the threshold value is about 450 units.

9. The portable system (100) as claimed in claim 1, wherein the signal (110) saturates at 17 seconds of exposure to pulsating monochromatic light (108) and the value is proportional to the quantity of target nucleic acid present in the sample.

10. The portable system (100) as claimed in claim 1, wherein the display unit includes:

a multi-colour lamp (120) configured to emit a first colour indicating the presence of the targeted amplified nucleic acid and a second colour indicating the absence of the targeted nucleic acid; and

a display panel (122) configured to display the numerical values.

11. A method for endpoint detection of a amplified nucleic acid, the method comprising:

transferring, to a bio-fluidic sample holder (104), a target sample; emitting, by an excitation source (106), a pulsating monochromatic light

(108) of a first wavelength on the target sample;

detecting, by a light sensor (114), a fluorescence light (110) of a second wavelength emitted by the target sample upon absorption of the monochromatic light (108) and generating a signal corresponding to the second wavelength of the fluorescence light (110);

processing, by a signal processing unit (116), said electrical signal and detecting the presence or absence of the nucleic acid based on the second wavelength of the fluorescence light (110); and displaying, by a display unit, the presence or absence of the targeted amplified nucleic acid.

12. The method as claimed in claim 10, wherein the method further comprises filtering, by a fluorescence optical filter (112), the fluorescence light (110) before detecting the fluorescence light (110).

13. The method as claimed in claim 11, wherein the fluorescence optical filter (112) is specific to a range of 510 to 530 nm.

14. The method as claimed in claim 10, wherein, before transferring to the bio-fluidic sample holder (104), the target sample is amplified using fluorescence probes labelled with 6-carboxyfluorescein (6-FAM) Molecule.