Loop-mediated isothermal amplification (LAMP) -based assays for detecting microorganisms
阅读说明：本技术 用于检测微生物的基于环介导等温扩增(lamp)的测定 (Loop-mediated isothermal amplification (LAMP) -based assays for detecting microorganisms ) 是由 黄啸 迈克尔·R·霍夫曼 于 2018-12-31 设计创作，主要内容包括：公开了一种用于检测疑似含有病原体的样品中的微生物病原体的方法和系统。该方法包括将环介导等温扩增(LAMP)试剂和聚合物凝胶(诸如水凝胶)与样品合并以形成混合物。使凝胶在短时间内聚合,将病毒颗粒固定在混合物内。如果样品中存在靶DNA/RNA,则产生扩增子。通过视觉检测扩增子的存在或不存在来检测靶微生物。靶微生物浓度可以基于反应后荧光扩增子点的数目使用智能手机或荧光显微镜确定。该方法可以用于快速且低廉地以高灵敏度对环境水样品中的微生物病原体定量。(A method and system for detecting a microbial pathogen in a sample suspected of containing the pathogen is disclosed. The method includes combining a loop-mediated isothermal amplification (LAMP) reagent and a polymer gel (such as a hydrogel) with a sample to form a mixture. The gel is allowed to polymerize in a short time, immobilizing the viral particles within the mixture. If the target DNA/RNA is present in the sample, an amplicon is produced. The target microorganism is detected by visually detecting the presence or absence of the amplicon. The target microorganism concentration can be determined using a smartphone or a fluorescence microscope based on the number of fluorescent amplicon points after the reaction. The method can be used to rapidly and inexpensively quantify microbial pathogens in environmental water samples with high sensitivity.)
1. A method of detecting a target microorganism in a sample suspected of containing the target microorganism, the method comprising:
combining a loop-mediated isothermal amplification (LAMP) reagent, a polymer gel, and the sample to form a mixture, the polymer gel for immobilizing the microorganism within the mixture; and
detecting the presence or absence of the target microorganism in the mixture, detecting the presence or absence of one or more amplicons produced by a LAMP reaction that amplifies DNA/RNA of the target microorganism, wherein the presence of the amplicon is indicative of the presence of the target microorganism in the sample and the absence of the amplicon is indicative of the absence of the target microorganism in the sample.
2. The method of claim 1, wherein the polymer gel is a hydrogel selected from the group consisting of: polyethylene glycol (PEG) gels and polyacrylamide gels.
3. The method of claim 1, further comprising:
quantifying the concentration of virus in the sample based on the number of amplicons detected to be present after the LAMP reaction.
4. The method of claim 1, wherein each of the target microorganisms corresponds to one amplicon.
5. The method of claim 1, wherein the microorganism is a virus, bacteriophage, coliphage, protozoan, or bacterium.
6. The method of claim 1, further comprising:
allowing the polymer gel to polymerize in the mixture at a predetermined temperature for a predetermined period of time; and
incubating the mixture after the polymer gel polymerization step.
7. The method of claim 1, further comprising:
to the mixture, a primer-dye and a primer-quencher duplex are added.
8. The method of claim 1, further comprising:
the mixture is dyed with a dye.
9. The method of claim 1, further comprising:
placing the mixture on a slide; and
visually detecting the presence or absence of amplicons on the slide using a smartphone camera or a fluorescence microscope.
10. The method of claim 1, wherein the cross-linking of the polymer gel is adjusted to form a predetermined mesh size and a predetermined molecular weight between cross-linkers in the polymer gel.
11. The method of claim 1, wherein the sample is selected from the group consisting of: ambient water, soil, feces, urine, blood, and any combination of the foregoing.
12. A method of detecting a bacteriophage in a sample suspected of containing the bacteriophage, the method comprising:
combining a loop-mediated isothermal amplification (LAMP) reagent, a polymer gel, and the sample to form a mixture, the polymer gel for immobilizing the bacteriophage within the mixture;
incubating the mixture; and
detecting the presence or absence of the bacteriophage in the mixture, detecting the presence or absence of one or more amplicons generated by a LAMP reaction that amplifies DNA/RNA of the bacteriophage, wherein the presence of the amplicon is indicative of the presence of the bacteriophage in the sample and the absence of the amplicon is indicative of the absence of the bacteriophage in the sample.
13. The method of claim 12, wherein the bacteriophage is escherichia coli bacteriophage MS2 infecting escherichia coli (escherichia coli).
14. The method of claim 12, wherein the sample is selected from the group consisting of: ambient water, soil, feces, urine, blood, and any combination of the foregoing.
15. The method of claim 12, wherein the polymer gel is selected from the group consisting of: polyethylene glycol (PEG) gels and polyacrylamide gels.
16. The method of claim 12, the method further comprising:
allowing the polymer gel to polymerize in the mixture at a predetermined temperature for a predetermined period of time; and
incubating the mixture after the polymer gel polymerization step.
17. The method of claim 12, the method further comprising:
to the mixture, a primer-dye and a primer-quencher duplex are added.
18. The method of claim 12, the method further comprising:
placing the mixture on a slide; and
the presence or absence of amplicons on the slide is visually detected using a cell phone camera or a fluorescence microscope.
19. A system for detecting a target microorganism in a sample suspected of containing the target microorganism, the system comprising:
a reactant comprising a loop-mediated isothermal amplification (LAMP) reagent and a polymer gel;
a chamber for combining the reagent and the sample to form a mixture; and
a slide configured to receive the mixture to allow visual detection of the presence or absence of the target microorganism in the mixture, the presence or absence of one or more amplicons generated by a LAMP reaction that amplifies DNA/RNA of the target microorganism, wherein the presence of the amplicon is indicative of the presence of the target microorganism in the sample and the absence of the amplicon is indicative of the absence of the target microorganism in the sample.
20. The system of claim 20, further comprising a fluorescent marker and a camera, the fluorescent marker being applied to the mixture to form the amplicon; the camera is configured to visually detect the presence or absence of amplicons in the mixture received on the slide.
Technical Field
The present disclosure relates generally to techniques for detecting a microorganism of interest in an environmental sample, and more particularly, to loop-mediated isothermal amplification (LAMP) -based detection techniques.
Background
Water borne (waterworne) diseases are disorders caused by pathogenic microorganisms (e.g., viruses, bacteria, and protozoa). According to the World Health Organization (WHO), water-borne diarrhea disease alone causes 180 million deaths each year, which makes it a major cause of disease and death worldwide. Due to their small size and great diversity, detection of microbial pathogens in environmental water is challenging.
Currently, there are a variety of methods available for the detection of water-borne microbial pathogens. These methods can be divided into two categories. The first category is the traditional culture-based method, which is based on providing a combination of nutritional and physicochemical conditions that will support the growth of the microorganism of interest. However, providing a similar environment as a warm-blooded host of a pathogen may be difficult or, in some cases, impossible. Additionally, culture-based methods are often time-consuming (days to weeks) and labor-intensive, and therefore can only be performed in a self-contained laboratory by trained technicians. Another class is molecular-based methods such as Polymerase Chain Reaction (PCR), immunoassays, and various biosensors. PCR, and in particular the quantitative PCR (qpcr) method, is the most widely used method of pathogen detection and is considered a new gold standard test. qPCR provides a much shorter sample-to-result time (3 to 5 hours). However, although qPCR is widely accepted, it is limited by relying on standard reference substances (standard curves) for quantification. Unreliable and inconsistent commercial standard reference materials may also affect the accuracy of qPCR quantification. In addition, qPCR is susceptible to inhibition by naturally occurring substances in environmental samples (e.g., heavy metals and organic matter), leading to inaccurate or false negative results in target quantification.
Compared to qPCR, recent digital PCR techniques have proven to be more robust solutions for the detection of microbial pathogens in environmental samples. Digital PCR is based on partitioning (partioning) and poisson statistics, so no external quantification standards need to be compared to quantify samples of unknown concentration. However, implementing digital PCR methods for use with point-of-use applications (point-of-use applications) can be challenging. This is because digital PCR requires expensive instrumentation (i.e., Bio-rad droplet digital PCR), a fully equipped laboratory environment, and trained technicians to perform the assays. These factors severely limit the accessibility and applications of digital PCR in resource-limited contexts.
Thus, a rapid, simplified, low cost assay for detecting microorganisms is desired to provide the benefits of molecular assays outside of a centralized laboratory, for example, where on-site point-of-use testing of environmental water in resource-limited locations is required.
SUMMARY
In addition to PCR-based nucleic acid amplification and detection techniques, isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), Multiple Displacement Amplification (MDA), and Rolling Circle Amplification (RCA), offer the opportunity to provide the benefits of molecular assays outside of centralized laboratories. Isothermal reactions are more suitable for integration with miniaturized, portable, and battery-powered "lab-on-a-chip" platforms because no thermal cycling is required. LAMP, originally described in 2000, has now become the most prevalent isothermal amplification technology, covering almost all microbial pathogens important in environmental hygiene (contamination). LAMP is capable of templating target DNA at temperatures of about 65 ℃ in less than
Portable devices have been developed that facilitate the use of LAMP in point-of-care disease diagnosis. However, most of these assays are qualitative and therefore not suitable for quantitative environmental water quality monitoring. Although several quantitative LAMP assays in real-time or digital format have been reported, these assays require either complex instruments (i.e., handheld real-time fluorescence detection devices) or custom microfabricated consumables (e.g., Slipchip and DropChip).
LAMP-based techniques and systems are disclosed herein that can provide quantitative, low-cost, and rapid bacteriophage detection and/or monitoring tools that can be readily employed in resource-limited settings. Inspired by early work on in situ PCR, immobilization of microorganisms in hydrogels, PCR amplification in polyacrylamide gels, and MDA amplification in polyethylene glycol (PEG) hydrogels, a novel in-gel lamp (g lamp) technique is described herein that is capable of quantifying microbial pathogens in environmental water samples within 30 min. The g lamp system does not require microfluidic chips and has limited personnel training. Although the disclosed exemplary embodiments may focus on coliphage (virus-infected bacteria) detection, the gmlamp technology may also be adapted to detect and/or monitor other microbial pathogens (e.g., e.coli, Salmonella) in other background water or food samples. The system can also be used to detect and quantify microorganisms in other matrices (e.g., stool, urine, and blood) by simple sample pre-treatment (DNA/RNA extraction and purification).
According to exemplary embodiments of the g lamp technique, one or more methods and systems are disclosed for detecting a target microorganism (including viral particles, bacterial cells, or target DNA/RNA) in a sample suspected of containing the target microorganism. The method includes combining a loop-mediated isothermal amplification (LAMP) reagent and a hydrogel with a sample to form a mixture. The hydrogel polymerizes within a short time to immobilize the target within the mixture. If the target is present in the sample, amplicons are generated by LAMP amplification during the heat incubation. The target is detected by visually detecting the presence or absence of the amplicon. The concentration of the target can be determined based on the number of fluorescent amplicon spots after the reaction using a smartphone or a fluorescence microscope.
The foregoing summary does not define limitations on the claims that follow. Other aspects, embodiments, features and advantages will be or will become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional features, embodiments, aspects and advantages be included within this description and be protected by the accompanying claims.
Brief Description of Drawings
It is to be understood that the drawings are solely for purposes of illustration and are not intended as a definition of the limits of the appended claims. Furthermore, the components in the drawings are not necessarily to scale. In the drawings, like reference numerals designate corresponding parts throughout the different views.
Fig. 1 is a schematic diagram of an exemplary in-gel LAMP assay system.
Fig. 2 is a flow diagram illustrating an exemplary in-gel LAMP method for detecting viral particles and/or microbial pathogens in a sample.
Fig. 3A-3F are photographic images showing exemplary amplicon spot spectra resulting from certain experimental in-gel LAMP tests on ambient water.
FIGS. 4A-4B are graphs of exemplary experimental results showing the effect of reaction time and template concentration on the results of an example of the in-gel LAMP method.
Fig. 5A-5B are graphs of exemplary experimental results showing the effect of certain sample pretreatments on examples of the in-gel LAMP method (fig. 5A) and a count comparison between examples of the in-gel LAMP method and conventional plaque assays (fig. 5B).
Fig. 6A-6C are graphs of exemplary experimental results of exemplary direct g lamp assays when processing certain environmental water samples.
FIG. 7 is a table (SEQ ID NOs: 1-11) showing primer and probe sequences of an example of the in-gel LAMP technique disclosed herein.
Detailed Description
One or more examples of assay systems, kits, and methods based on loop-mediated isothermal amplification in gel (g LAMP) test samples are described and illustrated in the following detailed description, which refers to and incorporates the accompanying drawings. These examples, which are shown and described in sufficient detail to enable those skilled in the art to practice the claimed subject matter, are not provided to be limiting but are merely intended to illustrate and teach embodiments of the assays and systems of the present invention. Accordingly, the description may omit certain information known to those of skill in the art, where appropriate, in order to avoid obscuring the present invention. The disclosure herein is not to be interpreted as an example of an over-limiting scope that may be based on any patent claims to which the application is ultimately entitled.
The word "exemplary" is used throughout this application to mean "serving as an example, instance, or illustration. Any system, method, device, technique, feature, or the like described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other features.
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific examples of suitable materials and methods are described herein.
Further, the use of "or" means "and/or" unless otherwise indicated. Similarly, "comprise", "comprises", "comprising", "including" and "including" are interchangeable and are intended to be non-limiting.
It will also be understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that in certain specific instances embodiments may be alternatively described using the language "consisting essentially of" or "consisting of.
As discussed above, water borne diseases are disorders caused by pathogenic microorganisms (e.g., viruses, bacteria, and protozoa). For example, human pathogenic enteroviruses that are transmitted through environmental water are the major cause of many water-borne diseases, including hepatitis, gastroenteritis, meningitis, fever and conjunctivitis. Due to their small size and great diversity, detection of microbial pathogens in environmental water is challenging. For example, direct monitoring of a particular viral pathogen is often impractical for public health protection due to methodological limitations. Similar to bacterial indicators, coliphage (a virus that infects escherichia coli) can be used as an indicator organism to predict viral contamination of water. While the disclosed exemplary embodiments may focus on coliphage (a virus that infects bacteria) detection, the g lamp technique may also be applicable to the detection and/or monitoring of other microbial pathogens (e.g., gram positive and gram negative bacterial species, viruses, and protozoa) in water, clinical, or food samples. In addition to ambient water, the systems and methods disclosed herein can also be used for detection and quantification of microorganisms in other matrices (e.g., stool, urine, and blood) by sample pre-treatment (DNA/RNA extraction and purification).
Loop-mediated isothermal amplification (LAMP or Loopamp) is an isothermal DNA amplification procedure that uses a set of 4 to 6 primers that specifically recognize the target DNA sequence: from 2 to 3 "forward" and from 2 to 3 "reverse" (see Nagamine et al, nucleic acids Res. (2000)28: e 63; Nagamine et al, Clin. chem. (2001)47: 1742-43; U.S. Pat. No. 6,410,278; U.S. Pat. App. No. 2006/0141452; No. 2004/0038253; No. 2003/0207292; and 2003/0129632; and European patent application No. 1,231,281). Briefly, a set of primers was designed such that the primer sequence of about 1/2 was the positive strand and the primer sequence of another 1/2 was the negative strand. After strand displacement amplification by polymerase, a nucleic acid construct is generated with a hairpin loop on each side. From this construction, proceedRepeated rounds of amplification produce products of different sizes. A by-product of this amplification is the formation of magnesium pyrophosphate, which forms a white precipitate, resulting in turbidity of the reaction solution. The presence of such turbidity means a positive reaction, while the absence of turbidity is a negative reaction. Additional additives, such as calcein, allow other visualization phenomena to occur; as for calcein, it enables fluorescence detection. The amplification reaction takes place under isothermal conditions (at about 65 ℃) and continues to accumulate 10 in less than one hour9Copies of the target.
Reagents for LAMP are known (e.g., Bst polymerase, dNTPs, buffer, etc.). In addition, the skilled person can easily design and identify primer pairs using known sequence information. For example, a preparation reagent (reagent preparation) for loop-mediated isothermal amplification of nucleic acids comprises at least one polymerase (wherein the enzyme is capable of strand displacement), a target-specific primer set and deoxynucleotide triphosphates (dntps). In some embodiments, the polymerase capable of strand displacement is Bst enzyme. If the target is RNA, the preparation reagent further comprises a reverse transcriptase. In some embodiments, the reverse transcriptase is an AMV reverse transcriptase.
The term "microorganism" as used herein includes bacterial, fungal, protozoan and viral organisms. The methods and systems of the present disclosure may be used to detect and identify the presence of such microbial life forms.
Bacterial microorganisms that can be detected using the methods and systems of the present disclosure include both gram-negative and gram-positive bacteria. For example, bacteria that may be affected include staphylococcus aureus (staphylococcus aureus), Streptococcus pyogenes (group a), Streptococcus species (Streptococcus sp.) (group viridans group), Streptococcus agalactiae (group B), Streptococcus bovis (s.bovis), Streptococcus (Streptococcus) (anaerobic species), Streptococcus pneumoniae (Streptococcus pneumoniae), and Enterococcus species (Enterococcus sp.); gram-negative cocci, such as, for example, Neisseria gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis) and Branhamella catarrhalis (Branhamella catarrhalis); gram-positive bacilli, such as Bacillus anthracis (Bacillus ankhrasis), Bacillus subtilis (Bacillus subtilis), propionibacterium acnes (p.ace), Corynebacterium diphtheriae (Corynebacterium diphtheriae) and hypopharyomyces (Corynebacterium) species (aerobic and anaerobic), listeria monocytogenes (listeriology), Clostridium tetani (Clostridium tetani), Clostridium difficile (Clostridium difficile), Escherichia coli (Escherichia coli), Enterobacter species (Enterobacter), Proteus mirabilis and other species, Pseudomonas aeruginosa (Pseudomonas aeruginosa), Klebsiella pneumoniae (pneerulea), salmonella, Shigella (Shigella), and Campylobacter (Campylobacter). In particular, the methods and systems of the present disclosure may be used to detect any pathogen. Diseases that may result from infection with one or more of these bacteria, such as bacteremia, pneumonia, meningitis, osteomyelitis, endocarditis, sinusitis, arthritis, urinary tract infections, tetanus, gangrene, colitis, acute gastroenteritis, impetigo, acne, ace pos, wound infections, birth infections (born infections), fasciitis, bronchitis and various abscesses, nosocomial infections and opportunistic infections.
Fungal organisms may also be detected by the methods and systems of the present disclosure, for example, microsporidia canis (Microsporum canis) and other Microsporum species (Microsporum sp.); and Trichophyton species (Trichophyton sp.), such as Trichophyton rubrum (t. rubrum) and Trichophyton mentagrophytes (t. mentagrophytes), yeast (yeasts) (e.g., Candida albicans, Candida tropicalis (c. tropicalis), or other Candida species), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Torulopsis glabrata (Torulopsis glabrata), Epidermophyton floccosum (Epidermophyton floccosum), Malassezia furfur (Malassezia furfur) or Pityrosporum ovale (p. ovale)), cryptococcus neoformans, Aspergillus fumigatus, Aspergillus nidulans and other Aspergillus species (Aspergillus sp.), Zygomycetes (zygomycotes) (e.g., Rhizopus (Rhizopus), Mucor (Mucor)), Paracoccidioides brasiliensis, Blastomyces dermatitidis (Blastomyces dermatitidis), Histoplasma capsulatum (Histoplasma capsulatum), coccidioidomycosis immitis and trichosporoides (Sporothrix schenckii).
Viral organisms that can be detected by the methods and systems of the present disclosure include, but are not limited to, Human Immunodeficiency Virus (HIV), Junin Virus (Junin Virus), BK Virus, Marigold Virus, Varicella zoster Virus (Varicella zoster Virus), alphavirus (alphavirus), Colorado tick fever Virus (Colorado tick fever Virus), rhinovirus and coronavirus, cytomegalovirus (cytomegavirus), dengue Virus (Degueevirus), Ebola Virus (Ebola Virus), Enterovirus (Enterovirus) species, herpes simplex Virus-1, herpes simplex Virus-2, hepatitis A Virus, hepatitis B Virus, hepatitis C Virus, hepatitis D Virus, hepatitis E Virus, measles Virus, Mumps Virus (Mumps Virus), Norovirus (Norirus), respiratory syncytial Virus (respiratory syncytial Virus), Rotavirus (Rollera Virus), Rollera Virus (Rollera Virus), SARS coronavirus, West Nile Virus (West Nile Virus), and Zika Virus (Zika Virus).
Fig. 1 is a schematic diagram of an exemplary in-gel lamp (g lamp)
In some embodiments, the hardware included in
Typically, during operation of the
The g lamp assay is significantly faster than other available methods, taking less than 30 minutes, compared to 2-4 hours qPCR and traditional culture-based methods of more than 24 hours. The amplified gel slides can be stored at room temperature for more than one month without affecting the fluorescent spot visualization. This indicates that the gel matrix provides good protection for the amplicons, which will allow for the transport of the sample in case further analysis is required. In view of the outstanding simplicity, sensitivity and rapidity of the gLAMP technology disclosed herein, the gLAMP technology disclosed herein has great potential for water quality analysis of microorganisms, particularly in resource-limited contexts.
Furthermore, the results of the exemplified experiments show that the gLAMP assay results show a good correlation with the traditional plaque assay counts (R)2＝0.984,p<0.05) and achieved sensitivity (1 PFU/reaction) similar to real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the experimental results show that the gLAMP technique shows high tolerance to the inhibitors present in the toilet wastewater in which RT-qPCR is completely inhibited. Considering the simplicity, sensitivity and rapidity of gLAMP, gLAMP provides a greatly improved technique for the water quality analysis of microorganisms, especially in the background of limited resources.
The g
Fig. 2 is a flow diagram illustrating an exemplary in-gel lamp (g lamp)
The gel may comprise, consist of, or consist essentially of: any suitable polymer gel or combination of different gels. For example, in some exemplary embodiments, the polymer gel used as the matrix for the g lamp may be a polyacrylamide gel. For example, polyacrylamide gels can be formed by using ammonium persulfate (Bio-Rad, Hercules, Calif.) as an initiator to catalyze the crosslinking between acrylamide and bisacrylamide (40% (w/v) solution acrylamide/bisacrylamide 19:1) (Bio-Rad, Hercules, Calif.). In other exemplary embodiments, the polymer gel used as a matrix for the g lamp may be a polyethylene glycol (PEG) hydrogel. For example, a PEG gel may be formed by a michael addition between a four-arm PEG acrylate [ Molecular Weight (MW)10K ] and thiol-PEG-thiol (MW3.4K) (Laysan Bio, Arab, AL) in a molar ratio of 1: 2.
In the gLAMP assayIn certain exemplary embodiments, 25 μ L of the hydrogel reaction mixture may comprise, consist of, or consist essentially of: 12.5 μ L2 ×
Following amplification, the LAMP dye may then optionally be used (included in)
In step 56, the presence or absence of amplification products (e.g., amplicons) in the polymerized mixture is visually determined. For this purpose, E-Gel may be usedTMA safety imager (Invitrogen, Carlsbad, CA) illuminates the slide and records the amplicon spots with a smartphone (e.g., iPhone6s plus or similar). Alternatively, the slide can be visualized using a fluorescence microscope (e.g., Leica DMi8, Leica co., Germany).
To demonstrate the efficacy of
In embodiments of the gLAMP assay using polyacrylamide gels, clear polyacrylamide gels can form relatively quickly in 10-15 minutes. In embodiments of the g LAMP assay using PEG gels, PEG gel formation can generally be faster, requiring only 3-5 minutes at basic pH (the pH of the LAMP reaction mixture can be about 8.8). Neither of these two types of gels generally showed a fluorescent background, and in either case the gLAMP assay could be successfully performed (see FIG. 3).
Figure 3 is a photographic image showing an exemplary amplicon spot spectrum generated by certain experimental g lamp tests of ambient water. Photographs a and B of fig. 3 show the amplicon spots during the assay experiment using polyacrylamide hydrogel. Photographs C-F of fig. 3 show the amplicon spots during the assay experiment using PEG hydrogel. In photographs A-D, amplicons were stained with 0.5 × LAMP dye after incubation, and in photographs E-F with QUASR primers without post-reaction staining. Photograph A, C, E was taken by a fluorescence microscope and photograph B, D, F was taken by an iPhone6S Plus. The reaction time for image measurement was 25 minutes.
When the size of the amplicon spot is less than 20 μm (diameter), its detection may require a fluorescence microscope. To facilitate reading of the results with a smartphone camera while still maintaining the actual assay dynamic range, a dot size of 50-200 μm may be used. The size of the amplicon spot may be determined primarily by the restriction of the gel matrix.
In some embodiments of the assay, a gel matrix can be formed that allows free diffusion of small molecules (MW <100kDa), such as water, ions, primers (<50bp, MW <15kD), and enzymes (Bst ═ 67kDa), but limits diffusion of DNA/RNA templates/amplicons (>150bp, MW >100 kDa). This can be achieved by manipulating the degree of crosslinking of the gel to adjust key parameters such as mesh size and molecular weight between the crosslinking agents in order to alter macroscopic properties (i.e. diffusion). Previous studies have found that in polyacrylamide hydrogels, 514bp, 234bp and 120bp templates yield uniform PCR amplicon spots of 100 μm, 400 μm and 800 μm, respectively. It should be noted that unlike single length PCR amplicons, the products of LAMP are a mixture of concatemers of target regions with different sizes. Based on agarose gel electrophoresis patterns, the shortest MS2LAMP amplicon can be about 90bp, while the longest amplicon can be as much as several thousand bp. During the g LAMP assay, longer amplicons may be retained by the hydrogel, but shorter amplicons may diffuse away from the initial template (center of the spot) and serve as templates for further amplification until they reach the diffusion limit or near LAMP reagents (e.g., enzymes, primers, dntps) are depleted. Due to the random nature of LAMP, spots of different sizes can be generated in the hydrogel.
The mesh sizes of polyacrylamide and PEG hydrogels may be similar and in the range of 20-25 nm. However, the amplicon spots in the PEG gel (see, e.g., fig. 3, panel C) may be smaller and more uniform than the amplicon spots formed in the polyacrylamide gel (fig. 3, panel a). Thus, PEG gels may have better confinement for smaller amplicons. Thus, in addition to size exclusion, other interactions (i.e., charge interactions) between the polymer and the DNA template may also affect the diffusion coefficient.
A clear amplicon spot spectrum can be obtained by post-reaction gel staining with, for example, an intercalating LAMP dye (fig. 3, photograph A, B). This highlights the feasibility of adapting traditional qualitative LAMP assays for other microorganisms (e.g., e.coli, enterococci, and salmonella) to quantitative assays by the g LAMP technique.
In some embodiments, normal LAMP primers can be used for post-reaction staining of the g LAMP mixtures. Referring to FIG. 7, the final concentrations of F3/B3, FIP/BIP and LF/LB were 0.2. mu.M, 1.6. mu.M and 0.4. mu.M, respectively.
In another embodiment of the gLAMP assay, a primer-dye/primer-quencher duplex, also known as QUASR (quenching unincorporated amplification signal reporter), can be used to label the amplification products without the need for post-reaction staining. In QUASR, the Forward Inner Primer (FIP) is labeled at the 5 'primer end with a fluorophore (5' FAM-FIP). The probe was provided with a quencher at the 3' primer end (Iowa)FQ) was quenched (qFIP-3' IBFQ). Due to the melting temperature (T) of the compomerm) 5-10 ℃ lower than the reaction temperature (65 ℃), so that 5' FAM-FIP is released during the LAMP reaction and it behaves like conventional FIP. When the target template is present in the sample, 5' FAM-FIP is incorporated into the LAMP amplicon. After the reaction, the free-flowing 5'FAM-FIP was quenched again by the complementary quenching primer qFIP-3' IBFQ. In contrast, 5' FAM-FIP incorporated into LAMP amplicons will not be quenched because they have formed a stable double stranded DNA structure during the LAMP reaction.
For the QUASR probe, a Fluorophore (FAM) can be attached to FIP at the 5' end; the quencher IBFQ may be attached to the quenching probe qFIP at the 3' end. IBFQ may be lowaFQ (Integrated DNA Technologies, Coralville, IA) with a broad absorption spectrum ranging from 420nm to 620nm with a peak absorption at 531 nm. The quencher can be used with fluorescein and other fluorescent dyes that emit in the green to pink spectral ranges. In QUASR gLAMP, FIP (FIG. 7) can be replaced by 5'FAM-FIP, and 2 XqFIP-3' IBFQ (3.2. mu.M) can be added to the reaction mixture.
QUASR significantly reduces the problem of false positives associated with LAMP assays compared to non-specific DNA intercalating dyes (i.e. LAMP dyes). However, QUASR cannot be converted to a quantitative determination of the real-time LAMP protocol, since the fluorescence intensity of the reaction mixture does not increase gradually during the heat incubation, but is always at the highest level (all 5' FAM-FIP are released). Therefore, QUASR can only be used as a qualitative measure of endpoint determination.
In some embodiments of the g lamp assay, a higher concentration of quenching primers (2x complementary probe primers) may be used to maintain a clean gel background at the end of the g lamp reaction. PEG gels can allow dye-labeled short oligonucleotides to move freely, even though the diffusion coefficient in the gel may be smaller than the diffusion coefficient in solution. Since 5' FAM-FIP is incorporated into the amplicon and accumulates around the initial template, bright and well-defined amplicon spots can be visualized directly with the smartphone camera after blue light exposure.
FIGS. 4A-4B are graphs of exemplary experimental results showing the effect of reaction time and template concentration on QUASR amplicon point results for an exemplary gLAMP method. The gLAMP assay experiment was performed using extracted MS2 viral RNA. These figures show box plots of left-hand amplicon spot diameters. According to one-way ANOVA followed by Tukey post hoc testing, the different letters (a, b, c) indicate significant differences at the p, 0.05 level. For the graph of fig. 4A, the template concentrations are defined as follows: low-1-20 copies/gel, medium-20-200 copies/gel, and high-200-2000 copies/gel. The graph of fig. 4A uses medium template concentrations and the reaction time of the graph of fig. 4B is 25 minutes. The extracted MS2RNA was used as a template in the sample.
The experimental results show that the amplicon spots were visible under the fluorescence microscope as early as 20 minutes (fig. 4A). After 25min, the spot developed to approximately 156 ± 33 μm (diameter) and the fluorescence intensity was strong enough to be detected with a smartphone camera (fig. 3). Although the size of the amplicon spot remained increased and reached 212 ± 50 μm after 30 minutes, the spot count remained similar to that at 25 minutes. Thus, for the exemplary MS2 g lamp assay, 25 minutes may be the desired reaction time. The amplicon spot size did not show significant differences at low template concentrations (1-20 copies/gel) and medium template concentrations (20-200 copies/gel) (fig. 4B). Under these conditions, the amplicon spots are relatively far apart from each other and have limited interactions. The dot size represents the maximum size that an amplicon can develop within a given reaction time, while dimensional variability in a single gel can be caused by variable initial template conformation, degree of template denaturation, or local heterogeneity of the hydrogel structure due to free pendant ends of the macromers (macromers), self-looping (self-looping) or entanglement. In contrast, the size of the amplicon spot at high concentration (200-. The smaller amplicon size at higher template concentrations may be due to overall self-contained fabrication, particularly due to a decrease in pH. Local competition for enzymes, primers and dntps may also contribute because a clear separation is revealed between amplicons in close proximity to each other. Smaller amplicon size plus cut (washed) that appears at higher template concentrations is beneficial to the dynamic range of the assay by increasing the detectability of the fluorescent spots. For smartphone camera readings, the dynamic range of the assay may be 1-1000 points/gel. When reading results using a fluorescence microscope, each gel can accommodate up to 5000 spots without compromising accuracy.
In some embodiments of the g lamp assay, manual amplicon counting may be used. In other embodiments of the g lamp assay, automated amplicon analysis of the microscope and smartphone images can be performed by an automated amplicon counter, such as CellProfiler 2.2.0. With appropriate threshold settings, the difference between the automatic and manual counts may be less than 5%.
In some embodiments of the g LAMP assay, the sample may be subjected to nucleic acid extraction and purification prior to the LAMP reaction. In qPCR assays, it is often necessary to extract and purify nucleic acids to improve detection. Many commercial extraction kits have been optimized for procedures that provide high quality DNA/RNA for downstream qPCR analysis, and these kits can be used with the gillamp technology. However, this process takes 30-90 minutes and these kits are relatively expensive.
In other embodiments of the g LAMP assay, the sample may be subjected to a heat pretreatment prior to the LAMP reaction. In some embodiments of the g lamp assay, direct detection of viral particles is achieved in an untreated sample without prior nucleic acid extraction or pre-heating treatment.
Fig. 5A-5B are graphs of exemplary experimental results showing the effect of certain sample pretreatments on an example of the g lamp method (fig. 5A) and a count comparison between an example of the g lamp method and a conventional plaque assay (fig. 5B).
Referring to fig. 5A, the graph shows the experimental results of the g lamp method when various sample pretreatments were employed. In the diagram of FIG. 5A, virions refer to the direct detection of viral particles without sample pretreatment; heating means that the sample is preheated to 95 ℃ for 5 minutes for denaturation; and the extracted RNA represents the pre-treatment of the extracted RNA using a commercially available AllPrep Power Viral DNA/RNA kit. These experiments were performed in Phosphate Buffered Saline (PBS); thus, the extraction kit only acts to release viral RNA. Purification is limited because very low concentrations of inhibitors in the buffer system are expected. Figure 5A shows that the direct g lamp assay (without sample pretreatment) yielded similar amplicon counts as compared to the heat-pretreated g lamp assay and the g lamp assay using samples pretreated with commercial viral RNA extraction kits. No significant differences were found between these treatments in amplicon spot size and fluorescence intensity. It was previously reported that simple heat pretreatment improves bacterial detection in LAMP assays because damaged cell membranes are more permeable to LAMP reagents, while denatured DNA can promote strand displacement activity of Bst enzyme. However, the presented results show that LAMP primers and enzymes (RTx reverse transcriptase and Bst 2.0DNA polymerase) are able to penetrate the viral capsid at the reaction temperature (65 ℃) and that a denaturing heating step may not be necessary because the viral genome is much smaller compared to the bacterial genome.
To assess the sensitivity of direct g lamp, it can be compared to traditional plaque assays and RT-qPCR (as shown by the graph of fig. 5B). Direct gLAMP determination of amplicon counts showed good correlation with plaque assay counts (R)2＝0.984,p<0.05). The regression line (slope 1.036, intercept-0.290) indicates that 1 gel amplicon spot is nearly equal to (closely equivalent to)1 Plaque Forming Unit (PFU). The direct gLAMP assay reached a similar lower limit of detection (0.7 PFU/reaction) compared to the lower limit of detection of RT-qPCR (0.4 PFU/reaction), while RT-qPCR still showed the advantage of a larger upper limit of detection. The assay sensitivity and dynamic range were compared to RT-qPCR using Eppendorf RealPlex2(Hamburg, Germany).
The dynamic range of the g lamp assay can be increased by decreasing the amplicon spot size. Accommodating more amplicon spots in a single gel may be desirable for applications such as mutation detection and in-gel sequencing. However, the ability to distinguish amplicon spots from other contaminating fluorescent signals (contaminating DNA/RNA fragments or particles) may be affected when the spot size is small. Therefore, at low concentrations of (<20 copies/reaction) can suffer. For environmental monitoring purposes, the concentration of MS2 in water samples rarely exceeds the upper detection limit of the disclosed gmlamp assay (10)6PFU/mL). Furthermore, high concentration samples can be easily diluted. Indeed, concentrating small amounts of MS2 from large water samples (e.g., groundwater) is more challenging. Therefore, maintaining accuracy at low concentrations may be valuable for the disclosed gmlamp assay.
Fig. 6A-6C are graphs of exemplary experimental results of an exemplary direct g lamp assay for processing certain environmental water samples. Enzyme-driven nucleic acid amplification processes are susceptible to a variety of inhibitory substances (e.g., organic matter and heavy metals) commonly found in environmental samples. An example of a direct g lamp assay was tested using: toilet waste water that is yellowish brown and has a Chemical Oxygen Demand (COD) level of 821mg/L, representing highly contaminated water; clear and less contaminated (COD 75mg/L) pond water; and PBS control. Direct gLAMP assays were successfully performed in two samples of environmental water spiked with MS2 (toilet and pool water). No significant differences were observed in amplicon spot counts (p >0.05) (fig. 6A) and spot morphology compared to control experiments in PBS buffer. In contrast, slight inhibition was observed in the tank water g lamp assay, as the detection time was delayed from 23min in PBS to 27min in the tank water. Generally, LAMP assays have more robust chemistry than PCR for processing complex samples because: (1) the LAMP assay uses six primers to initiate amplification compared to two primers in PCR, (2) the smaller 67kDa Bst polymerase used in LAMP is likely to enter the target cells/viral particles more easily than the 94kDa Taq DNA polymerase used in PCR, and (3) the yield of LAMP (10-20. mu.g/reaction) is about 50-100 times higher than the yield of PCR (0.2. mu.g/reaction). However, some known LAMP assays are qualitative or semi-quantitative. The use of a crude sample may not compromise the lower detection limit (which is still detectable), but it typically results in increased detection time or reduced signal-to-noise ratio at the end of the reaction.
The concentration of humin-like soluble organic matter (DOM) in the toilet wastewater may be 10-15 times higher than the concentration in the pond water. The toilet waste water may also contain low levels of proteinaceous matter and inhibitors in the toilet waste water may be of organic origin, similar to those present in urine and faeces samples. The presence of urea in urine samples is known to prevent non-covalent binding of polymerases and to interfere with primer annealing. LAMP tolerance to urea is reported to be as high as 1.8M. Inhibitors derived from feces are highly variable depending on nutrition, intestinal flora and lifestyle. Especially the complex polysaccharides, bile salts, lipids and urates present in the stool sample cause inhibition of PCR. The inhibition mechanism is complex, including binding to polymerase (complex polysaccharides, bile salts), competition with template (bilirubin) and Mg as a cofactor for amplification2+Chelating (phytic acid).
The better performance of the g lamp assay in toilet waste water when compared to the PCR method may be attributed to the fact that the gel matrix may have a more important role in enhanced tolerance to inhibitors in toilet waste water. First, like digital PCR, the g lamp assay is an end-point amplification detection assay by counting the final amplification products, and thus, its quantification is less affected by amplification efficiency. Second, since the DNA/RNA templates are spatially separated in the g lamp assay, interference between DNA/RNA molecules and substrate competition can be minimized during amplification. Furthermore, depending on their molecular weight, the movement of large molecular weight organic inhibitors may be limited by the gel matrix and, therefore, the concentration of local inhibitors close to the template may be reduced.
Any of the assay systems (e.g., system 10) or methods (e.g., method 50) disclosed herein can be used to effectively detect and/or monitor microorganisms, such as viral particles, viruses, bacteriophages, protozoa, bacteria, or any combination of the foregoing, etc., in any suitable sample (e.g., a water or food sample or a biological sample).
Examples
Model E.coli phage MS2 preparation. Escherichia coli phage MS2(ATCC15597-B1) was selected as the model virus for the development of the process. For phage propagation, 0.1mL [10 ]7Plaque Forming Unit (PFU)/mL]MS2 was inoculated into 20mL of an actively growing E.coli-3000 (ATCC15597) host suspension in LB medium. The infected bacteria were continuously aerated at 37 ℃ for 36 h. The host bound MS2 suspension was then centrifuged at 3000g for 10min to pellet bacterial cells and debris. The supernatant containing MS2 virions was further purified by passing through a 0.2 μm syringe filter (GE Whatman, Pittsburgh, PA). The filtrate was diluted 1000 ×, in 1 × PBS (pH 7.5) (Corning, New York, NY) and used as a stock solution for MS2 for vaccination studies. The concentration of the MS2 stock solution was titrated by the agar double layer method (double-agar method). MS2RNA extraction was performed using the AllPrep PowerViral DNA/RNA kit (Qiagen, Germanown, Md.) according to the manufacturer's protocol.
gLAMP assay design. Two types of hydrogels were initially tested as substrates for the g lamp. Polyacrylamide (PA) gels were formed by using 0.05% (w/v) ammonium persulfate (Bio-Rad, Hercules, CA) as an initiator (initiator) and catalyzing the crosslinking between acrylamide and bisacrylamide (acrylamide/bisacrylamide 19:1) (BioRad, Hercules, CA) via 0.05% (w/v) Tetramethylethylenediamine (TEMED) (Bio-Rad). PEG gels are formed by the Michael addition between a four-arm PEG acrylate [ Molecular Weight (MW)10000] and a thiol-PEG-thiol (MW 3400; Laysan Bio, Arab, AL) in a molar ratio of 1: 2. In this study, MS2LAMP primers and probes originally developed by Ball et al were used and optimized. For each g lamp assay (25 μ L), the optimized hydrogel reaction mixture had the following composition: 10% (w/v) hydrogel, 12.5. mu.L of 2 × WarmStart LAMP master mix (Bst 2.0 blend of WarmStart DNA polymerase and WarmStartRTx reverse transcriptase; New England Biolabs, Ipshich, MA), 1.25. mu.L of 20 × Virus primer mix (F3/B3, final concentrations of FIP/BIP and LF/LB of 0.2. mu.M, 1.6. mu.M and 0.4. mu.M, respectively) and 2. mu.L of MS2RNA template or 2. mu.L of water sample. For reactions using complementary fluorescent probes and quenching primers, when fluorophore-labeled primers (5'FAM-FIP) were used instead of the conventional FIP primers, quenching primers (qFIP-3' IBFQ) were added (final concentration 3.2 μ M). The above 25. mu.L of hydrogel reaction mixture was loaded into an in situ PCR frame-sealed chamber (9X 9 mm; Bio-Rad) on a glass slide and then covered with a clear qPCR membrane (Sorenson, Salt Lake City, UT). The hydrogel was polymerized at room temperature (21 ℃) for 5-15min and then incubated on a PCR instrument (MJ Research PTC-100, Watertown, Mass.) or a small dry bath (Benchmark, Edison, NJ) for 25min at 65 ℃. After amplification, the gel was stained with 0.5 × LAMP dye (included in the WarmStart LAMP kit) in the dark for 15min, and then washed twice with 2 × TE buffer (pH 7.8; Corning, New York, NY). For reactions using complementary fluorescent probes and quenching primers, post-reaction staining is not required. Slides were irradiated with an E-Gel safety imager (Invitrogen, Carlsbad, Calif.) and amplicon spots were recorded with iPhone6s Plus. To verify the sensitivity of the smartphone detection system, the slides were also imaged using a fluorescence microscope (Leica DMi 8; Leica Co., Germany).
And (5) gLAMP determination optimization. The initial gLAMP development was performed using extracted MS2 viral RNA. The assay was performed to find the optimal staining strategy (post-reaction staining with LAMP dye or using fluorescent probes), incubation time (20min, 25min and 30min) and assay dynamic range (low, 1-20 copies/reaction; medium, 20-200 copies/reaction; and high, 200-2000 copies/reaction). Subsequently, to simplify the RNA extraction procedure, we also explored simple heating (95 ℃, 5min) as a pre-treatment procedure or direct detection of MS2 virions without RNA extraction. The assay sensitivity and dynamic range were compared to RT-qPCR using Eppendorf RealPlex2(Hamburg, Germany).
Tolerance of the g lamp to inhibitors present in environmental water samples. A total of three samples of ambient water were tested to assess the tolerance of the gillamp to inhibitors naturally present in the ambient water. Lake Water (LW) is collected from Echo Park Lake (losengles, CA) and is used primarily as a lagoon in urban storm drainage while providing recreational values (recreational functions) and wildlife habitats. Pool Water (PW) was collected from Turtle Pond, Calif. Institute of Technology (Calif.). The wastewater (WW) was collected from the settling and storage tanks of a pilot scale solar powered portable toilet system, also located on the Caltech campus. The WW is composed of urine, feces, and hand and toilet water. More details on the design and operating conditions of toilet systems have been reported in previous studies (Huang et al, wall Res., 92: 164-. Table 1 summarizes the basic water quality parameters of these samples.
TABLE 1: water quality parameters of environmental samples:
lake water
Pool water
Waste water from toilet
First-stage water outlet
pH
8.45
7.75
9.16
8.02
EC(mS/cm)
0.794
0.926
14.59
1.377
COD(mg/L)
60
74.7
821.3
1110
DOC(mg/L)
30
101
430
352
Dissolved Organic Matter (DOM) in environmental samples was characterized by excitation-emission matrix (EEM) using fluorescence spectroscopy (Shimadzu RF-6000, Kyoto, Japan). The corrected EEM is generated from the original scan (excitation wavelength: 250-550nm, 5nm interval; emission wavelength: 300-600nm, 2nm interval) and is used to estimate the various DOM components. The concentration of intrinsic MS2 (without preconcentration) in all of these samples was below the detection limit for plaque assay (1PFU/mL) and RT-qPCR (1 plaque forming unit/reaction). Thus, pure cultured MS2 was cultured at
Detection of MS2 in the primary effluent sample. To demonstrate the detection of MS2 in unincorporated natural water, a primary effluent wastewater sample was collected from a local sewage treatment plant servicing 15 million people. A20 mL sample of water was filtered through a 0.22 μm syringe filter (GE Whatman, Pittsburgh, Pa.) to remove bacteria and debris, and then further analyzed. For the double-layer plaque assay, F-specific e.coli phages were counted using e.coli temp (ATCC 700891) as the bacterial host, while total (somatic and F-specific) e.coli phages were counted using e.coli C3000(ATCC 15597). A total of 15mL of the filtrate was further concentrated to 150. mu.L using an Amicon Ultra-15 centrifugal filter (30kDa nominal molecular weight limit) (Millipore, Burlington, Mass.). Viral RNA was extracted from 100 μ L of the concentrate using the AllPrep PowerViral DNA/RNA kit (Qiagen, Germantown, MD) and then analyzed by g lamp and RT-qPCR.
And (4) gel selection. Clear polyacrylamide gels formed in 10-15min, whereas PEG gels formed faster, requiring 3-5min at basic pH (pH of LAMP reaction mixture 8.8). Neither gel showed a fluorescent background and in either case gLAMP was successfully performed. Experiments have shown that when the amplicon spot size is less than 20 μm (diameter), fluorescence microscopy will be required for detection. To facilitate reading of the results with a smartphone camera while still maintaining the actual (practical) assay dynamic range, dot sizes between 50 μm and 200 μm were used in this study. The size of the amplicon spot is mainly determined by the restriction of the gel matrix. Ideally, the gel matrix should allow free diffusion of small molecules (molecular weight (MW) <100kDa), such as water, ions, primers (<50bp, MW <15kDa) and enzymes (Bst:67kDa), but restrict movement of DNA and RNA templates and amplicons (>150bp, MW >100 kDa). This can be achieved by controlling the gel mesh size, and thus the macroscopic gel properties (i.e. diffusion), by adjusting the degree of crosslinking of the gel and the length of the crosslinking agent. It should be noted that unlike single length PCR amplicons, the products of LAMP are a mixture of concatemers of target regions with different sizes. Based on agarose gel electrophoresis patterns, the shortest MS2LAMP amplicon is about 90bp, while the longest amplicon is up to several thousand base pairs. During g LAMP, longer amplicons are retained by the hydrogel matrix, but shorter amplicons diffuse from the original template (the center of the spot) and serve as templates for further amplification until they reach the diffusion limit or nearby LAMP reagents (e.g., enzymes, primers, and dntps) are depleted. Due to the random nature of LAMP, spots of different sizes are generated in the hydrogel. According to previous studies, the mesh size of these 2 hydrogels was similar and in the range of 20-25 nm. However, the amplicon spots in the PEG gel were significantly smaller and more uniform than those formed in the polyacrylamide gel. The results show that PEG gels have a better restriction on smaller amplicons. Thus, in addition to size exclusion, other interactions (i.e., charge interactions) between the polymer and the DNA template may also affect the diffusion coefficient. PEG hydrogels were chosen for further method development in view of better template restriction.
gLAMP amplicon staining strategy. Clear MS2 amplicon spot spectra were obtained by gel staining after reaction with intercalating LAMP dye. Similar profiles were obtained in the gLAMP assay for E.coli and Salmonella. The results highlight the feasibility of converting an established qualitative LAMP assay into a quantitative assay by the g LAMP system. However, opening the frame-sealed chamber and staining the gel after amplification adds additional complexity to the assay and may lead to contamination of the ambient working environment with amplicons.
To develop a simpler gLAMP that did not require post-reaction staining, the present study adopted and optimized primer-dye and primer-quencher duplexes (QUASR). In QUASR, the Forward Inner Primer (FIP) is labeled at the 5 'primer end with a fluorophore (5' FAM-FIP). The probe was quenched with a complementary primer (qFIP-3 'IBFQ) with a quencher (Iowa Black FQ) at the 3' primer end. Since the melting temperature (Tm) of the complex is 5-10 ℃ lower than the reaction temperature (65 ℃), 5' FAM-FIP is released during the LAMP reaction and behaves like conventional FIP. When the target template is present in the sample, 5' FAM-FIP is incorporated into the LAMP amplicon. After the reaction, additional unincorporated 5 'FAMFIP was quenched again by the complementary quenching primer qFIP-3' IBFQ. In contrast, 5' FAM-FIP incorporated into LAMP amplicons will not be quenched because they have formed a stable double stranded DNA structure during the LAMP reaction. QUASR significantly reduces the problem of false positive results associated with LAMP assays compared to non-specific DNA intercalating dyes (i.e., LAMP dyes). However, QUASR cannot be converted to a quantitative determination of the real-time LAMP protocol because the fluorescence intensity of the reaction mixture is always at the highest level (all 5' FAM-FIP is released) rather than increasing gradually during the heat incubation. Therefore, QUASR can only be used as a qualitative measure of endpoint determination. In preliminary experiments, this study showed that the QUASR primer did not reduce the efficiency of the gillamp amplification, but a higher concentration of quenching primer (2x complementary probe primer) was required to maintain a clear gel background at the end of the gillamp reaction. These results indicate that PEG gels allow dye-labeled short oligonucleotides to move freely, even though the diffusion coefficient in the gel matrix may be smaller than in solution. Since the 5' famipip is incorporated into the amplicon and accumulates around the original template, bright and well-defined amplicon spots can be directly visualized with the smartphone camera after blue light exposure. Therefore, QUASR primers were used for further gillamp optimization.
And (5) gLAMP optimization. The amplicon spots were visible as early as 20min under the fluorescence microscope. After 25min, the spot developed to approximately 156 ± 33 μm (diameter) and the fluorescence intensity was strong enough to be detected with a smartphone camera. Although the size of the amplicon spots remained increased and reached 212 ± 50 μm after 30min, the number of spots remained similar to that at 25 min. Therefore, for MS2 g lamp, 25min was chosen as the optimal reaction time. The amplicon spot size did not show significant differences at low template concentrations (1-20 copies/reaction) and medium template concentrations (20-200 copies/reaction). Under these conditions, the amplicon spots are far from each other and have very limited interactions. The dot size represents the maximum size that an amplicon can develop in a given reaction time, while dimensional variability in a single gel can be caused by variable initial template conformation, degree of template denaturation, or local inhomogeneity in the hydrogel structure (due to free pendant ends of the macromer, looping or entangling itself). In contrast, the size of the amplicon spot at high concentration (200-. In g lamp, there is local competition for enzymes, primers and dntps because a clear separation is revealed between amplicons that are close to each other. The smaller amplicon size plus a clear border, which is apparent at higher template concentrations, facilitates the dynamic range of the assay by increasing the detectability of the fluorescent spots. For smartphone camera reading, the optimal measurement dynamic range is 1-1000 points/reaction. When reading results using a fluorescence microscope, each gel can accommodate up to 5000 spots without compromising accuracy. Automated amplicon analysis of microscope and smartphone images was achieved by CellProfiler 2.2.0. With appropriate threshold settings, the difference between the automatic and manual counts is less than 5%.
For nucleic acid-based detection methods, simple DNA and RNA extraction procedures are preferred in point-of-use applications. In the gLAMP analysis of the MS2 spiked PBS solution, the crude sample, the sample after simple heat (95 ℃, 5min) pretreatment, and the sample extracted with the commercial RNA extraction kit showed no significant differences in amplicon spot count, spot size, and amplicon fluorescence intensity. The current results indicate that LAMP primers and enzymes (RTx reverse transcriptase and Bst 2.0DNA polymerase) are able to penetrate the viral capsid at the reaction temperature (65 ℃) and denaturation may not be necessary because the viral genome is much smaller compared to the bacterial genome.
To assess the sensitivity of direct g LAMP, the method was compared to traditional plaque assays and RT-qPCR. gLAMP amplicon counts showed good correlation with plaque assay counts (R)2＝0.984，p<0.05). The regression line (slope 1.036, and intercept-0.290) indicates that 1 gel amplicon spot is nearly equal to 1 PFU. gLAMP reached a lower limit of detection (0.4 plaque forming units/reaction) compared to RT-qPCRSimilar lower detection limit (0.7 plaque forming units/reaction), while RT-qPCR still showed the advantage of a larger upper detection limit. As discussed previously, the dynamic range of g lamp (1-1000 plaque forming units/reaction) can be increased by decreasing amplicon spot size. Accommodating more amplicon points in a single gel would be desirable for applications such as mutation detection and in-gel sequencing.
Tolerance to inhibitors. Enzyme-driven nucleic acid amplification processes are susceptible to a variety of inhibitory substances (e.g., organic matter and heavy metals) commonly found in environmental samples. WW is yellow-brown and has a Chemical Oxygen Demand (COD) level of 821mg/L, representing highly contaminated water. LW and PW are clear and contain fewer organic contaminants with COD levels of 63mg/L and 75mg/L, respectively. All samples of environmental water spiked with MS2 (spiking level 2X 10)3To
The major fluorescent DOM peaks of PW and WW are the C and M peaks associated with the humoid component. The concentration of the humoid DOM in the WW was 10-15 times higher than that in the LW and PW, which is consistent with the COD and DOC data. WW also contains low levels of proteinaceous matter, as represented by the B and T peaks. Considering the source of WW, inhibitors may be of organic origin, similar to those found in urine and fecal samples. The presence of urea in urine samples is known to prevent non-covalent binding of polymerases and to interfere with primer annealing. In PCR, urea is inhibited at concentrations as low as 50mM, whereas LAMP is reported to be tolerant to urea up to 1.8M. Nevertheless, the better performance of gillamp in the WW cannot be simply attributed to the more robust LAMP chemistry. It is likely that the gel matrix plays a more important role in enhancing tolerance to inhibitors in the WW. First, like digital PCR, gollamp is an end-point amplification detection assay that counts the final amplification products. Therefore, its quantification is less affected by amplification efficiency. Second, because the DNA template and RNA template are spatially separated, substrate competition during amplification should be minimized. Furthermore, depending on their molecular weight, the mobility of large molecular weight organic inhibitors will be limited by the gel matrix and, therefore, the local inhibitor concentration close to the template is reduced.
MS2 in the primary effluent. MS2 (7.8. + -. 7.7PFU/mL) was successfully detected by gLAMP in RNA extracted from the primary effluent sample. Similar results were obtained in RTqPCR (1.13 ± 0.98PFU/mL), confirming the sensitivity and specificity of the g lamp assay. Escherichia coli C3000[ (6.9. + -. 0.4). times.10 ] was used3PFU/mL]And Escherichia coli Famp [ (2.6. + -. 0.7). times.10)3PFU/mL]Culture-based plaque assays as host cells yielded much higher counts. This difference is due to the sensitivity of the bacterial host used in the culture-based plaque assay to the wide variety of coliphage contained in the sample, whereas the g-LAMP and RT-qPCR assays are specific for MS 2.
The foregoing description is illustrative and not restrictive. While certain exemplary embodiments have been described, other embodiments, combinations, and modifications relating to the present invention will readily occur to those of ordinary skill in the art in view of the foregoing teachings. Accordingly, the present invention is to be limited only by the following claims, which cover at least some of the disclosed embodiments, as well as all other such embodiments and modifications, when read in conjunction with the above specification and the accompanying drawings.
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