Surviable cell detection and embodiments thereof

Abstract

A method for detecting viable cells, such as sterility detection, comprising the steps of: obtaining a sample; pre-treating the sample by maximizing the viability of at least one RNA-based biomarker, wherein the RNA-based biomarker is preferably versatile, rich and short-lived, and a method for preparing the same, wherein the method comprises the steps of: pre-treating the sample by maximizing the viability of at least one RNA-based biomarker, wherein the RNA-based biomarker is preferably versatile, rich and short-lived; processing the sample to facilitate a reagent to contact the RNA-based biomarker; amplifying the at least one nucleic acid sequence of the RNA-based biomarker using an amplification reagent; interacting with an amplification sequence of the RNA-based biomarker to produce a readout signal; and detecting the readout signal and determining the presence of viable cells in the sample. A pharmaceutical regimen for a short-lived drug implements the method for detecting viable cells, the drug being administered to a patient after confirmation of safety within three hours of obtaining the sample from the patient.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of and claims the benefit of provisional patent application No. 63 / 434,641, filed on December 22, 2022, the entire contents of which are incorporated herein by reference in their entirety. Statement Regarding Federally Funded Research

[0002] This invention was developed in part with government support under Grant No. R43FD006914 awarded by the U.S. Food and Drug Administration. The government has certain rights in this invention. Background of the Invention Technical Field

[0003] The present invention generally relates to the detection of viable cells and applications in microbial testing, such as sterility testing, and to industrial solutions for the manufacture and implementation of pharmaceuticals, medical treatments, foods, and beverages, as well as kits and systems for performing the same. Background Art

[0004] The industry currently utilizes culture-based microbial testing and its implementation methods for sterility testing. Culture-based techniques are time-consuming, requiring several days and requiring both manpower and infrastructure. Culture-based techniques target culturable, viable cells, including active microorganisms, dormant cells, and spores, but exclude dead cells (i.e., cells that are unable to reproduce). Viable cells are typically defined as living cells, such as those that synthesize proteins and have intact cell membranes.

[0005] Sterility refers to the absence of any viable microorganisms. As long as at least one viable cell, or a single spore that can develop into a viable cell, is present in a sample, the sample is not sterile. A given sample is considered sterile when it is demonstrated by recognized protocols that it contains even a single viable cell or spore.

[0006] Sterilization processes are used on products across a range of industries, including food and beverage manufacturing, but primarily in the pharmaceutical and medical fields, where sampling and testing of sterile products remains an important routine task for microbiologists.

[0007] Sterility assurance is crucial in the manufacturing of many pharmaceuticals and other medical products and is strictly regulated worldwide. Products bearing a sterility claim typically require some form of sterility testing prior to release to validate the claim. Ideally, a sterilization process (such as heat or ionizing radiation) is applied to the product in its final container at the end of the manufacturing process. However, some products contain heat- or radiation-sensitive ingredients that cannot be terminally sterilized in packaging. These products are typically sterilized by filtration followed by aseptic filling.

[0008] Sterility testing in the pharmaceutical and medical product sector is essentially a test to assess whether a pharmaceutical or medical product, after sterilization, is free from contaminating microorganisms, usually performed by incubating all or part of the product with a nutrient medium.

[0009] Sterility testing has important applications in monitoring the microbiological quality of filter-sterilized and aseptically filled products and serves as a final inspection of terminally sterilized items. When product sterility claims are made, sterility testing remains a crucial tool for pharmaceutical microbiology laboratories to determine compliance. Pharmaceutical regulations, such as the United States Pharmacopoeia, the European Pharmacopoeia, or the Japanese Pharmacopoeia, specify the procedures that must be followed. They detail the sample sizes and conditions to be used in specific situations. While pharmaceutical industry regulations may require or recommend sterility testing, it plays a relatively minor role in sterility assurance. By far, the greatest contribution to sterility comes from the validation and control of sterilization processes and / or aseptic processing procedures.

[0010] Today's traditional, culture-based sterility testing has several significant limitations for pharmaceutical and medical product quality control. Most notably, it's time-consuming, with traditional methods typically requiring 14 days to complete. It also requires both manpower and infrastructure. Clearly, such delays are becoming increasingly unacceptable in modern manufacturing operations.

[0011] Culture-based technologies are not suitable for pharmaceutical or medical products with a short shelf life and / or intended for immediate use. These pharmaceutical products include, for example, positron emission tomography (PET) tracers, CAR-T cells and novel cell therapy products, platelet transfusion products, gene therapy, and tissue engineering products. These products are often administered to patients before standard sterility testing is completed.

[0012] Initiatives such as Process Analytical Technology (PAT) and parametric product release are challenging the requirement to complete sterility testing before product release. Both the FDA and the EMEA are encouraging the adoption of new analytical technologies to help ensure final product quality. In the United States, the FDA's Center for Biologics Evaluation and Research (CBER) is proposing fundamental changes to sterility testing requirements for biological products, although not yet covering all drug products, to promote the use of rapid microbiological methods (RMMs) as an alternative to traditional sterility testing methods. This change in the regulatory environment has rekindled interest in RMMs for sterility testing, and a variety of technologies are now commercially available.

[0013] ATP-bioluminescence (adenosine triphosphate (ATP) bioluminescence) is a well-established, rapid method that utilizes a specific combination of substrate and enzyme (luciferin / luciferase) to decompose microbial ATP in growing cells and produce visible light that can be measured using a photometer. Several commercial systems have been developed for a variety of pharmaceutical testing applications (including sterility testing), particularly for filterable samples where non-microbial ATP in the sample is less of a concern. Because microbial growth in the culture medium is detected by ATP bioluminescence rather than visible turbidity, testing time can be significantly shortened. Typically, results comparable to pharmacopoeial tests are available in approximately 7 days.

[0014] Millipore Rapid microbial detection and enumeration ( The Rapid Microbiology Detection and Enumeration system also uses ATP bioluminescence to detect microbial cells and is designed for monitoring microbial contamination in filterable samples. It is automated and uses image analysis technology to detect microcolonies growing on the membrane filter surface immediately after adding a bioluminescent reagent. While the system is designed to be quantitative, methods have been developed and validated for rapid sterility testing with an incubation time of 5 days.

[0015] Colorimetric growth assays rely on the color change of the growth medium due to metabolism (usually CO2 production) during microbial growth. An example of a commercial colorimetric assay system that can be used for sterility testing is the bioMerieux 3D Dual-T Microbial Detection System. This automated system uses sensitive colorimetric detection and analysis technology to provide results in as little as three days. It detects aerobic and anaerobic bacteria, as well as yeast and mold.

[0016] Cell counting systems that do not rely on microbial growth but instead use cell labeling techniques to detect viable microorganisms for contamination detection have been proposed. This approach has the potential to detect a wide range of organisms, including yeasts and molds, in a matter of minutes. Commercial systems utilize fluorescent cell markers in conjunction with flow cytometry or solid phase cytometry to detect viable microbial cells. Typically, cells are labeled with a fluorescent dye or a non-fluorescent substrate that is converted to a fluorescent pigment in viable cells. The labeled cells are detected by laser scanning in a flow cell (flow cytometry) or on a solid phase platform such as a membrane filter (solid phase cytometry). AES Chemunex has developed a solid phase cytometry detection system. The company's (also known as ChemScanRDI) system is claimed to be able to detect 1 CFU per sample and has been evaluated as a possible sterility test RMM. Sterility testing system.

[0017] Viability PCR (vPCR) assesses cell membrane integrity. Propidium monoazide (PMA) covalently binds to DNA in cells with compromised cell membranes and inhibits PCR amplification upon photoactivation. In this method, false-positive results are expected for inactivated cells with intact cell membranes. False-negative results may occur for spores and competent cells. Furthermore, DNA is not abundant enough to allow detection of low cell concentrations (starting at 1 cfu / mL) (Nocker & Camper, 2009) (Emerson et al., 2017).

[0018] Sterility tests and other rapid methods for selective detection of live microorganisms are not commercially available. Published methods were developed for microbiome research and are used in conjunction with ribosomal rRNA sequencing (e.g., 16S RNA). Other methods have been developed to detect pathogens in samples, where the mere presence of the pathogen (without distinguishing between live and dead) in clinical samples is evidence of infection.

[0019] The above is a brief overview of RMM technology. There is still a need in the industry for methods that can quickly and sensitively detect low concentrations (starting at 1-5 CFU / mL) of viable prokaryotic and eukaryotic cells without the need for a culture step (cell division typically takes several hours to 14 days). A variety of RRM methods have been developed for microbial detection for different purposes, but no method has yet been developed that can efficiently, effectively and consistently achieve ultra-rapid detection of low concentrations (1-100 cells / mL) of viable microorganisms with versatility and the goal of detecting only viable cells. Since tests within 7 days are sometimes considered to be the most rapid in the art, in this application, the phrase "ultra-rapid" related to viable cell detection or sterility testing refers to a process that produces results within 2 hours, which obviously does not require a culture process. This solution to the ultra-fast processing requirements will enable breakthrough engineering technologies that can be applied to multiple fields of medicine and science, as well as industries that have long been waiting for ultra-fast sterility testing solutions. It will also enable ultra-fast bioburden analysis in the pharmaceutical industry and provide quality solutions for food and water safety control and diagnostics and testing. Summary of the Invention

[0020] The various embodiments and examples of the present invention presented herein should be understood as illustrative rather than restrictive of the present invention and should not be construed as limiting the scope of the present invention.

[0021] One embodiment of the present description provides a method for detecting viable cells, comprising the steps of: obtaining a sample for detection; pre-treating the sample for detection by promoting the abundance of at least one universal, short-lived RNA-based biomarker; performing a reaction to generate a readout signal from at least one nucleic acid sequence of the RNA-based biomarker; detecting the readout signal indicating the presence or absence of at least one viable cell in the sample; and analyzing the readout signal and reporting whether the method detects viable cells in the analyzed sample.

[0022] According to the method, the provided analysis sample may include viable cells to be detected, non-viable cells, a combination of viable cells and non-viable cells to be detected, or no cells. The analysis sample is reacted to detect abundant short-lived RNA molecules from viable cells in the analysis sample, wherein at least one nucleic acid sequence of the RNA-based biomarker is amplified using an amplification reagent. To detect abundant short-lived RNA molecules, reaction data can be obtained and analyzed. Analysis of the data determines one of two results: (1) the presence of viable cells in the analysis sample (regardless of whether the analysis sample contains non-viable cells), which is indicated by the presence of short-lived RNA molecules; (2) the absence of viable cells or the absence of cells in the analysis sample. When a threshold amount of reaction product (such as an amplification product) is detected directly or indirectly, the presence of viable cells in the analysis sample is indicated.

[0023] Another embodiment of the present description provides a method for sterility testing of a pharmaceutical product, comprising the following steps: obtaining a sample of the pharmaceutical product to be tested; pretreating the sample by promoting the abundance of at least one universal RNA-based biomarker; amplifying at least one nucleic acid sequence of the RNA-based biomarker using an amplification reagent; detecting a readout signal indicating the presence or absence of at least one viable cell in the sample; and determining the sterility of the sample and the pharmaceutical product based on the detected readout signal.

[0024] Another embodiment of the present disclosure provides a pharmaceutical regimen for administering a drug to a patient, comprising the steps of: obtaining a sample of the drug; pre-treating the sample for testing by promoting the abundance of at least one RNA-based biomarker; amplifying at least one nucleic acid sequence of the RNA-based biomarker using an amplification reagent; detecting a readout signal indicating the presence or absence of at least one viable cell in the sample; and, after determining the absence of at least one viable cell in the sample, administering the drug to the patient. In some embodiments, the drug is administered to the patient within 12 hours of obtaining the sample from the drug. Another embodiment of the present disclosure provides a kit for detecting viable cells in an analyte sample. The kit includes one or more pre-treatment reagents that promote conditions in the analyte sample such that viable cells present in the analyte sample are enriched for short-lived RNA molecules. The kit includes one or more RNA acquisition components for acquiring short-lived RNA biomarkers present in viable cells in the analyte sample, the components comprising: i) a lysis reagent for acquiring short-lived RNA molecules in the analyte sample; ii) a release reagent for acquiring short-lived RNA molecules in the analyte sample; iii) a capture agent for acquiring short-lived RNA molecules in the analyte sample; and physical reagents for acquiring short-lived RNA molecules in the analyte sample. One or more detection agents for detecting the presence of a short-lived RNA biomarker, wherein the detection agent is detectable directly or indirectly to indicate the presence of a short-lived RNA that is present in viable cells and absent in non-viable cells.

[0025] According to another embodiment of the present invention, the test kit may include an imaging agent for detecting the presence of short-lived RNA biomarkers. Other components and devices may also be included in the test kit, including one or more RNA acquisition reagents, one or more positive or negative control reagents, and reagents and materials for nucleic acid amplification. In some embodiments, the test kit may include a staining agent or one or more oligonucleotide probes for RT-PCR or RT-LAMP, which target short-lived RNA biomarkers as detection agents. In some embodiments, the biomarker is a non-coding short-lived RNase P RNA subunit (RPR), and the probe is specific for the conserved regions (CR1, CR2, CR3, CR4, CR5) in the RPR, or the variable region between CR1 and CR5.

[0026] Another embodiment of the present invention provides a kit for determining the presence of viable cells in an analytical sample, the kit comprising: an RNA acquisition component for acquiring short-lived RNA molecules present in viable cells in the analytical sample, the component comprising: i) one or more pretreatment reagents that provide conditions in the analytical sample such that viable cells present in the analytical sample are enriched with short-lived RNA molecules; and ii) a lysis reagent, a release reagent, a perforation consumable, or a capture agent for acquiring short-lived RNA molecules in the analytical sample; and one or more RT-PCR or RT-LAMP primer sets targeting a short-lived RNA biomarker, wherein the biomarker is RNase P RNA (RPR), wherein the primer set anneals to the entire or partial sequence of the conserved regions of CR1, CR2, CR3, CR4, and CR5, and wherein the RT-PCR or RT-LAMP primer set can be detected directly or indirectly to indicate the presence of biomarker RNA that is present in viable cells and absent in non-viable cells.

[0027] Also described herein are systems for detecting viable cells in a sample that can comprise the kits described herein, as well as other components, including one or more reaction vessels, an area for changing the temperature of one or more reaction vessels, an area for manipulating cells to allow access to cell contents, and an area for biomarker detection.

[0028] In some embodiments, the methods, protocols, and kits and systems described herein are used for ultra-rapid viable cell detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description, appended claims and accompanying drawings, in which like reference numerals represent like elements, and in which:

[0030] Figure 1 is a schematic flow chart of a viable cell detection method according to one embodiment of the present invention.

[0031] Figure 2 is a schematic flow chart of a pharmaceutical regimen for administering a drug to a patient according to one embodiment of the present invention.

[0032] Figure 3 FIG. 4 is a schematic flow chart of a method for detecting viable cells by LAMP amplification according to one embodiment of the present invention.

[0033] Figure 4 Kits and systems for practicing the methods of the present invention are schematically shown.

[0034] Figure 5is a schematic flow chart of a pharmaceutical regimen for administering a drug to a patient according to one embodiment of the present invention.

[0035] 6 is a schematic diagram of an at least partially automated system for implementing the described method.

[0036] Figure 7 is a table showing exemplary universally conserved regions of RNaseP RNA, with relevant regions highlighted to indicate common nucleotides.

[0037] Figure 8 are images showing gel electrophoresis analysis of RNaseP PCR products.

[0038] Figure 9 is an image showing gel electrophoresis analysis of RNaseP RNA subunit RT-LAMP products.

[0039] Figure 10 The graph shows qPCR curves of RT-qPCR experiments performed with dilutions of RNA obtained from resuscitated microorganisms (Gram-negative bacteria, Gram-positive bacteria, fungi).

[0040] Figure 11A and 11B The graph shows LOD data for detection sensitivity of RNAse P RNA subunits in pre-treated (revived) bacterial and fungal cells. DETAILED DESCRIPTION

[0041] According to the present description, a method 200 is provided for detecting the presence of viable cells in a sample 20. The presence of viable cells in the sample 20 is determined by detecting a short-lived RNA biomarker 40 indicative of the presence of viable cells in the sample 20. Also described are protocols 220, detection agents, kits 250, and systems 230, 280 for detecting the presence of short-lived RNA biomarkers 40, and detection agents.

[0042] The methods 200, protocols 220, assays, kits 250, and systems 230, 280 described herein improve the ability of pharmaceutical manufacturers to produce drug products 150 that meet patient safety requirements, thereby improving clinical practice. Also described are ultra-rapid methods 200 and protocols 220, as well as assays, kits 250, and systems 230, 280 for use in the ultra-rapid methods and protocols, which allow for rapid (within 1 hour) sterility testing, providing true pre-release sterility results for drug products 150 with short shelf lives and / or intended for immediate use. This description provides a paradigm shift for patient safety in PET pre-release sterility testing. Beyond PET, this description represents a significant improvement in the drug release testing process. The quality control (QC) process implemented in this description for sterile, short-lived products (completed rapidly within 1 hour) can be rapidly adopted in the pharmaceutical industry, and the same method can be extended to other areas requiring rapid detection and widespread identification of viable microorganisms. Furthermore, the availability of a 1-hour 1-CFU sterility test will challenge and transform current practices well beyond the realm of short-lived products, enabling, for example, traditional pharmaceutical manufacturers to eliminate the 14-day quarantine period for their products.

[0043] The methods 200, protocols 220, assays, kits 250, and systems 230, 280 described herein, including assay primers and ultra-rapid ultra-sensitive sterility tests, address an unmet need in the industry as they meet customer demands for 1-hour testing in the context of FDA 1-CFU requirements and positively impact patient safety, sterility compliance, and workflow efficiency.

[0044] Reference Figure 1 , provides a schematic flow chart of a sterility testing method 200 according to one embodiment of the present disclosure, which tests for viable cells in a sample 20. The method 200 described herein to detect viable cells and sterility has one or more target biomarkers 40 present on short-lived RNA molecules, i.e., short-lived RNA biomarkers 40. The short-lived RNA biomarkers 40 described herein are substantially abundant in viable cells and substantially absent in non-viable cells. Therefore, detection of a threshold amount of short-lived RNA biomarkers 40 indicates the presence of viable cells and indicates that the sample is not sterile.

[0045] The present invention provides a method 200 for detecting viable cells, such as a sterility test method, comprising the following steps: obtaining 10 a sample 20 for detection; pre-treating 30 the sample 20 by promoting the abundance of at least one short-lived RNA biomarker 40 (which can also be expressed as maximizing the abundance of RNA-based biomarkers), wherein the short-lived RNA biomarker 40 is universal (i.e., present in more than one biological kingdom, preferably at least three or four biological kingdoms), abundant (i.e., present in sufficient amounts in cells) and short-lived (i.e., having a lifespan of less than 18 s in living cells). 0 minutes, preferably less than 120 minutes, more preferably less than 60 minutes); or absent in cells that have died for 60 minutes, preferably absent in cells that have died for less than 30 minutes; treating 60 the sample 20 with a detection agent 70 and contacting it with a short-lived RNA biomarker 40; amplifying 80 at least one nucleic acid sequence of the short-lived RNA biomarker 20 using an amplification reagent 70; interacting 90 with the amplified sequence of the RNA-based biomarker 40 with the detection agent to produce a readout signal 110; and detecting 100 the readout signal and determining sterility (i.e., whether viable cells are present in the sample). It should be noted that viable cells can be stored at low temperatures, freeze-dried, and under other conditions in which short-lived RNA can still be present after a longer period of time.

[0046] Conditions in the analyte sample 20 are provided such that the viable cells to be detected (when present in the analyte sample 20) have short-lived RNA molecules. Optionally, a pretreatment reagent is provided in the pretreatment step 30 and combined with the analyte sample 20 to provide at least one RNA-based biomarker 40. The sample 20 is treated (step 60) to facilitate contact of the reagent 70 with the short-lived RNA-based biomarker 40. The treatment 60 may include combining the analyte sample 20 with an RNA acquisition component 50 (not shown) and manipulating the sample to acquire the short-lived RNA biomarker 40. The analyte sample 20 is then combined with a detection agent (i.e., an amplification reagent 70) that interacts with the manipulated sample to amplify 80 at least one nucleic acid sequence of the short-lived RNA-based biomarker 20. At step 90, the combined analyte sample 20 also includes a detection agent and an optional control reagent. The detection agent generates a readout signal 110, which is detected and analyzed at step 100 to detect the presence of the short-lived RNA biomarker 40 in the sample 20. The presence of short-lived RNA biomarker 40 detected in the analytical sample 20 indicates the presence of viable cells, or a combination of viable and non-viable cells, in the analytical sample 20. As described above, when the detection signal of the short-lived RNA biomarker 40 is above a threshold value determined by the negative control signal, the method results indicate the presence of viable cells and indicate that the sample is not sterile. When the copy number of the short-lived RNA biomarker in viable cells is estimated to be at least 300 copies / cell, or preferably at least 500-1000 copies / CFU, then when the sample 20 contains less than 1000 CFU of dead cells or no cells, the readout signal 110 will be absent or below the threshold amount. The presence of signal 110 indicates the presence of viable cells. In the method flow, the detection of short-lived RNA biomarker nucleic acid sequences in total RNA obtained from viable cells above the threshold signal can also be considered a reflection of the sensitivity of the method. Above a selected threshold signal, method 200 can be used to detect nucleic acids of short-lived RNA biomarkers as low as 1 CFU, and thus method 200 can be accurately defined as implementing a readout signal 110 that is capable of indicating the presence or absence of at least one viable cell within sample 20.

[0047] Exemplary viable cells to be detected include microorganisms, more preferably pathogenic microorganisms, examples of which include gram-positive bacteria, gram-negative bacteria and fungi. Other cells to be detected may include tumor cells, and may also include cells that have undergone cell therapy. Exemplary bacteria / true bacteria include gram-positive bacteria (Bacillus, etc.), gram-negative bacteria (Pseudomonas aeruginosa (P.aeruginosa), etc.), mycobacteria (Mycobacterium smegmatis (M.smegmatis), etc.), mycoplasmas, and bacteria including aerobic bacteria (Escherichia coli (E.coli), etc.) and anaerobic bacteria (Clostridium). Other exemplary species include molds (Aspergillus, etc.), yeasts (Candida, etc.), protozoa (such as amoeba), unicellular eukaryotes (such as protozoan species), photosynthetic eukaryotes other than plants, chromoalgae species, and archaea / archaea (including species associated with the human microbiome).

[0048] The analytical sample 20 can be a drug 150 or a radiopharmaceutical sample, such as a PET tracer, or a drug 150 or radiopharmaceutical sample that is delivered 180 to a patient by injection. Other embodiments include analytical samples 20 for rapid detection of viable microorganisms, such as for biohazard detection. Still other embodiments of the analytical sample 20 include cells treated with a pharmaceutical agent 150 to determine the sensitivity of the cells to the pharmaceutical agent 150, such as a drug or cancer therapeutic agent.

[0049] The analysis sample 20 can be 1 to 100 microliters, or have a larger volume. In certain embodiments, the analysis sample 20 is 1 to 10 microliters. In some embodiments, the sample 20 is a membrane filtered sample, a surface swab, or an air sampling method sample. In other embodiments, the sample 20 is a solid tablet, a pharmaceutical product, or a supplement. The sample 20 can also be a medical device, an implantable object, or an extract thereof. In some embodiments, the sample 20 is a food or beverage product. The sample 20 can also be a human specimen, such as a nasopharyngeal swab, sputum, and normally sterile samples, such as cerebrospinal fluid, blood, and urine, as well as tissue bank samples. The sample 20 can be a bacterial or eukaryotic cell culture, whether natural or a culture that has been treated to inactivate or disinfect cells.

[0050] 10 Get sample 20

[0051] The first step of the viable cell test method 200 of the present disclosure is to obtain a suitable sample 20 of a test subject (such as a drug 150) in step 10 herein. Obtaining a suitable sample 20 includes conditions that minimize accidental contamination. As is known in the art, it is crucial to obtain accurate results and minimize the accidental contamination introduced during the test. For example, in the sterility test application of the inventive method 200, a false positive result inevitably means that the object / product of the tested batch or batch group will be judged as non-sterile. Therefore, obtaining or collecting 10 the sample 20 of the method 200 described herein preferably follows a high level of contamination control. For example, at least the level of an aseptic filling facility is equivalent. This can be relevant to an ISO 5 class clean room or equivalent facilities, or an isolator can be used to provide a barrier between a laboratory environment and the test subject or product.

[0052] The pharmacopoeial method for sterility testing requires that the sample be cultured in two different culture media. These are typically fluid thioglycollate broth (FTM) (for culturing anaerobes and some aerobic bacteria) and soy casein digest broth (SCDM) (for culturing fungi and aerobic bacteria). The cultures are incubated at 32.5°C and 22.5°C for 14 days and then examined. Any turbidity in the culture medium may indicate growth and must be investigated. There are two recommended methods for performing the test. The first is direct inoculation, where a relatively small amount of sample is aseptically removed from the sample unit and inoculated directly into a suitable volume of growth medium, which is then incubated. Direct inoculation in traditional processes has some significant disadvantages. First, only a small amount of product can be inoculated into the culture medium, limiting the sensitivity of the test. The present method 200 does not have these disadvantages because it is effective for small volumes, so small volume samples 20 are completely acceptable and even preferred.

[0053] Returning to the traditional method, to overcome the shortcomings, the recommended sample acquisition method is membrane filtration whenever possible. In membrane filtration, the sample is passed through a 0.45 μm membrane filter and the filter is then transferred to a culture medium for incubation. Membrane filtration allows the entire sample or composite sample to pass through a single filter and is therefore likely to be much more sensitive than direct inoculation. Filtration also provides the opportunity to rinse out components in the sample that may cause turbidity and any growth inhibitors (such as antibiotics or preservatives) that may be present. The membrane filtration method can be performed using a traditional "open" filtration system or one of the commercially available closed systems in which the sample is never exposed to the test environment, thereby minimizing the chance of contamination and false positive results. A closed membrane filtration system can be formed by a connecting device and a piping system to aseptically extract samples from ampoules, collapsible bags and other containers without exposing the sample to the external environment. As discussed above, membrane filtration can be used to obtain sample 20 in method 200 of the present invention.

[0054] In an embodiment of method 200, method 200 is used for ultra-rapid detection, preferably within 2 hours, but in other embodiments, ultra-rapid detection of viable cells is completed within 15 minutes, 30 minutes, 45 minutes, 1 hour, 1 hour and 15 minutes, 1 hour and 30 minutes, or 1 hour and 45 minutes.

[0055] 30 Pretreatment samples 20

[0056] Obtaining the sample 20 is followed by a step 30 of pre-treating the sample 20 to favor the abundance (which can be described as maximizing the abundance, i.e., concentration) of the RNA biomarker 40. In the pre-treatment step 30, conditions are provided in the sample 20 for analysis such that any viable cells in the sample 20 will be enriched for the short-lived biomarker 40.

[0057] This pretreatment 30 primarily requires placing the sample 20 in pretreatment reagents (such as culture media and reagents) that provide conditions for the cells, dormant organisms, and spores of interest to "wake up" and metabolize for a short period of time, typically 20-30 minutes. This pretreatment step 30 is used to stimulate cell activity (viability) without waiting for replication (as required for most traditional tests), which makes the pretreatment step 30 short and significantly different from culturing. This step 30 can also be called a recovery step 30 for RNA-based biomarkers 40 to ensure that the analyzed sample 20 is rich in short-lived RNA molecules 40. The recovery step 30 can also include a step to remove preservatives that may hinder the stimulation of RNA-based biomarkers 40. For some samples 20, particularly solid samples 20, it may be necessary to dissolve, or expose the solid object to the pretreatment.

[0058] The pretreatment step 30 provides the optimal pH, temperature and nutrients required for normal metabolism of viable cells (if present). The process is preferably short (less than 4, 6, 10, 20, 30, 40, 50 minutes, or less than 1 hour).

[0059] The enriched medium used in the resuscitation step 30 can be a natural or synthetic cell culture medium, but it does not have the same requirements as traditional growth medium, which is designed to achieve optimal growth and proliferation during the culture period (greater than 14 days). The enriched medium of the resuscitation step 30 is used to resuscitate dormant cells rather than to provide nutrients to the sample for culture and cell growth. If the test is to be changed to a different type of cell, a different type of culture medium can be used. When the sample 20 is divided into two or more parts, two or more pretreatment reagents can also be used to pretreat the cells in the analysis sample 20 30. The resuscitation step 30 may include a resuscitation period of 5-80 minutes at an elevated temperature (e.g., 25-40°C).

[0060] In some embodiments, different types of culture media are used to culture different types of cells. In other embodiments, at least a portion of the analysis sample 30 is divided into at least two or more portions for pretreatment 30 .

[0061] In other embodiments, the cell culture medium is a sterile cell culture medium or a cell culture medium known to be sterile. In other embodiments, two or more pretreatment reagents are provided for pretreating cells in the analysis sample 20.

[0062] In some embodiments, the method includes a short "pretreatment" step 30 in which the sample 20 is incubated with one or more pretreatment reagents (such as a nutrient-rich universal medium), followed by rapid lysis 60 or disruption of the membranes of the cells (if any are present), and then analysis of the products of the amplification step in a contamination-free platform 280.

[0063] The methods can also be used to perform pretreatment 30 and / or lysis 60 procedures optimized for specific types of organisms, including but not limited to aerobic and anaerobic bacteria, including but not limited to eukaryotic cells, including but not limited to fungal cells and spores.

[0064] Examples of pretreatment agents include growth media for culturing fastidious aerobic and anaerobic bacteria. Examples of suitable enrichment media include brain heart infusion (BHI) broth, minimum essential medium, Dulbecco's modified Eagle's medium, Iscove's modified Dulbecco's medium, and RPMI-1640.

[0065] Brain Heart Infusion (BHI) broth is a medium containing bovine brain extract and disodium phosphate. It has many variations. Another exemplary pre-treated medium includes a pepsin digest of sheep blood (which can be added to aid the growth of fastidious microorganisms). The addition of 0.1% agar can be used to reduce oxygen tension to enhance the growth of anaerobic organisms. This also has many variations.

[0066] Additional components may be added to the culture medium used, such as buffers to support growth, and additives to neutralize the sample that may act as preservatives or in any way prevent cellular metabolism (e.g., sodium citrate, sodium benzoate, ethanol, etc., which may be neutralized by adding magnesium ions or dilution). Under certain conditions, an agent that lyses the fungal and / or bacterial cell wall, such as, but not limited to, ready-to-use lysozyme, may be added to the pretreatment reagent.

[0067] The pretreatment or resuscitation step 30 can resuscitate dormant cells and spores. Data show that spores activate RNA synthesis and protein synthesis within the first 15-30 minutes of exposure to the resuscitation enrichment medium, which is essential for germination. Further data also show that permeable (active) spores regain metabolic activity after being exposed to the enrichment medium for less than 10 minutes. Therefore, the pretreatment step 30 for resuscitating microorganisms ensures that the selected essential target RNA 40 is expressed. In some embodiments, a brief nutritional stimulus (pretreatment 30) is provided to the sample 20 at a physiological temperature (equal to or less than 25°C, 33°C, 35°C, 40°C) for a short period of time (equal to or less than 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 60 minutes, 80 minutes).

[0068] RNA-based biomarkers40

[0069] A key aspect of the present invention is the use of short-lived RNA biomarkers 40 as universal biomarkers for viable cells in the method 200 of the present invention. It is worth noting that not every molecule in a cell can be used as a universal biomarker for viable cells. Short-lived RNA molecules are identified as target biomarkers for detecting viable cells and performing rapid sterility tests. These short-lived RNA biomarkers 40 provide sterility tests with high sensitivity and universal microbial applicability. The short-lived RNA biomarkers 40 of the present invention are: (1) ubiquitous in a large number of living microorganisms, that is, present in a wide range of biological groups, especially in multiple biological kingdoms (optionally, at least three or four); (2) short-lived and essentially absent in dead (non-viable) cells; (3) abundant in living cells to allow for high sensitivity of assays using nucleic acid amplification; and (4) the target biomarker sequences are satisfactorily conserved, allowing for the design of degenerate universal primers for amplification.

[0070] Organisms are traditionally divided into three domains, which are further subdivided into one of six kingdoms. Organisms are assigned to these categories based on similarities or shared characteristics. The two main cell types are prokaryotes and eukaryotes. Prokaryotes (such as the domains Archaea and Fungi) lack a nucleus, while eukaryotes do. Eubacteria are the organisms usually referred to when discussing bacteria, while Archaea (originally thought of as bacteria) are significantly different from the other two domains. The Eukaryota domain includes a very diverse group of organisms, some with animal characteristics (protozoa) and others that resemble plants (algae) or fungi (slimemolds).

[0071] The RNA biomarkers 40 used in the method 200 of the present invention are preferably universal, meaning that they are present in a wide range of biological groups, particularly in more than one kingdom, preferably at least three kingdoms, and even more preferably at least six kingdoms. In some embodiments, the RNA biomarkers may have regions in their sequences that are universally conserved among different species in a single phylogenetic domain and may be present in multiple species in each of three different phylogenetic domains.

[0072] Furthermore, within the meaning of the present application, the "short lifespan" of a short-lived RNA biomarker 40 refers to a lifespan of less than 180 minutes (preferably less than 120 minutes) in living cells, or absence after 60 minutes, preferably 30 minutes, of cell death. Not every molecule in a living cell can be detected at low concentrations within a short time. The present invention utilizes a type of nucleic acid testing (NAT) that identifies a short-lived and abundant portion of nucleic acids in living cells, thereby distinguishing them from non-viable (dead) cells. NAT differs from other tests in that it interacts with genetic material (RNA or DNA) rather than antigens or antibodies. The testing method is extremely sensitive, allowing detection of fewer than 10 copies in a reaction, but this typically means that multiple copies are present in each analyzed sample. Due to the low efficiency of extraction, transfer, reverse transcription, and template amplification, this sensitivity can be difficult to achieve in conventional tests. Therefore, the nucleic acid target in the present invention should be highly expressed or abundant, in other words, a nucleic acid target with a large number of molecules per cell. In the meaning of the present application, abundant means that the nucleic acid target is present in at least 300 copies within a cell.

[0073] The method 200 of the present invention focuses on identifying viable cells, and short-lived RNA biomarkers 40 have been found to be substantially abundant in viable cells. For the purposes of this application, "substantially abundant RNA" refers to RNAs present in an average prokaryotic cell, numbering at least 300-1500 molecules. However, RNAseq data are widely available, and substantially abundant RNAs can often be quantified by comparison with other molecules and spike-in internal controls in available RNAseq experiments. For more information, see https: / / rationumbers.hms.harvard.edu / search.aspx.

[0074] There are several functional RNAs that meet the requirements of universal conservation, short lifespan, and abundance, and can effectively form the biomarker 40 of the present invention. These include ribozymes (RNAzymes), tRNAs, some small regulatory RNAs, some transfer messenger RNAs (such as SsrAtmRNA in bacteria, which acts as both tRNA and mRNA, releasing stalled protein biosynthesis), and some signal recognition particles (such as 4.5S RNA in bacteria). There are many functional RNAs that meet the requirements of abundance, universality, and short lifespan, and can effectively form the biomarker 40 of the present invention. These include some mRNAs encoding ribosomal subunit proteins, such as 50S and 30S ribosomal subunit proteins, such as L33, L30, L27, L35, and S15. Some mRNAs meet the desired parameters only under specific conditions. For example, mRNAs encoding proteins involved in amino acid biosynthesis, such as the mRNA encoding N-acetylglutamate synthase (ArgA), are present at 28 molecules per cell when cells are in rich medium, but at 1129 molecules per cell in glucose-supplemented minimal medium, where enzyme activity is highly required. Many RNAs also do not meet the requirements, most notably ribosomal RNAs (such as 16S rRNA) because they are stable in dying cells.

[0075] Exemplary abundant RNAs have housekeeping functions that are always required in living cells. Examples include: ribozymes (excluding RNAse P RNA and ribosomal RNA above), regulatory RNAs, and tRNAs are examples of abundant RNA molecules with functions other than encoding proteins. Other examples include: mRNAs encoding abundant, highly conserved essential housekeeping proteins, such as the small and large subunits of ribosomal proteins, and short-lived abundant RNA molecules that perform the same basic essential functions and are therefore ubiquitous in cells of more than one biological kingdom. Examples include RNAse P RNA (because RNA is involved in the maturation of the cellular translation machinery) and tRNA (which is directly used for translation in all living cells). Other examples include short-lived abundant RNA molecules that perform the same basic essential functions and are ubiquitous in cells of more than one biological kingdom (and therefore have conserved regions in the nucleic acid structure that are related to performing the essential functions). In particular, the conserved regions in the nucleic acid sequence should have a consensus sequence shared by a large number of microorganisms and cells. For example, RNAse P RNA subunits, types, and structures are described in the rfam database https: / / rfam.org, searching for the query "RNase P".

[0076] RNA analysis was used to develop biomarkers. 40 Ribosomal RNA (rRNA) was identified as a potential biomarker because it has a highly conserved universal sequence and is highly abundant, thus allowing for high sensitivity in microbial detection. However, because rRNA can also be found in dead cells, other target biomarkers were identified to avoid the unintended detection of dead cells by targeting rRNA. The ribozyme ribonuclease P (RNAse P) 12 RNA subunit was identified as a biomarker for the presence of viable microorganisms because RNAse P is short-lived, highly abundant in living (viable) cells, and essentially absent in dead (non-viable) cells. For example, RNase E ribonuclease is present at approximately 1500 copies per cell, and the number of RNA polymerases is estimated to be ~4,500 per cell. RNAse P is expected to be present at similar concentrations, but it has the specific advantage that each copy of RNAse P carries a copy of enzymatically active RNA that can be amplified and detected.

[0077] Databases confirm that RPR subunits are universally conserved across species. The Rfam database contains 9,332 collated RPR sequences and their alignments; seed alignment sequences were obtained from the RNase P database, while a more comprehensive alignment was obtained by searching Genbank (NCBI) using an in-house search engine. The results showed that bacterial RNase P is represented by at least 7,091 species; 6,324 species have type A RNase P, and 767 species have type B sequences. Fungal nuclear RNase P RNA subunits are represented by a total of 392 species and 493 sequences. Most fungal sequences in the database are associated with two major Dikarya groups: (1) Ascomycota types (263 species and 276 sequences in the classes Saccharomycetes, Sordariomycetes, Eurotiomycetes, Pneumocystidomycetes, Dothideomycetes, Pezizomycetes, Leotiomycetes, Taphrinomycetes, Xylonomycetes, and other closely related types); and (2) Basidiomycota (83 species and 162 sequences for seed alignment), which includes notable types such as Agariomycetes and Tremellomycetes. Other fungal phyla with highly consistent RNase P RNA subunits included Microsporidia (21 sequences, 21 species), Mucoromycota (16 sequences, 13 species), Fungiincertaesedis, Blastocladiomycota, Chytridiomycota, and Zoopagomycota. Thus, the selected RPR target biomarkers demonstrated satisfactory universality through in silico analysis.

[0078] Similar assessments can be performed on other genes to allow identification of alternative biomarkers 40 .

[0079] In the embodiment, the target RNA biomarker 40 is a non-coding short-lived RNA molecule selected from the group consisting of RPR, tRNA or small non-coding RNA. In an exemplary embodiment, the RNA subunit of ribonuclease P (RNase P) present in living organisms is selected as a short-lived RNA biomarker to distinguish living cells from dead cells, because RNase P RNA subunits (RPR) are commonly (i.e., substantially abundant) present in living organisms of all life domains, including archaea, eukaryotic nuclei and mitochondria. The RNA subunit structure is functionally conserved in different bacteria, showing two main types (A and B), and is conserved in fungal species. Figure 7 is a table showing an alignment of universally conserved regions of RNAse P RNA, as described in RNA (1997), 3:557-560 (Cambridge University Press), the entire contents of which are incorporated herein by reference. The alignment includes RNase P RNAs from three eukaryotes (E), three archaea (A), three bacteria (B), and one mitochondrial. The highlighted nucleotides represent Figure 7 The nucleotides shared by all 10 RNase P RNAs shown in are shown in Figure 3. The CR sequence is indicated above the alignment with a bar. The number between each CR is the number of nucleotides omitted from the alignment. The secondary structure associated with the CR is indicated below the alignment. In an embodiment, the short-lived RNA biomarker 40 is the complete or partial sequence of one or more of the conserved regions CR1, CR2, CR3, CR4, and CR5 of the RNase P RNA subunit.

[0080] 60 processed samples 20

[0081] The method 200 of the present invention may include a step 60 of treating the sample 20 to facilitate contact of the reagent 70 with the short-lived RNA biomarker 40 prior to amplifying 80 at least one nucleic acid sequence of the RNA-based biomarker 40. Essentially, this is a step of manipulating the cells to enable interaction with the biomarker 40. To acquire the target biomarker 40, the analysis sample 20 may be manipulated by chemical, biological, and physical forces to allow interaction with the cell contents. Acquisition may be achieved by using an RNA acquisition component (including reagents and devices that provide for acquisition of short-lived RNA biomarkers present on cells in the analysis sample). The RNA acquisition component may include a lysis reagent, a release reagent, a capture agent, and a physical reagent.

[0082] Physical methods for cell lysis used in method 200 of the present invention include mechanical disruption of the cell membrane, such as repeated freezing and thawing, sonication, pressurization, or filtration, which can also be referred to as lysis. Another physical method for cell lysis is sonication, which uses ultrasound to generate high and low pressure areas, resulting in cavitation and, in turn, cell lysis. Examples of hardware devices for RNA acquisition components include electroporation cells, bead mills, spin columns, negative pressure devices, and magnets.

[0083] Chemical cell lysis methods used in method 200 of the present invention include chemical destruction cell lysis, i.e., chemically destroying / dissolving proteins and lipids present in the target cell membrane (this is the most commonly used lysis process in the laboratory and is well known). Chemical cell lysis methods used in method 200 of the present invention include enzymatic lysis, i.e., using enzymes such as lysozyme or protease to disintegrate the cell membrane 80.

[0084] Additional conditions for cell lysis used in method 200 of the present invention include: using a lysis reagent (non-ionic detergent) and adding additives to the lysis buffer that allow minimizing adsorption to plastic (1 mg / mL BSA (more than 0.01 mg / mL)) and chemicals that allow preservation of RNA (2 mM DTT (more than 0.02 mM)), as well as a ribonuclease inhibitor that is active at high temperatures (SUPERAseln 0.5 u / uL (more than 0.005 u / uL)), all components are compatible with downstream nucleic acid detection methods without the need for intermediate purification.

[0085] The cell lysis method used in the method 200 of the present invention can divide the analysis sample 20 into two or more parts to manipulate the analysis sample 20 for obtaining the content of viable cells to be detected.

[0086] Amplify sample 20, process 90 and detect 100

[0087] After processing 60 the sample 20, at least one nucleic acid sequence of the RNA-based biomarker 40 is amplified 80, typically using an amplification reagent. In the following discussion, it is noted that a portion of the total lysed sample can be used for amplification.

[0088] Since short-lived RNA biomarkers 40 are generally conserved, they can be used as binding sites for universal primers, referred to herein as detection agents, detection primers, or amplification reagents for detecting viable cells. The amplification products reacted by the amplification reagent with the short-lived RNA biomarker 40 identify the presence of viable cells. The target biomarker nucleic acid sequence 40 may be present in nominal amounts in non-viable cells, for example, 1 copy to tens of copies of the target nucleic acid sequence can be found in the DNA of non-viable cells. However, since the target nucleic acid sequence is substantially abundant in viable cells and not abundant in non-viable cells (for example, a ratio of 300: 1, 1000: 1, 1500: 1), the amplification products reacted by the amplification reagent with the analysis sample can be used to identify the presence of viable cells (even in the presence of non-viable (dead) cells), and can be distinguished from samples containing only non-viable cells or samples without cells. For example, in an analysis sample 20 comprising viable cells, or a combination of viable cells or cells, the amplification product can be detected at a threshold amount to identify the presence of viable cells. In an analysis sample 20 containing nonviable cells or no cells, some amplification products may be detected, but not in a threshold amount. Therefore, the amplification products resulting from the reaction of the amplification reagent 70 with the analysis sample 20 can be used to detect the presence or absence of viable cells in the analysis sample 20.

[0089] Provided is a detection agent 70, also referred to as an amplification reagent, or more specifically, a detection primer, for detecting the presence of a viability biomarker 40 (such as a short-lived RNA biomarker) present on a short-lived RNA molecule on a viable cell. When an analysis sample 20 containing a viability biomarker 40 is combined with a detection agent 70, a reaction product (such as an amplification product) is detected directly or indirectly in a threshold amount, indicating the presence of a viability marker from a viable cell, thereby indicating the presence of a viable cell in the analysis sample 20. Non-viable cells may contain a nominal amount of viability biomarker or a portion thereof, for example, a ratio of viable cells to non-viable cells of a viability biomarker of 1000:1. However, the reaction product of the reaction of the detection agent 70 with a sample 20 containing viable cells or a combination of viable cells and non-viable cells can be distinguished from the reaction product of a sample 20 containing non-viable cells or cells, because when there are no viable cells, a threshold amount of detection reaction product will not be detected. In one embodiment, the detection agent 70 comprises one or more primer sets (i.e., detection primers) that target the nucleotide sequence of the viability biomarker.

[0090] One method for amplifying 80 samples is reverse transcription (sometimes called transcriptase) polymerase chain reaction (RT-PCR), a PCR technique that involves reverse transcription of RNA into DNA in vitro, followed by enzymatic amplification of the DNA. RT-PCR combines the reverse transcription process with conventional PCR. The polymerase chain reaction (PCR) is a temperature-dependent nucleic acid amplification technique used for enzymatically amplifying DNA or RNA in vitro. Developed in the mid-1980s, PCR is considered one of the most important tools in modern biology (molecular biology and genetics). During RT-PCR, sample RNA is first converted into double-stranded DNA (complementary DNA) by reverse transcriptase during reverse transcription. The cDNA is then thermally decomposed into two single-stranded DNA templates. Within these single-stranded DNA templates, primers anneal to their complementary sequences based on the principles of nucleic acid hybridization. DNA polymerase then extends the primers by sequentially adding nucleotides to their 3' ends, generating double-stranded DNA (dsDNA) based on the principles of DNA replication. These three steps (denaturation, annealing, and extension) are repeated in a cyclical manner, regulating the reaction temperature and producing millions of copies of the cDNA. New ultrafast PCR instruments using multiple heating zones offer an alternative to rapid PCR, completing up to 30 cycles in 10–15 minutes, enabling faster amplification and detection than previously possible. In some cases, samples can be treated with DNase, designed to remove DNA, before the RT step. This can be useful for samples where high concentrations of nonviable organisms may be expected.

[0091] One process for amplifying 80 samples is reverse transcription followed by isothermal amplification, such as loop-mediated isothermal amplification (LAMP). LAMP is an isothermal nucleic acid amplification technique. Unlike PCR (PCR) techniques (wherein the reaction is carried out through a series of alternating temperature steps or cycles), isothermal amplification is performed at a constant temperature and does not require a thermal cycler. This makes amplification faster because it is not limited by hardware temperature changes. The use in the method 200 of the present invention can be described as a reverse transcription LAMP process (RT-LAMP), which combines LAMP with a reverse transcription step to allow detection of RNA.

[0092] In the LAMP process of the present invention (such as Figure 3In one example (shown schematically in FIG), a target sequence or specifically an RNA-based biomarker 40 is amplified at a constant temperature of 60-65°C (140-149°F) using two or three sets of primers 70 and a polymerase with high strand displacement and replication activity. Typically, four different primers are used to amplify six different regions on the target gene, which increases specificity. Additional "loop primer" pairs can further accelerate the reaction. The amount of RNA-based biomarker 40 produced during the LAMP process is typically higher than the PCR-based amplification 80 discussed above. The LAMP process can be modified to target RNA templates rather than genomic DNA. For example: excluding the B3 primer from RT-LAMP and using a reverse transcriptase with RNAseH activity.

[0093] Primer design for primer 70 can be performed using a variety of programs, such as PRIMEREXPLORER, MORPHOCATCHER, and NEB LAMP PRIMER DESIGN TOOL. The combination of PRIMEREXPLORER and MORPHOCATCHER may be particularly useful.

[0094] The amplification products of the LAMP process of amplification 80 can be directly detected photometrically (step 100) by measuring the turbidity caused by the precipitation of the amplification byproduct magnesium pyrophosphate in the solution (here signal 110). This allows for simple photometric detection 100 of signal 110 even for small volumes.

[0095] The amplification products of the LAMP process of amplification 80 can be monitored in real time by measuring turbidity as discussed above, or by interacting 90 an imaging agent with the amplified sequence of the RNA-based biomarker 40 to produce a readout signal 110, and detecting 100 the readout signal 110 and determining the presence or absence of viable cells in the sample 20. The interaction 90 with the sample 20 can be performed by fluorescence using an intercalating dye such as SYTO 9. Dyes such as SYBR green can be used to produce a visible color change as a signal 110 that can be accurately measured or detected 100 by an instrument.

[0096] Detection agents / imaging systems are provided for detecting the presence of amplification products from reactions with biomarkers. For example, imaging agents can include colorimetric detection systems, fluorescence detection systems, electrochemical detection systems, mass spectrometry detection systems, antibody detection systems, or pH detection systems. Examples of detection agents / systems include 5'6-FAM TM 、 5'SUN, 5'TEX 615, 5'TYE TM 563、5'TYE TM 665, 5'HEX TM,5'Yakima TET 539, YAK 549, SUN554, HEX 555, and biotin.

[0097] Dye molecules intercalate or directly label RNA-based biomarkers 40, which can then be correlated with the number of copies initially present. Thus, LAMP amplification 80 can also produce quantitative results.

[0098] Detection 100 following LAMP-based amplification 80 may be achieved by using manganese-loaded calcein to interact 90 with the sample 20, which becomes fluorescent upon complexation of manganese with pyrophosphate during in vitro DNA synthesis.

[0099] Another approach to detection 100 following LAMP-based amplification 80 is the ability of RNA-based biomarkers 40 to hybridize with complementary gold nanoparticle-bound (AuNP) single-stranded DNA (ssDNA). Thus, upon interaction 90 with sample 20, RNA-based biomarkers 40 hybridize with complementary gold nanoparticle-bound (AuNP) single-stranded DNA (ssDNA), preventing the normal color change from red to violet-blue that would otherwise occur during salt-induced gold particle aggregation, thereby providing a signal 110 for detection 100. Thus, LAMP-based amplification 80 combined with AuNP amplicon detection 100 offers advantages over other methods in terms of shortened assay time, confirmation of amplicons by hybridization, and the use of simpler equipment (i.e., no thermal cycler, electrophoresis equipment, or UV transilluminator is required).

[0100] LAMP-based amplification 80 has the potential to enable method 200 to be used by clinicians as a simple screening assay at the point of care or on-site. LAMP has been observed to be less sensitive (more resistant) to inhibitors in complex samples than PCR. Another advantage of our proposed RT-LAMP-based amplification is that the B3 primer can be removed to maximize amplification from the RNA molecules of the biomarker rather than genomic DNA.

[0101] A process of sample amplification 80 integrates clustered regularly interspaced short palindromic repeats (CRISPR). Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the fields of genome editing and transcriptional regulation. Advances in CRISPR-Cas technology have also promoted the molecular detection of a variety of targets, from nucleic acids to proteins. Combining the CRISPR-Cas system with various nucleic acid amplification strategies (PCR, isothermal amplification, and ambient temperature amplification) can generate amplified detection signals, enrich low-abundance molecular targets, improve analytical specificity and sensitivity, and develop point-of-care (POC) diagnostic technologies. These amplification and detection processes utilize specific features of various Cas proteins, including RNA-guided nuclease activity, sequence-specific recognition, multiple rounds of trans-cleavage activity of Cas12 and Cas13, and the unwinding and cleavage ability of Cas9. Due to the RNA-guided recognition of specific sequences in the amplicon, the integration of the CRISPR-Cas system after nucleic acid amplification improves detection specificity. Integration of CRISPR-Cas before nucleic acid amplification can achieve the enrichment of rare and low-abundance nucleic acid targets and remove unnecessary abundant nucleic acids. Using CRISPR-Cas9 to unwind dsDNA into ssDNA at moderate temperatures helps to achieve isothermal exponential amplification of nucleic acids. The integration of CRISPR technology with nucleic acid amplification technology achieves high sensitivity and rapid detection of RNA-based biomarkers 40. For more details on the amplification 80 method of integrating CRISPR, see Feng W, Newbigging AM, Tao J, Cao Y, Peng H, Le C, Wu J, Pang B, Li J, Tyrrell DL, Zhang H, Le XC. CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules. Chem Sci. 2021 Mar 2; 12 (13): 4683-4698.

[0102] Other known amplification 80 methods may also be used, including NASBA amplification starting directly from RNA sequences. In the processes discussed above, LAMP-based amplification 80 provides a cost-effective and efficient solution.

[0103] Amplification reagents 70 are discussed above in conjunction with LAMP-based amplification 80. As described above, PCR amplification employs two main reagents 70: primers (ssDNA fragments) and a DNA polymerase.

[0104] With respect to detection 100, real-time and endpoint fluorescence detection 100 can be performed using fluorescent probes (fluorescein, Cy3, TAMARA, JOE, TYE, ATTO dyes, IRDye, Rhodamine, Alexa Fluor, etc.) or fluorescent dyes (DNA intercalating dyes (Syto 9) or dyes that respond to chemical changes during amplification (calcein). End-point readout or detection 100 can be achieved using colorimetric detection, for example, using hydroxynaphthol blue dye or phenol red pH indicator. Real-time detection and endpoint detection agents can be combined in the same master mix (step 90), and colorimetric and fluorescence-based readouts or detection 100 can be used separately or together.

[0105] One process of sample amplification 80 incorporates the use of a biomarker 40 to detect the enzymatic activity of the biomarker. This is possible when using non-coding RNA / RNAzyme or a ribonucleoprotein complex comprising a non-coding RNA / RNAzyme. For example, the enzymatic activity of RNAse P is to hydrolyze a specific phosphodiester bond in a provided substrate (e.g., pre-tRNA). Fluorescent probe-quencher pairs can be developed that utilize the hydrolysis of the RNAse P bond to generate a signal.

[0106] The signal assessment of step 100 is analyzed to determine whether the sample contains viable cells. In embodiments, the reaction product and the imaging agent that detection agent (such as detection primer) produces with the analysis sample reaction in the assessment sample, and the amount of reaction product can be detected so that the viable cells in the sample can be distinguished from the sample comprising non-viable cells or not containing cells. The presence of at least a threshold amount of detection agent is interpreted as indicating the presence of viable cells in the analysis sample, or the combination of viable cells and non-viable cells. The absence of at least a threshold amount of detection agent indicates the absence of viable cells or not containing cells. As used herein, the threshold amount is determined according to reagents used, imaging agent and system. However, the data indicating the threshold amount of reaction product detected from the sample with viable cells are generally higher than the negative control, and can be distinguished from the detection reaction product with non-viable cells or the sample without cells, and when appropriate, a control sample is adopted.

[0107] Drug therapy regimen for medication 150 200

[0108] As discussed above, culture-based techniques are not suitable for pharmaceutical or medical products with a short shelf life or intended for immediate use. The method 200 of the present invention solves this problem. Therefore, the method 200 of the present invention can provide an ultra-rapid sterility testing method for drug products 150, including, for example, positron emission tomography (PET) tracers, CAR-T cells and new cell therapy products, platelet transfusion products, gene therapy and tissue engineering products. Another subcategory or group of drug products 150 for which the method 200 is particularly suitable is regenerative medicine therapy, which is one of cell therapy, gene therapy and tissue engineering medical products. In the scheme 220 of the present invention, these products can be administered 180 to the patient before the standard sterility test is completed. The results obtained from the execution of 200 are used to evaluate the sterility of the drug sample and make a release decision.

[0109] Figure 4 Schematically illustrates a system 230 and a kit 250 for implementing the method 200 of the present invention, a bedside sterility testing method for a product 150, and a pharmaceutical regimen 220 for administering the pharmaceutical product 150 to a patient according to the present invention. The system may include a qPCR instrument 230 with an improved heating block insert and a method for implementing Figure 1 The components of the kit 250 for all steps shown are: a) a bottle 254 containing concentrated sterile recovery medium; b) a sterile syringe 252 for obtaining and / or transferring the test sample 20 to the bottle 252 containing recovery medium; c) a sterile syringe 256 preloaded with lysis buffer; d) a nuclease-free labeled sterile PCR tube 258 preloaded with amplification reagents and controls.

[0110] One aspect of the present invention provides a method for testing the sterility of a pharmaceutical product 150, comprising the steps of obtaining 10 a sample 20 of the pharmaceutical product 150 to be tested; pre-treating 30 the sample 20 by promoting or maximizing the abundance of at least one universal RNA-based biomarker 40; amplifying 80 at least one nucleic acid sequence of the RNA-based biomarker 40 using an amplification reagent 70; and detecting 100 a readout signal 110 indicating the presence or absence of at least one viable cell within the sample; and, determining the sterility of the sample 20 and the pharmaceutical product 150 based on the detected readout signal 110.

[0111] Figure 52 is a schematic flow diagram of a drug regimen 220 for administering (at 180) a drug 150 to a patient according to one embodiment of the present invention. The drug regimen 220 for administering 180 the drug 150 to the patient includes obtaining 10 a sample of the drug 150 using a syringe 252 and transferring the sample 20 to a vial 254 containing an enrichment medium. The vial 254 containing the sample 20 and the enrichment medium is incubated for 15-30 minutes to pre-treat 30 the sample 20 being tested by promoting (or maximizing) the abundance of at least one RNA-based biomarker 40. Following pre-treatment 30, a lysis buffer is added to the vial 254 to process 60 the sample 20. The sample 20 is then transferred to a testing strip 258, and LAMP amplification 80 is performed to amplify at least one nucleic acid sequence of the RNA-based biomarker 50 using amplification reagents within the strip 258. Amplification 80 includes incubation at 55-60°C for 5 minutes for the RT step of the process, and incubation at 60-62°C for 15-30 minutes for the remainder of LAMP amplification 80. The method includes reading a signal 110 via endpoint fluorescence detection 100, the reading of which indicates the presence or absence of at least one viable cell in sample 20. After confirming the sterility or safety of sample 20, and thus the drug product 150, the drug product 150 is administered to the patient within 3 to 12 hours of obtaining the sample from the pharmacy. Obviously, if the method fails to confirm the absence of at least one viable cell in sample 20, treatment of the patient with drug 150 is postponed 180 until an alternative drug 150 is found and tested.

[0112] Kit 250

[0113] According to the present description, a kit 250 for determining the presence of viable cells in an analytical sample is provided. The kit 250 may include one or more pretreatment reagents for the sample 20 pretreatment step 30. The materials in the kit 250 may be one or more components of the sample 20 used to provide access to biomarkers in step 60. These physical materials may be hardware devices such as electroporation cells, bead mills, spin columns, negative pressure devices, and magnets. The kit 250 may also include reagents and consumables for amplifying 80 the sample 20, treating 90 with a detection agent, and detecting 100. Examples of detection agents include fluorescent labels, luminescent labels, chemiluminescent labels, radioactive labels, mass tag labels, optical or electrochemical labels.

[0114] In certain embodiments, the materials of step 30 and step 60 are combined into a single step, and the mixture of the required materials provides a one-step pretreatment and lysis reagent without the need for intermediate steps.

[0115] The kit 250 may also include one or more reaction containers; one or more positive or negative control reagents. A detection agent may also be included in the kit 250. Examples of detection agents include fluorescent labels, luminescent labels, chemiluminescent labels, radioactive labels, mass tags, optical or electrochemical labels as described herein.

[0116] system

[0117] According to the present description, a system for determining the presence or absence of viable cells in a sample is provided, such as the at least partially automated system 280 of FIG6 . The system 280 can include the components and reagents of the kit 250 described herein, and can also include one or more reaction vessels, a region for changing the temperature of one or more reaction vessels, a region for manipulating cells to allow access to cell contents, and a region for biomarker detection. The region for biomarker detection can be a device for performing nucleic acid amplification using isothermal or single or multiple zones that change temperature.

[0118] System 280 can be automated and embodied as a device in which a user adds a sample 20 to a cartridge and the process proceeds automatically. The device comprises multiple zones where the user first places the sample 20. The sample is transferred to a zone where pre-treatment steps 30 are performed and then to a zone where lysis 60 is performed. Amplification 80 reagents are mixed and then transferred to a detection zone 100.

[0119] In preferred embodiments, system 280 performs real-time measurements of biomarkers 40. In other embodiments, system 280 is fully or partially automated, and in other embodiments, the RNA acquisition component is a hardware device.

[0120] Method 200 may additionally incorporate analysis of amplified sequences to determine the species of any cells found within analyzed sample 20. Although biomarkers 40 contain highly conserved regions, portions thereof may be highly variable between species, allowing for the generation of a unique signature for each species.

[0121] The present invention is not limited to use with drugs 150, but has a wide range of applications, such as in food or beverage production, tissue banking, medical implants, medical device manufacturing, among others.

[0122] The present invention will also be applied in environmental testing, such as water quality, sludge treatment, facility testing, etc.

[0123] The present invention can be used as a rapid test to test the sensitivity of a microorganism of interest to a disinfectant / therapeutic, or on cells treated with a pharmaceutical agent to determine the sensitivity of the cell to the pharmaceutical agent (eg, a drug or cancer therapeutic).

[0124] The sample may include bacteria or eukaryotic cell cultures, whether native or subjected to treatments designed to inactivate or sterilize the cells.

[0125] The present invention can be used for the analysis of animal specimens, such as nasopharyngeal swabs, sputum, and normally sterile samples, such as cerebrospinal fluid, blood, and urine.

[0126] Another modification of the invention mentioned above is to use the amplification reaction products after the method is applied to identify specific sequences of a specific pathogen of interest.

[0127] Implementation

[0128] Because the methods, kits, systems, and assays described herein detect viable cells, a simple sterility test method is provided. The methods, kits, systems, and assays described herein also provide ultra-fast, ultra-sensitive sterility testing. Furthermore, the methods, kits, systems, and assays described herein achieve the sensitivity required for small numbers of organisms.

[0129] According to the present description, a rapid test workflow is provided that can detect viable cells (including active microorganisms, dormant cells and spores) with high sensitivity, but not dead cells. As described herein, only viable cells are detected (because dead cells do not result in colony forming units). To achieve sensitivity and speed in the detection of target biomolecules, a three-step sample-to-answer "Ultra-Rapid Sterility Analysis" workflow is provided by integrating microbial recovery, lysis, isothermal amplification and detection without the need for intermediate cleanup. Again refer to Figure 3 , showing a three-step workflow: 1) Samples are pretreated to ensure the presence of RNAse P. 2) Cells are lysed and ribonucleoproteins are denatured. 3) Target RNA sequences are reverse transcribed and detected through rapid nucleic acid amplification.

[0130] To promote RNA synthesis in the organism, the test sample is mixed with a universal culture medium suitable for fastidious and non-fastidious aerobic and anaerobic microorganisms. This brief nutritional stimulation ("pretreatment step") ensures the presence of RNAse P in living microorganisms and initiates spore recovery. Then, a one-step lysis suitable for single cells is performed (without intermediate purification), followed by amplification using degenerate primers targeting conserved regions of RPR, and multiplexing in six groups to detect specific microbial groups. This workflow allows universal detection of microorganisms in less than an hour. Due to the use of RPR as a target biomolecule, a single microorganism will have an estimated 1000 sequence copies for amplification, allowing high test sensitivity to be achieved.

[0131] Nucleic acid amplification techniques (such as RT-PCR) are listed as embodiments of the sterility test according to this description due to their extremely high sensitivity, the ability to amplify specific target sequences, and the ability to detect the sequences in a short period of time. A challenge of PCR-based technology is that, for low-abundance targets, even for rapid RT-PCR, current detection times may be longer than 40-45 minutes. Therefore, loop-mediated isothermal amplification (LAMP) can be used to allow even faster (15-30 minutes), more specific, and comparable sensitivity target sequence amplification. RT-LAMP can be performed using a thermal cycler or a simple heating block, and is known to be robust to many common DNA polymerase inhibitors in test samples.

[0132] Exemplary embodiments of kits and systems according to the present description are as follows Figure 4 As shown. Figure 4 As shown, a qPCR instrument with a custom heating block insert is provided. Also provided is a Figure 3 Kit components for the steps shown include: a) a bottle of concentrated sterile recovery medium; b) a sterile syringe for transferring a test sample to the bottle of recovery medium; c) a sterile syringe preloaded with lysis buffer; and d) a nuclease-free, labeled, sterile PCR strip tube preloaded with amplification reagents and controls.

[0133] An exemplary embodiment of the use of the system, method and reagents is shown in Figure 6, which is a schematic diagram of an apparatus for illustrating the system and method of the present description. As shown in the process of Figure 6, 1) the user injects the test sample 20 into the provided sterile, nuclease-free sample area, which is pre-loaded with concentrated sterile recovery medium (pretreatment reagent) 30. 2) The user loads the recovery bottle onto the instrument and starts the pretreatment protocol (30-35°C, 25°C). After the pretreatment step, the protocol is paused. 3) In the RNA acquisition step 50, the user uses a provided sterile syringe pre-loaded with lysis buffer to add the lysis reagent to the bottle containing the pretreated sample. The user then starts the protocol for the lysis step (25°C, 60°C, 68-72°C, 4°C). In the amplification and readout steps, 4) the user uses a repeating pipette to transfer the lysed sample to a sterile, nuclease-free PCR tube pre-loaded with frozen amplification reagent 70. Sample loading starts from the test well and ends with the positive control well. 5) The user transfers the tubes to the thermal cycler and initiates the RT-LAMP protocol (55°C-60°C, then 60°C-67°C for the specified period). 6) The instrument provides real-time feedback (data) on the analysis and provides the user with an answer as to whether viable cells are present in the sample, preferably within one hour. The analysis is validated using positive and negative controls to ensure the accuracy of the system and data reporting.

[0134] Instructions for performing the test are provided with the kit. Other components include racks for the safe handling of standard resuscitation vials and standard PCR tubes, waste containers for solid waste and sharps, instructions for the disposal of all solid waste, and engineered solutions to ensure sterility and radioactivity safety. To achieve this goal, this project required two major developments.

[0135] Example

[0136] Assessing Amplification Interference:

[0137] Different PET tracers were evaluated for their potential sources of interference with PCR due to their composition. The PET tracers used in this experiment were expired and therefore non-radioactive. The effects of the presence of three PET tracers (the estradiol-based PET tracers 16α-[18F]-fluoro-17β-estradiol (FES), [18F]flurbetaben (FBB), and [18F]fluorodeoxyglucose (FDG)) on the performance of two different DNA polymerases: One-step Taq Path (Applied Biosystems) and PlatinumSuperFi (ThermoFisher). The PET tracers were added to the PCR reaction mixture at 10% and 30%. In addition, FDG was used at a concentration of 15%.

[0138] The results showed that FDG had no effect on the amplification data in qPCR mode. We then clarified the effect of all three PET tracers on the observed results by possible quenching of the fluorescence signal in the FAM channel (EvaGreen, Biotium 31090). DNA amplicons in FDG and FBB drug products were observed by qPCR and gel electrophoresis (E-Gel EX agarose gel, 2%, Invitrogen) ( Figure 8 ). Figure 8 The figure shows gel electrophoresis analysis of RNase P PCR products to test DNA polymerase inhibition by varying volume percentages of PET tracers (2% agarose). Lane M: E-Gel 50bp DNA ladder. Sizes range from 50bp to 2500bp, with reference bands at 2500, 800, and 350bp; Lane 1: FES 30%; 2: FES 10%; 3: FBB 30%; 4: FBB 10%; 5: FDG 30%; 6: FDG 15%; 7: FDG 10%; 8: Negative control; 9: No PET tracer added, positive control.

[0139] Only the FES sample showed an effect on DNA polymerase performance. We hypothesize that inhibition occurs due to the presence of up to 15% by volume of ethanol in the FES formulation, a known PCR inhibitor. Therefore, the methods and reagents described herein can be used for sterility testing of PET tracers and other pharmaceutical compositions described herein.

[0140] Primer design:

[0141] In an exemplary embodiment, primers were developed for the five universal conserved regions of RNase P. Pan-bacterial degenerate multiplex RT-PCR primers can be developed for this sequence and structure that has both type A and type B. However, the problem of using the structure to design RT-LAMP primers was solved by excluding the B3 primer from the primer set, which helped us to start the reverse transcription from the B2 portion of the BIP primer. By excluding the B3 primer, we also made it impossible for B3 to perform chain displacement. Therefore, accidental amplification from genomic DNA was prevented, and only living cells were detected. Using a reverse transcriptase with RNase H activity, single-stranded cDNA can be separated from its original RNA template without a denaturation step. The cDNA product reverse transcribed using the BIP primer has a B1 tail at the 5' end to continue the amplification reaction.

[0142] Development of a workflow that achieves 1 CFU / mL sensitivity in less than 1 hour:

[0143] After identification of the target biomarker and development of the initial primer set for the molecular viability test, a rapid lysis protocol and amplification conditions were developed and optimized to allow for a highly sensitive assay.

[0144] For RT LAMP, reagents were selected from three groups of enzymes (WarmStart RT-LAMP (NEB), RTx reverse transcriptase (NEB), and BST2 polymerase (NEB), as well as the Superscript IV RT-LAMP system (Invitrogen)) to be compatible with our three-step workflow of pretreatment, lysis, and amplification without the need for intermediate cleanup.

[0145] Amplification conditions were optimized, a reverse transcriptase with RNase H activity was selected to target RNA in RT-LAMP, and the amplification step was then integrated into the designed pipeline. Figure 9Shown are Gram-negative bacterial RNA amplified using a universal set of G-RT-LAMP primers (2% agarose). M: E-Gel 50bp DNA ladder. Band sizes range from 50bp to 2,500bp, with reference bands at 2,500, 800, and 350bp. 1) Negative control; 2) E. coli RNA; 3) Target RNA (P. aeruginosa) without magnesium addition after lysis; 4, 5, 6) Commercially available quantitative P. aeruginosa DNA loaded without detection in RT-LAMP without a denaturation step; 7) E. coli RNA obtained and detected during the test workflow; 8) E. coli RNA (1 / 100 dilution); 9) P. aeruginosa RNA obtained and detected during the test workflow; 10) E-Gel 50bp DNA ladder. Our results showed that the signal-to-noise ratio of positive samples (over 40,000 RFU) was approximately 1000-fold higher than that of negative controls (less than 300 RFU) within 1 hour. We then achieved the target sensitivity by RT-PCR and RT-LAMP amplification in Gram-negative viable microorganisms, with an LOD of 1 CFU ( Figure 10 ). Figure 10 This is a gel electrophoresis analysis of RT-LAMP products of the RNaseP RNA subunit. The complete workflow protocol takes approximately 1 hour, with the following steps: a brief nutrient stimulation using brain heart infusion (BHI) broth as a recovery medium (15-25 minutes), followed by rapid lysis in the presence of monovalent cations, an RNAse inhibitor, a nonionic detergent, and lysozyme (3-6 minutes), and finally a rapid RT-LAMP amplification (35 minutes).

[0146] Figure 11A and 11B The graph shows LOD data for the sensitivity of RNase P RNA subunit detection in pre-treated (revived) bacterial and fungal cells. Figure 11A and 11B Shown are the detection sensitivity of RNA from a quantitative bacterial pool recovered (A) obtained in the developed recovery / lysis / amplification test workflow or (B) obtained by standard extraction using silica columns and diluted for use as template for RNAase P detection by RT-qPCR. Figure 11A Figure 2 shows the LOD data for the detection of RNAse P RNA subunits in the mini-pack freezer of Pseudomonas aeruginosa ATCC#9027 tested in the custom testing workflow. Specific RNAse P RNA subunit amplicons were detected in 1 cfu of RNA loaded per RT-PCR reaction. Figure 11A shown. Figure 11BLOD data for detection of RNAse P RNA subunits in C. albicans ATCC#10231 frozen stocks tested in custom recovery and commercial lysis using silica gel columns are shown. Specific RNAse P RNA subunit amplicons were detected in ~1.4 cfu / mL RNA loaded in each RT-PCR reaction. Figure 11B The complete workflow protocol takes less than 1 hour (for RT-LAMP) and approximately 2.5 hours (for RT-PCR), with the following steps: a brief nutrient stimulation using brain heart infusion (BHI) broth as a recovery medium (15-25 minutes), followed by rapid lysis in the presence of monovalent cations, an RNAse inhibitor, a nonionic detergent, and lysozyme (3-6 minutes), and finally, rapid RT-LAMP amplification (15-35 minutes) or RT-PCR amplification (1 hour 15 minutes to 1.5 hours) of the released target RNA in a single biochemical pipeline without any intermediate cleanup.

[0147] Although the foregoing written description enables one of ordinary skill in the art to make and use what is presently believed to be the best mode, those of ordinary skill in the art will understand and appreciate that there are variations, combinations, and equivalents of the specific embodiments, methods, and examples herein. Other embodiments will be apparent to those skilled in the art from consideration of this specification and practice of the embodiments disclosed herein. It is intended that the description and examples be considered exemplary, with the true scope and spirit of the embodiments being indicated by the following claims.

Claims

1. A method for detecting viable cells, comprising the following steps: a) obtaining a sample for testing; b) pre-processing the sample by promoting the abundance of at least one universal, abundant RNA-based biomarker; c) performing a reaction to generate a signal from at least one nucleic acid sequence of the RNA-based biomarker; d) detecting a readout signal indicative of the presence or absence of at least one viable cell within the sample; as well as e) analyzing the readout signal and reporting whether the method detected viable cells in the analyzed sample.

2. The method for detecting viable cells according to claim 1, wherein the RNA-based biomarker is short-lived and has a lifespan of less than 180 minutes in living cells. 3 . The method for detecting viable cells according to claim 1 , wherein the sensitivity of the detection method is such that less than 9 viable cells can be detected in a sample volume of at most 1 ml.

4. The method for detecting viable cells according to claim 1, further comprising the step of treating the sample to facilitate contact of a reagent with the RNA-based biomarker before the step of amplifying the at least one nucleic acid sequence of the RNA-based biomarker. The method for detecting viable cells according to claim 1 , wherein steps ae) are performed in less than 2 hours. 6 . The method for detecting viable cells according to claim 1 , further comprising the step of allowing a dye to interact with the amplification component of the RNA-based biomarker to generate the readout signal. The method for detecting viable cells according to claim 1 , wherein the RNA-based biomarker is a ribozyme.

8. The method for detecting viable cells according to claim 1, wherein the step of performing a reaction to generate a signal from at least one nucleic acid sequence of the RNA-based biomarker comprises using an amplification reagent, and wherein the amplification reagent is constructed to target at least one conserved region of the RNA-based biomarker.

9. The method according to claim 1, wherein the reaction of the analysis sample to generate a signal uses an amplification reagent and includes nucleic acid amplification.

10. The method of claim 9, wherein the nucleic acid amplification comprises polymerase chain reaction (PCR). The method of claim 9 , wherein the nucleic acid amplification comprises a loop-mediated amplification (LAMP) reaction.

12. The method of claim 9, wherein the nucleic acid amplification comprises one of RT-PCR, RT-LAMP, RCA, or NASBA.

13. The method of claim 9, wherein the nucleic acid amplification comprises isothermal amplification. The method according to claim 9 , wherein reacting the analysis sample to generate a signal comprises a reverse transcription step. The method of claim 9 , wherein the nucleic acid amplification comprises a cascade reaction for signal amplification.

16. The method of claim 1 , further comprising providing a detection agent or detection system for detecting the presence of RNA biomarkers from viable cells in the sample, wherein the detection agent or detection system comprises a colorimetric detection system, a fluorescence detection system, an electrochemical detection system, a mass spectrometry detection system, or a pH detection system.

17. The method of claim 1, wherein the sample comprises cells treated with a pharmaceutical agent to determine the sensitivity of the cells to the pharmaceutical agent.

18. The method of claim 1, wherein the sample is obtained from a pharmaceutical product.

19. The method of claim 1, wherein analyzing the sample further comprises reporting that the sample is free of viable cells, wherein the sample contains nonviable cells or contains no cells.

20. The method of claim 19, wherein data indicating the presence of viable cells in the sample are distinguishable from data indicating the presence of non-viable cells in the sample in the analysis.

21. A sterility testing method for a drug product comprising the following steps: a) obtaining a sample of the drug product for testing; b) pre-treating said sample for testing by promoting the abundance of at least one universal RNA-based biomarker; c) amplifying at least one nucleic acid sequence of the RNA-based biomarker using an amplification reagent; d) detecting a readout signal indicative of the presence or absence of at least one viable cell within the sample; and e) determining the sterility of the sample and the drug product based on the detected readout signal.

22. The sterility test method of claim 21, wherein the test method has a sensitivity of detecting less than 5 viable cells in a sample volume of at most 1 ml, and wherein steps ae) are performed in less than 1 hour, and wherein the RNA-based biomarker is short-lived and abundant.

23. The sterility testing method of claim 21, further comprising the step of allowing a dye to interact with the amplification component of the RNA-based biomarker to produce the readout signal.

24. The sterility testing method of claim 21, wherein the amplification reagent is configured to target at least one conserved region of the RNA-based biomarker.

25. A pharmaceutical regimen for administering a drug to a patient comprising the steps of: a) obtaining samples of the drug; b) pre-treating said sample for testing by promoting the abundance of at least one RNA-based biomarker; c) amplifying at least one nucleic acid sequence of the RNA-based biomarker using an amplification reagent; d) detecting a readout signal indicative of the presence or absence of at least one viable cell within the sample; e) administering the medicament to the patient after determining the absence of at least one viable cell in the sample.

26. The pharmaceutical regimen according to claim 25, wherein the sensitivity of the test method is to detect less than 5 viable cells in a sample volume of at most 1 ml, and wherein steps ad) are performed in less than 1.5 hours.

27. The pharmaceutical regimen of claim 25, wherein the RNA-based biomarker is a ribozyme.

28. The pharmaceutical regimen of claim 25, wherein the amplification reagent is configured to target at least one conserved region of the RNA-based biomarker.

29. The drug regimen of claim 25, wherein the drug is one of the group consisting of a positron emission tomography (PET) tracer, a CAR-T cell and cell therapy product, a platelet transfusion product, a gene therapy product, and a tissue engineering product.

30. The pharmaceutical regimen of claim 25, wherein the pharmaceutical is a regenerative medicine therapy and is one of a cell therapy, a gene therapy, and a tissue engineered medical product.

31. The pharmaceutical regimen of claim 25, wherein the sensitivity of the test method is to detect less than 5 viable cells in a sample volume of at most 1 ml, and wherein steps ad) are performed in less than 1 1 / 2 hours, and further comprising the step of allowing a dye to interact with the amplification component of the RNA-based biomarker to produce the readout signal.

32. The pharmaceutical regimen of claim 25, wherein the pharmaceutical is administered to the patient after determining the absence of at least one viable cell in the sample and within 12 hours of obtaining the sample from the patient.

33. A kit for determining the presence of viable cells in an assay sample, the kit comprising: a) one or more pretreatment reagents that promote conditions in the assay sample such that viable cells present in the assay sample are enriched for short-lived RNA molecules; b) one or more RNA acquisition components for providing acquisition of short-lived RNA biomarkers present in viable cells in the analysis sample, the components comprising: i) a lysis reagent for obtaining short-lived RNA molecules in the analysis sample; ii) a release reagent for obtaining short-lived RNA molecules in the analysis sample; iii) a capture agent for acquiring short-lived RNA molecules in the analysis sample; and iv) a physical reagent for obtaining short-lived RNA molecules in the analysis sample; c) a detection agent for detecting the presence of the short-lived RNA biomarker, wherein the detection agent is capable of being detected directly or indirectly to indicate the presence of a short-lived RNA that is present in viable cells and absent in non-viable cells.

34. The kit of claim 33, wherein the kit further comprises one or more reaction vessels.

35. The kit of claim 33, wherein the kit further comprises one or more positive or negative control reagents.

36. The kit of claim 33, wherein the detection agent is a fluorescent label, a luminescent label, a chemiluminescent label, a radioactive label, a mass tag label, an optical or electrochemical label.

37. The kit of claim 33, wherein the lysis reagent comprises a sterile syringe pre-loaded with a lysis buffer for obtaining short-lived RNA molecules in the analysis sample.

38. The kit of claim 33, wherein the detection agent comprises a material for nucleic acid amplification.

39. The kit of claim 33, wherein the detection agent comprises one or more primer sets targeting RPR, tRNA, or small non-coding RNA.

40. The kit of claim 33, wherein the pretreatment reagents and RNA acquisition component are provided as reagents combined into a single bottle.

41. A kit for determining the presence of viable cells in an assay sample, the kit comprising: a) one or more pretreatment reagents that provide conditions in the assay sample such that viable cells present in the assay sample are enriched for short-lived RNA molecules; and b) one or more RT-PCR or RT-LAMP primer sets that target a short-lived RNA biomarker, wherein the biomarker is RNase P RNA (RPR), wherein the primer set anneals to the entire sequence or a portion of the conserved region of CR1, CR2, CR3, CR4, and CR5, and wherein the RT-PCR or RT-LAMP primer set is capable of being detected, directly or indirectly, to indicate the presence of the biomarker RNA that is present in viable cells and absent in non-viable cells.

42. The kit according to claim 41 further comprises an RNA acquisition component for providing acquisition of short-lived RNA molecules present in viable cells in the analysis sample, wherein the component comprises one or more lysis reagents, release reagents, perforation consumables or capture agents for acquiring short-lived RNA molecules in the analysis sample.

43. A system for determining the presence or absence of viable cells in a sample, comprising the kit for determining the presence of viable cells in an analytical sample according to claim 41, and further comprising: a) one or more reaction vessels; b) an area for changing the temperature of one or more reaction vessels; c) an area for manipulating cells to allow access to cellular contents; and d) Area for biomarker detection.

44. The system of claim 43, wherein the RNA acquisition component is a hardware device.

45. The system of claim 43, wherein the area for biomarker detection comprises a device for nucleic acid amplification using a single area or multiple areas using isothermal or variable temperature.

46. ​​The system of claim 43, wherein the system measures the biomarker in real time.

47. The system of claim 43, wherein the system is at least partially automated.

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