EP-4070319-B1 - SYSTEM AND METHOD FOR IDENTIFYING ANALYTES IN ASSAY USING NORMALIZED TM VALUES

EP4070319B1EP 4070319 B1EP4070319 B1EP 4070319B1EP-4070319-B1

Inventors

ROBBINS, THOMAS CHARLES
WILSON, Theodore Allan
COX, CHARLES BENJAMIN
JONES, JAY
Galvin, Benjamin William
KISER, Amber Cooper

Dates

Publication Date: 20260513
Application Date: 20201203

Claims (11)

A method for generating an array-specific Tm window to be used for calling a sample positive or negative for a target nucleic acid sequence in a given array run, the method comprising: providing, in an array of a closed system, a control sample well with a control sample containing a control nucleic acid sequence, primers configured for amplifying the control nucleic acid sequence, a fluorescent dye, and components for amplification; amplifying the control sample by thermal cycling the control sample well; measuring, by an optical system, fluorescent data during or subsequent to the amplification of the control sample; generating, by a processor, a control melting curve using the fluorescent data; identifying, by the processor, based on the control melting curve, a Tm value for the control sample in the closed system; comparing, by the processor, the identified Tm value for the control sample in the closed system to an expected Tm value for the control nucleic acid sequence; calculating, by the processor, based on the comparing, a relationship between the identified Tm value for the control sample in the closed system and the expected Tm value for the control nucleic acid sequence; and generating, by the processor, the array-specific Tm window for the target nucleic acid sequence in the closed system by applying the calculated relationship between the expected Tm value for the control nucleic acid sequence and the identified Tm value for the control sample in the closed system to a pre-determined Tm window for the target nucleic acid sequence, wherein the array-specific Tm window for the target nucleic acid sequence in the closed system is narrower than the pre-determined Tm window for the target nucleic acid sequence; providing, in the array of the closed system, an experimental sample well with an experimental sample, primers configured for amplifying the target nucleic acid sequence, the fluorescent dye, and components for amplification; amplifying the experimental sample by thermal cycling the experimental sample well, wherein the control sample well and experimental sample well are thermal cycled during the same array run of the closed system; measuring, by an optical system, fluorescent data during or subsequent to the amplification of the experimental sample; generating, by the processor, an experimental melting curve using the fluorescent data; identifying, by the processor, based on the experimental melting curve, a Tm value for the experimental sample in the array of the closed system; and calling, by the processor, the experimental sample positive or negative for the target nucleic acid sequence based on whether the Tm value of the experimental sample falls within the array- specific Tm window for the target nucleic acid sequence in the closed system.
The method of claim 1, the method further comprising: comparing, by the processor, the Tm value for the experimental sample in the array of the closed system to one or more known Tm values associated with one or more respective nucleic acid sequences; and determining, by the processor, which of the one or more nucleic acid sequences is most likely to correspond to the experimental sample based on the comparison.
The method of any one of claims 1 or 2, the method further comprising: comparing, by the processor, the Tm value for the experimental sample in the array of the closed system to one or more known Tm values associated with one or more respective nucleic acid sequences; and determining, by the processor, based on the comparison, that the Tm value for the experimental sample in the array of the closed system differs from each of the one or more known Tm values by greater than a threshold Tm value amount; and determining, by the processor, that the experimental sample in the array of the closed system corresponds to a novel nucleic acid sequence that is distinct from any of the one or more nucleic acid sequences.
The method of any one of claims 1 or 2, the method further comprising: determining, by the processor, a number of positive calls for the target nucleic acid sequence in an institution population for an institution associated with the experimental sample; determining, by the processor, a number of negative calls for the target nucleic acid sequence in the institution population; determining, by the processor, based on the number of positive calls and negative calls in the institution population, a first rate of positive calls for the target nucleic acid sequence in the institution population; obtaining, by the processor, a number of positive calls for the target nucleic acid sequence in a community population for a community associated with the experimental sample; obtaining, by the processor, a number of negative calls for the target nucleic acid sequence in the community population; determining, by the processor, based on the number of positive calls and negative calls in the community population, a second rate of positive calls for the target nucleic acid sequence in the community population; determining, based on comparing the first rate to the second rate, whether a pathogen associated with the target nucleic acid sequence was likely acquired from the institution or from the community.
The method of claim 4, wherein the institution is a hospital or a hospital system.
The method of any one of claims 1-5, wherein the array-specific Tm window for the target nucleic acid sequence in the closed system is a normalized Tm window for the target nucleic acid sequence in the closed system.
The method of claim 6, wherein the array-specific Tm window for the target nucleic acid sequence in the closed system is normalized based on a linear relationship between previous Tm values of the target nucleic acid sequence and previous values of the control nucleic acid sequence from previous array runs of the closed system.
The method of any one of claims 1-7, wherein the pre-determined Tm window for the target nucleic acid sequence is calculated based on previous identified Tm values for the target nucleic acid sequence.
The method of any one of claims 1-5, further comprising: obtaining, by the processor, a general relationship between Cp and Tm for the target nucleic acid sequence based on a reference dataset including a plurality of Cp values and Tm values for the target nucleic acid sequence from a respective plurality of arrays; obtaining, by the processor, a reference Cp value for the target nucleic acid sequence; calculating, by the processor, a first estimated Tm for the target nucleic acid sequence by applying the general relationship to the reference Cp value; identifying, by the processor, an observed Cp value for the experimental sample in the array of the closed system; calculating, by the processor, a second estimated Tm for the target nucleic acid sequence by applying the general relationship to the observed Cp value for the observed experimental sample; calculating, by the processor, an array-specific relationship between Cp and Tm for the target nucleic acid sequence by comparing the first estimated Tm to the second estimated Tm for the target nucleic acid sequence; and generating, by the processor, a Cp-normalized array-specific Tm window for the target nucleic acid sequence in the closed system by applying the array-specific relationship between Cp and Tm for the target nucleic acid sequence to the generated array-specific Tm window for the target nucleic acid sequence in the closed system.
A system for generating an array-specific Tm window to be used for calling a sample positive or negative for a target nucleic acid sequence in a given array run of a closed system, the system comprising: an array of a closed system housing a plurality of sample wells, including a control sample well housing a control sample containing a control nucleic acid sequence and an experimental sample well housing an experimental sample; one or more temperature controlling devices configured to amplify the control sample by thermal cycling the control sample well and configured to amplify the experimental sample by thermal cycling the experimental sample well, wherein the control sample well and experimental sample well are thermal cycled during the same array run of the closed system; an optical system configured to detect an amount of fluorescence emitted by the control sample and configured to detect an amount of fluorescence emitted by the experimental sample; and a controller configured to: receive, from the optical system, data indicative of the amount of fluorescence emitted by the control sample during or subsequent to the amplification; generate a control melting curve using the data indicative of the amount of fluorescence emitted by the control sample during or subsequent to the amplification; identify, based on the control melting curve, a Tm value for the control sample in the closed system; compare the identified Tm value for the control sample in the closed system to an expected Tm value for the control nucleic acid sequence; calculate, based on the comparing, a relationship between the identified Tm value for the control sample in the closed system and the expected Tm value for the control nucleic acid sequence; generate the array-specific Tm window for the target nucleic acid sequence in the closed system by applying the calculated relationship between the expected Tm value for the control nucleic acid sequence and the identified Tm value for the control sample in the closed system to a pre-determined Tm window for the target nucleic acid sequence, wherein the array-specific Tm window for the target nucleic acid sequence in the closed system is narrower than the pre-determined Tm window for the target nucleic acid sequence; receive, from the optical system, data indicative of the amount of fluorescence emitted by the experimental sample during or subsequent to the amplification; generate an experimental melting curve using the data indicative of the amount of fluorescence emitted by the experimental sample during or subsequent to the amplification; identify, based on the experimental melting curve, a Tm value for the experimental sample; and call the experimental sample positive or negative for the target nucleic acid sequence based on whether the Tm value of the experimental sample falls within the array-specific Tm window for the target nucleic acid sequence in the closed system.
The system of claim 11, wherein the array of the closed system is part of a closed system sample vessel that also includes a sample preparation zone.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority to U.S. Provisional Patent Application No. 62/942,900, filed December 3, 2019, entitled "SYSTEM AND METHOD FOR IDENTIFYING ANALYTES IN ASSAY USING NORMALIZED TM VALUES,". FIELD OF THE DISCLOSURE The present disclosure generally relates to techniques used in PCR systems and, more particularly, to generating an array-specific range of Tm values to be used for calling a sample positive or negative for a target nucleic acid sequence in a given array. BACKGROUND In the United States, Canada, and Western Europe infectious disease accounts for approximately 7% of human mortality, while in developing regions infectious disease accounts for over 40% of human mortality. Infectious diseases lead to a variety of clinical manifestations. Among common overt manifestations are fever, pneumonia, meningitis, diarrhea, and diarrhea containing blood. While the physical manifestations suggest some pathogens and eliminate others as the etiological agent, a variety of potential causative agents remain, and clear diagnosis often requires a variety of assays to be performed. Traditional microbiology techniques for diagnosing pathogens can take days or weeks, often delaying a proper course of treatment. In recent years, the polymerase chain reaction (PCR) has become a method of choice for rapid diagnosis of infectious agents. PCR can be a rapid, sensitive, and specific tool to diagnose infectious disease. A challenge to using PCR as a primary means of diagnosis is the variety of possible causative organisms and the low levels of organism present in some pathological specimens. It is often impractical to run large panels of PCR assays, one for each possible causative organism, most of which are expected to be negative. The problem is exacerbated when pathogen nucleic acid is at low concentration and requires a large volume of sample to gather adequate reaction templates. In some cases, there is inadequate sample to assay for all possible etiological agents. A solution is to run "multiplex PCR" wherein the sample is concurrently assayed for multiple targets in a single reaction. While multiplex PCR has proven to be valuable in some systems, shortcomings exist concerning robustness of high level multiplex reactions and difficulties for clear analysis of multiple products. To solve these problems, the assay may be subsequently divided into multiple secondary PCRs. Nesting secondary reactions within the primary product often increases robustness. However, this further handling can be expensive and may lead to contamination or other problems. Fully integrated multiplex PCR systems integrating sample preparation, amplification, detection, and analysis are user friendly and are particularly well adapted for the diagnostic market and for syndromic approaches. The FilmArray® (BioFire Diagnostics, LLC, Salt Lake City, UT) is such a system, a user friendly, highly multiplexed PCR system developed for the diagnostic market. The single sample instrument accepts a disposable "pouch" that integrates sample preparation and nested multiplex PCR. Integrated sample preparation provides ease-of-use, while the highly multiplexed PCR provides both the sensitivity of PCR and the ability to test for up to 30 different organisms simultaneously. This system is well suited to pathogen identification where a number of different pathogens all manifest similar clinical symptoms. Current available diagnostic panels include a respiratory panel for upper respiratory infections, a blood culture panel for blood stream infections, a gastrointestinal panel for GI infections, and a meningitis panel for cerebrospinal fluid infections. Other panels are in development. Generally speaking, the temperature at which DNA strands denature depends on the sequence characteristics of the DNA. Accordingly, in many PCR systems, a range of Tm (the temperature at which half of the nucleic acid has melted) values are used to call a positive or a negative for an assay. If an identified Tm for a sample falls outside of the range of Tm values for a particular nucleic acid sequence, the sample is called negative for that nucleic acid sequence, but if the identified Tm for the sample falls within the range of Tm values for the nucleic acid sequence, the sample is called positive for that nucleic acid sequence. In some cases, this range of Tm values can be fairly broad and still be useful in distinguishing between DNA associated with different pathogen species, because different pathogen species typically have quite different Tm values. However, the Tm values for the DNA of related pathogens may vary only slightly. Consequently, when a Tm window is too broad, it may be difficult to distinguish between closely related pathogens due to overlap in Tm value ranges for each pathogen. For instance, some assays amplify nucleic acids multiple strains of a given pathogen, and some assays can amplify similar pathogen speci