The amplification and detection of diverse strains of an infectious virus or bacteria, or variants within a gene family is important for both clinical and basic research but can be difficult using conventional PCR. This report describes and illustrates a novel closed-tube method for amplifying and characterizing heterogeneous target sequences using members of the CTX-M beta-lactamase gene family. Different subgroups of CTX-M genes exhibit low sequence identity, but accurate and efficient detection of these variants is critical because they all confer resistance to penicillin, cefotaxime, and other antibiotics of the beta-lactam class. The method combines a single pair of "thermodynamic consensus primers" (tcPrimers) with one or more "initiator primers" (iPrimers), added at low concentration (5-10 nM). Each iPrimer improves the initial amplification of one or more variants because it has fewer mismatches to its intended target than the more abundant tcPrimers. As a result of initial amplification, each heterogeneous sequence is shifted stepwise toward a better match with the tcPrimers. As soon as the tcPrimer hybridization takes place, amplification proceeds with high efficiency. The tcPrimer pairs can be designed for symmetric PCR or for Linear-After-The-Exponential (LATE)-PCR. LATE-PCR offers the advantage of generating single-stranded DNA that can be characterized for different gene variants in the same closed tube, using low-temperature mismatch-tolerant fluorescent probes.

Background: We have constructed a highly informative, rapid, single-tube, single-color assay for distinguishing variants in the inhA promotor, and katG and rpoB gene targets responsible for isoniazid and rifampicin resistance, using technologies invented at Brandeis University. Methods & Materials: A multiplex LATE-PCR reaction is used to generate single-stranded DNA products for the Rifampicin Resistance Determining Region of the rpoB gene, the katG gene, and the inhA promotor, as well as an amplifiable internal control. These four amplicons are detected with four sets of Therma-Light probes all of which are labeled with Black Hole Quenchers with/without Quasar 670 fluorophors. Probe-target hybridization takes place at end-point over a wide range of temperatures below the reaction annealing temperature. The temperature ranges used for the four sets of Therma-Light probes are deliberately overlapped, making it possible to maximize the amount of information generated in a single color. The amplifiable internal control generates a specific very low temperature signal and an additional non-amplifiable internal control generates a second signal at a specific high temperature. Together these controls can be used to calibrate each reaction. Another proprietary reagent is added to the reaction mixture to improve primer specificity. Results: The present assay has been tested on twenty-two different archival strains which are reported to harbor many of the common alleles responsible for resistance to rifampin and isoniazid. Each strain displayed its own “fluorescent signature” reflecting the underlying alleles present in the three target sequences. But, not every fluorescent signature agreed with archival information on the mutational composition of the strain. These discrepancies were resolved by sequencing the reaction products. The results show that the fluorescent signatures consistently identify the alleles correctly. Many more strains of M. tuberculosis will soon be tested. Conclusion: This single color multiplex assay has both clinical and research applications and is just one of many possible assays for M(X)DR-TB that can be constructed using these technologies. For instance, Therma-Light probes labeled with fluorophores in other colors can be added for simultaneous analysis variants responsible for resistance to all other first and second line antibiotics. Support: Brandeis University and Hain Lifescience

Rapid identification of specific TEM-type beta-Lactamase genes in bacterial infections is important for determining appropriate clinical treatment. We report here the design and initial testing of a molecular diagnostic assay capable of amplifying a large segment of the bla(TEM) gene, as well as detecting widely spaced extended-spectrum beta-Lactamase (ESBL) mutations and inhibitor-resistant TEM (IRT) mutations (eg, clavulanic acid resistance). Single-stranded DNA is generated using linear-after-the-exponential PCR (LATE-PCR) and is analyzed at the endpoint, using a set of four fluorescently labeled and four quencher-labeled probes in a single closed tube. These Lights-on/lights-off probes work in concert to generate sequence-specific fluorescence contours over a temperature range from 25 degrees C to 75 degrees C. Mutant sequences from synthetic TEM gene variants and from TEM gene variants in bacterial strains generated large increases in fluorescent signal relative to that from the reference sequence for TEM-1. Clinical use of this convenient, single-closed-tube assay would make it possible to rapidly distinguish ESBL from non-ESBL variants and thereby to begin early treatment with suitable antibiotics.

Detection and identification of highly variable viral sequences is important for tracking infectious outbreaks and determining treatment regimens using targeted drug therapy. This report describes a single tube assay that is able to distinguish extensive sequence variation in hepatitis C virus (HCV) by using mismatch tolerant probes to analyze single-stranded amplicons generated with reverse transcription linear-after-the-exponential PCR (RT-LATE-PCR). Detection and identification of sequences from the 5' non-coding region (NCR) of 31 different HCV strains was first evaluated via hybridization of two fluorescently labeled, mismatch-tolerant probes to synthetic DNA strands. The resulting data were used to calculate the ratio of fluorescent signals for the two probes over a wide temperature range as well as the melting temperature (Tm) of each probe with the targets. Although the Tm measurements alone distinguished only 5 sequences from the others, fluorescent signal ratio analysis provided a unique set of values for 27 of the 31 strains. RT-LATE-PCR was then used to amplify Armored RNA (AR) containing the 5' NCR of five different strains of HCV. Melting analysis of the resulting single-stranded DNA with the two probes distinguished all five AR sequences. This assay can be expanded to include additional gene segments, and it points the way to construction of highly informative single-tube assays for HCV and other RNA viruses.

A novel molecular assay for Clostridium difficile was developed using Linear-After-The-Exponential polymerase chain reaction (LATE-PCR). Single-stranded DNA products generated by LATE-PCR were detected and distinguished by hybridization to fluorescent mismatch-tolerant probes, as the temperature was lowered after amplification in 5(°)C intervals between 65°C and 25°C. Single-tube multiplex reactions for tcdA, tcdB, tcdC, and cdtB (binary toxin) sequences were initially optimized using synthetic targets and were subsequently done using genomic DNA; each target was detected and characterized by hybridization to one or more probes of a different fluorescent color. In the case of tcdC, three probes, each labeled with a Quasar fluorophore, hybridize to different locations with known mutations, including the deletion at nucleotide 117 in ribotype 027 strains and the premature stop codon mutation at nucleotide 184 in ribotype 078 strains, each of which is associated with hypervirulent infections. These and other tcdC mutations were distinguished from the reference sequence, as well as from each other by changes in the fluorescent contour generated from the combined Quasar-labeled probes. Specific variations in tcdA and tcdB were also identified in the multiplex assay, including those that identified strains lacking toxin A production. This single closed-tube assay generates substantially more information about virulent C. difficile than currently available commercial assays and could be further expanded to provide strain typing.

A reverse transcription Linear-After-The-Exponential polymerase chain reaction (RT LATE-PCR) assay was evaluated for detection of foot-and-mouth disease virus (FMDV). This pan-serotypic assay targets highly conserved sequences within the 3D (RNA polymerase) region of the FMDV genome, and uses end-point hybridisation analysis of a single mismatch-tolerant low temperature probe to confirm the identity of the amplicons. An Armored RNA served as an internal control to validate virus negative results. The ability of the assay to identify FMDV was directly compared to a real-time RT-PCR assay routinely used by reference laboratories. The analytical sensitivity of the RT LATE-PCR assay was 10 genomic copies and the dynamic range of the test was identical to real-time RT-PCR based on decimal dilutions of an FMDV-positive sample. This pan-serotypic assay was able to detect FMDV in a broad range of clinical samples collected from field cases of FMD (n = 121), while samples of other viruses causing vesicular disease in livestock and genetic relatives of FMDV were negative. In addition to the laboratory-based utility of this diagnostic test, the RT LATE-PCR assay format has potential application for use in a portable ("point-of-care") device designed to achieve rapid detection of FMDV in the field.

Accurate detection of gene sequences in single cells is the ultimate challenge of PCR sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of Linear-After-The-Exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at -different concentrations, but with novel design criteria to insure high efficiency and specificity. LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATE-PCR signals provides a means for improving the accuracy of single-cell genetic diagnosis.

Aims:  A novel molecular assay for the detection of foot‐and‐mouth disease virus (FMDV) was developed using linear‐after‐the‐exponential polymerase chain reaction (LATE‐PCR).

Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)–PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80–250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CF Δ 508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells.

This protocol describes the design and execution of monoplex and multiplex linear-after-the-exponential (LATE)-PCR assays using a novel reagent, PrimeSafe, that suppresses all forms of mispriming. LATE-PCR is an advanced form of asymmetric amplification that uses a limiting primer and an excess primer for efficient exponential amplification of double-stranded DNA, followed by linear amplification of one strand. Each single-stranded amplicon can be quantitatively detected in real time or at end point. By separating primer annealing from product detection, LATE-PCR enables product analysis at low temperatures. Alternatively, each single strand can be sequenced by a convenient Dilute-'N'-Go procedure. Amplified samples are diluted with individual sequencing primers without the use of columns or spins. We have amplified and then sequenced 15 different single-stranded products generated in a single multiplexed LATE-PCR comprised of 15 pairs of unrelated primers. Dilute-'N'-Go dideoxy sequencing is more convenient, faster and less expensive than sequencing double-stranded amplicons generated via conventional symmetric PCR. The preparation of LATE-PCR products for Dilute-'N'-Go sequencing takes only 30 seconds.

Accurate detection of gene sequences in single cells is the ultimate challenge to polymerase chain reaction (PCR) sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of linear-after-the-exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at different concentrations, but with novel design criteria to ensure high efficiency and specificity. Compared with conventional PCR, LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATE-PCR signals provides a means for improving the accuracy of single cell genetic diagnosis.

Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but it currently is hampered by a labor-intensive sample preparation process designed to isolate single-stranded DNA from double-stranded products generated by conventional PCR. Here linear-after-the-exponential (LATE)–PCR is introduced as an efficient and potentially automatable method of directly amplifying single-stranded DNA for pyrosequencing, thereby eliminating the need for solid-phase sample preparation and reducing the risk of laboratory contamination. These improvements are illustrated for single-nucleotide polymorphism genotyping applications, including an integrated single-cell-through-sequencing assay to detect a mutation at the globin IVS 110 site that frequently is responsible for β-thalassemia.