Yiqun Liao

Fluorescent Probe-Based Lateral Flow Assay for Multiplex Nucleic Acid Detection

Here we report a rapid, low cost, and disposable dipstick-type DNA biosensor that enables multiplex detection in a single assay. The fluorescent probes labeled with different fluorophores were introduced into the lateral flow nucleic acid testing system. In combination with multiple immobilized probes arranged in an array formant on the membrane, a dual-color fluorescent lateral flow DNA biosensor was developed using a portable fluorescence reader. Up to 13 human papillomavirus types could be detected simultaneously by a single-step operation in less than 30 min after linear-after-the-exponential (LATE)-PCR. The sensitivity was determined to be 10-10(2) copies plasmid DNA/μL. The specificity study showed no cross-reactivity among the 31 different common HPV types. In the clinical validation, 95.3% overall agreement showed very good potential for this method in the clinical application when compared to a commercial kit.

Multiplex Fluorescence Melting Curve Analysis for Mutation Detection with Dual-Labeled, Self-Quenched Probes

Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

Combination of fluorescence color and melting temperature as a two-dimensional label for homogeneous multiplex PCR detection

Multiplex analytical systems that allow detection of multiple nucleic acid targets in one assay can provide rapid characterization of a sample while still saving cost and resources. However, few systems have proven to offer a solution for mid-plex (e.g. 10- to 50-plex) analysis that is high throughput and cost effective. Here we describe the combined use of fluorescence color and melting temperature (Tm) as a virtual 2D label that enables homogenous detection of one order of magnitude more targets than current strategies on real-time polymerase chain reaction platform. The target was first hybridized with a pair of ligation oligonucleotides, one of which harbored an artificial sequence that had a unique Tm when hybridized with a reporter fluorogenic probe. The ligated products were then amplified by a universal primer pair and denatured by a melting curve analysis procedure. The targets were identified by their respective Tm values in the corresponding fluorescence detection channels. The proof-of-principle of this approach was validated by genotyping 15 high-risk human papillomaviruses and 48 human single-nucleotide polymorphisms. The robustness of this method was demonstrated by analyzing a large number of clinical samples in both cases. The combined merits of multiplexity, flexibility and simplicity should make this approach suitable for a variety of applications.

Simultaneous Detection, Genotyping and Quantification of Human Papillomaviruses by Multicolor Real-Time PCR and Melting Curve Analysis

ABSTRACT Long-term infection with high-risk human papillomavirus (HPV) is the leading cause of cervical cancer, while infection with low-risk HPV is the major reason for condylomata acuminata. An accurate, rapid, and convenient assay that is able to simultaneously detect, genotype, and quantify HPV would be of great clinical value yet remains to be achieved. We developed a three-color real-time PCR assay that is able to analyze 30 predominant HPV types in three reactions. The amplification curves indicated the presence of HPV, melting curve analysis identified the HPV genotype, and the quantification cycle value determined the quantity. We applied this assay to 647 cervical swab samples, and the results were compared with those obtained with a commercial genotyping system. The proposed assay had a limit of detection of 5 to 50 copies per reaction and a dynamic range of 5 × 101 to 5 × 106 copies per reaction. A comparison study showed that the overall sample concordance with the comparison method was 91.6% and the type agreement was greater than 98.7%. The quantification study demonstrated that the loads of HPV type 16 in 30 samples with cervical intraepithelial neoplasia grade III (CIN III) lesions were significantly higher than those in samples with CIN I lesions or CIN II lesions, and the results were concordant with those of the comparison method. The increased information content, high throughput, and low cost would facilitate the use of this real-time PCR-based assay in a variety of clinical settings.

Rapid detection of HCV genotyping 1a, 1b, 2a, 3a, 3b and 6a in a single reaction using two-melting temperature codes by a real-time PCR-based assay

The genotype of the hepatitis C virus (HCV) is an important indicator for antiviral therapeutic response. We hereby described development of a rapid HCV genotyping approach that enabled the identification of the six most common HCV subtypes of Asia, i.e., 1a, 1b, 2a, 3a, 3b, and 6a, in a single reaction. Using two dual-labeled, self-quenched probes that target the core region of the HCV genome, the exact subtype could be accurately identified by two-melting temperature codes determined from the two respective probes in a real-time PCR assay. Analytical sensitivity studies using armored RNA samples representing each of the six HCV subtypes showed that 5 copies/reaction of HCV RNA could be detected. The assay was evaluated using 244 HCV-positive serum samples and the results were compared with sequencing analysis. Of the 224 samples, subtype 3a (127, 52.3%) was the dominant, followed by 1b (51, 20.9%), 3b (47, 19.3%), 2a (8, 3.3%), 6a (4, 1.6%) and the least was subtype 1a (1, 0.4%). Moreover, 6 (2.5%) mixed infection samples were also detected. These results were fully concordant with sequencing analysis. We concluded that this real-time PCR-based assay could provide a rapid and reliable tool for routine HCV genotyping in most Asian countries.

Multicolor Melting Curve Analysis-Based Multilocus Melt Typing of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis outbreaks. To track the source of these diseases in a timely manner, a high throughput typing method is critical. We hereby describe a novel genotyping method for V. parahaemolyticus, termed multilocus melt typing (MLMT), based on multilocus sequence typing (MLST). MLMT utilizes melting curve analysis to interrogate the allelic types of a set of informative single nucleotide polymorphisms (SNPs) derived from the housekeeping genes used in MLST. For each SNP, one allelic type generates distinct Tm values, which are converted into a binary code. Multiple SNPs thus generate a series of binary codes, forming a melt type (MT) corresponding with a sequence type (ST) of MLST. Using a set of 12 SNPs, the MLMT scheme could resolve 218 V.parahaemolyticus isolates into 50 MTs corresponding with 56 STs. The discriminatory power of MLMT and MLST was similar with Simpson’s index of diversity of 0.638 and 0.646, respectively. The global (adjusted Rand index = 0.982) and directional congruence (adjusted Wallace coefficient, MT→ST = 0.965; ST→MT = 1.000) between the two typing approaches was high. The entire procedure of MLMT could be finished within 3 h with negligible hands on time in a real-time PCR machine. We conclude that MLMT provides a reliable and efficient approach for V. parahaemolyticus genotyping and might also find use in other pathogens.

Simultaneous Identification of Ten Bacterial Pathogens Using the Multiplex Ligation Reaction Based on the Probe Melting Curve Analysis

Pathogenic Vibrio spp., Aeromonas spp. and Plesiomonas shigelloides are associated with human gastroenteritis and wound infections, as well as fish diseases. The comprehensive and accurate identification of these pathogens is crucial for the current public health. The present study describes the development of a multiplex assay for the simultaneous identification of ten bacterial pathogens in a single reaction by using a multiplex ligation reaction based on probe melting curve analysis (MLMA). The specificity for target genes was 100%, as assessed with a panel of 67 bacterial pathogens, which indicated no cross-reactions. The detection limit of this assay ranged from 0.8 × 107 CFU/mL to 1.5 × 108 CFU/mL at the pure bacterial culture level and from 0.1 ng to 1.0 ng at the DNA level. The MLMA assay was used to detect ten species of pathogens in 269 clinical and seafood samples, and for further validation, the results were compared with the conventional culture method. The results indicated greater than 90% sensitivity and 100% specificity for each bacterial pathogen tested, and the kappa correlation for all the pathogens ranged from 0.95 to 1.00. Overall, this assay is well suited for public health laboratories for its high throughput, accuracy, and low cost.

Dissemination and Molecular Characterization of Staphylococcus aureus at a Tertiary Referral Hospital in Xiamen City, China

Staphylococcus aureus is a global epidemic pathogen that causes heavy disease burden. The aim of this study was to determine which globally known S. aureus lineages are currently present in a hospital of Xiamen. Therefore, the 426 S. aureus strains were detected by Melting Curve Analysis (MCA) and genotyped by Pulsed Field Gel Electrophoresis (PFGE) as well as Multicolor Melting Curve Analysis-Based Multilocus Melt Typing (MLMT). In addition, Multilocus Sequence Typing (MLST) was used to identify 108 representative strains. In light of eighteen antibiotics except for Vancomycin (by Broth Dilution Method), we used the Kirby-Bauer disc diffusion method to assess antibiotic susceptibility of 426 S. aureus strains. Finally, PFGE analysis revealed 14 different patterns with three major patterns (C10, C8, and C11) that accounted for 69.42% of all S. aureus strains, and MT-1~MT-5 occupied most part of the strains by MLMT. MLST revealed 25 different STs with the predominant types being ST239, ST59, and ST188. There have been 8 antibiotics that showed more than 50% resistance of all S. aureus strains. In summary, we found several of the lineages are predominant in our hospital. And antibiotic resistance is still a severe problem that needs to be controlled in clinic.

A comparison of the MeltPro ® HPV Test with the Cobas ® HPV Test for detecting and genotyping 14 high-risk human papillomavirus types

The clinical performance of the newly developed MeltPro® HPV Test, based on multicolor melting curve analysis, was evaluated and compared with the commercially available Cobas® HPV Test for detection of HPV and genotyping of HPV-16 and HPV-18. A total of 1647 cervical samples were analyzed with both tests. The agreement values were 96.2% for HPV detection, 99.6% for HPV-16 identification, and 99.7% for HPV-18 identification. All genotyping results from MeltPro® HPV Test showed that HPV-52, HPV-58, and HPV-16 were the most common types in this study. Intra-laboratory reproducibility studies showed 97.8% agreement while inter-laboratory reproducibility studies showed 96.9% agreement for the MeltPro® HPV Test. The MeltPro® HPV Test and Cobas® HPV Test are highly correlative and are useful for monitoring HPV infection.

Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis

ABSTRACT Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 104 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than