Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method.

Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/µl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/µl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.

Restriction endonuclease-mediated real-time digestion-PCR for somatic mutation detection.

PCR is a powerful platform for clinical and diagnostic applications, but challenges remain in detecting somatic mutations, as mutant cells are often mixed with more numerous wild-type cells at the tissue-sample sites. Here, we describe a novel method that couples PCR with restriction endonuclease digestion (designated real-time digestion-PCR, or RTD-PCR) in a one-step reaction tube for detecting somatic mutations from a minority of cells. The PCR mixture contains a thermostable restriction enzyme that digests wild-type alleles during the PCR program, allowing selective amplification of the mutant alleles. To validate this method, we used real-time digestion-PCR for the specific detection of the EGFR (epidermal growth factor receptor) treatment resistance-inducing mutation, T790M, combining with three different platforms: Sanger sequencing, TaqMan probe PCR and Sequenom MassArray. From 78 clinical samples, seven T790M mutations were consistently detected on all three platforms, indicating that RTD-PCR may be a useful clinical tool for analyzing the T790M point mutation.

Rapid oligonucleotide suspension array-based multiplex detection of bacterial pathogens.

A gene-specific microsphere suspension array coupled with 15-plex polymerase chain reaction (PCR) was developed to screen bacterial samples rapidly for 10 strains of bacteria: Shigella spp. (S. flexneri, S. dysenteriae, and S. sonnei), Staphylococcus aureus, Vibrio cholerae (serology O1 and O139), Legionella pneumophila, and Clostridium botulinum (types A, B, and E). Fifteen sets of highly validated primers were chosen to amplify target genes simultaneously. Corresponding oligonucleotide probes directly conjugated with microsphere sets were used to specifically identify PCR amplicons. Sensitivity tests revealed that the array coupled with single PCR was able to detect purified genomic DNA at concentrations as low as 10 copies/µL, while the multiplex detection limit was 10-104 copies/µL. The assay was validated using water samples artificially spiked with S. aureus and S. dysenteriae, as well as water specimens from swimming pools previously identified to contain S. aureus.

A comparison of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and surface plasmon resonance for genotyping of high-risk human papillomaviruses.

BACKGROUND: Human papillomavirus (HPV) testing coupled with appropriate clinical management is associated with a significant decline in the rate of advanced cervical cancer and associated death. METHODS: In this present study, we evaluated the performance of 2 new HPV genotyping methods, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and surface plasmon resonance (SPR) in 30 kinds of HPV control materials and in 129 cases of cervical smears including 79 HPV-positive samples screened from 1,600 abnormal clinical samples and 50 cervical cytology samples. RESULTS: The HPV genotyping accuracy of both MALDI-TOFMS and SPR was 100% for the HPV genotyping of control materials. In the analysis of the 79 HPV-positive samples by MALDI-TOFMS, HPV positivity was 88.6% (70/79). Nine samples were non-high-risk HPV (non-HR-HPV), which were not targets of MALDI-TOFMS. In the analysis of the 50 cervical samples, the agreement of both tests was 84% with a κ value of 0.660. By using consensus results that mean agreement between 2 of 3 methods, the HR-HPV genotyping accuracy was 100% (77/77) by MALDI-TOFMS and 94.8% (73/77) by SPR in the 129 cervical samples. The sensitivity (88.2%; 82/93) and specificity (88.9%; 32/36) of MALDI-TOFMS were similar to those of SPR. CONCLUSION: These results support that MALDI-TOFMS is a sensitive, specific and feasible method for HR-HPV detection in clinical application, compared with the SPR method.

Multicenter assessment of shotgun metagenomics for pathogen detection.

BACKGROUND: Shotgun metagenomics has been used clinically for diagnosing infectious diseases. However, most technical assessments have been limited to individual sets of reference standards, experimental workflows, and laboratories. METHODS: A reference panel and performance metrics were designed and used to examine the performance of shotgun metagenomics at 17 laboratories in a coordinated collaborative study. We comprehensively assessed the reliability, key performance determinants, reproducibility, and quantitative potential. FINDINGS: Assay performance varied significantly across sites and microbial classes, with a read depth of 20 millions as a generally cost-efficient assay setting. Results of mapped reads by shotgun metagenomics could indicate relative and intra-site (but not absolute or inter-site) microbial abundance. INTERPRETATION: Assay performance was significantly impacted by the microbial type, the host context, and read depth, which emphasizes the importance of these factors when designing reference reagents and benchmarking studies. Across sites, workflows and platforms, false positive reporting and considerable site/library effects were common challenges to the assay's accuracy and quantifiability. Our study also suggested that laboratory-developed shotgun metagenomics tests for pathogen detection should aim to detect microbes at 500 CFU/mL (or copies/mL) in a clinically relevant host context (10^5 human cells/mL) within a 24h turn-around time, and with an efficient read depth of 20M. FUNDING: This work was supported by National Science and Technology Major Project of China (2018ZX10102001).

A novel RFLP-ARMS TaqMan PCR-based method for detecting the BRAF V600E mutation in melanoma.

To enable the rapid and sensitive screening of the BRAF V600E mutation in clinical samples, a novel method combining restriction fragment length polymorphism (RFLP) analysis with the popular amplification refractory mutation system (ARMS) TaqMan quantitative (qPCR) genotyping method in a single reaction tube was developed. A total of 2 primer pairs were designed to enrich for and genotype the BRAF mutational hotspot (RFLP primers and ARMS primers) and a restriction enzyme was used to remove the wild-type alleles. The analysis revealed that this method detected mutant alleles in mixed samples containing >0.1% mutant sequences. In a survey of 53 melanoma samples, this method detected 21 mutation-positive samples. This novel RFLP-ARMS TaqMan qPCR protocol may prove useful for detecting mutations in clinical samples containing only a small proportion of mutant alleles.

[EGFR and KRAS Gene Mutations in 754 Patients with Resectable Stage I-IIIa Non-small Cell Lung Cancer and Its Clinical Significance].

BACKGROUND: Epidermal growth factor receptor (EGFR) and KRAS gene are important driver genes of non-small cell lung cancer (NSCLC). The studies are mainly focused on detection of EGFR gene for advanced NSCLC, and the mutation feature of EGFR and KRAS gene in early NSCLC tissue is unknown. This study aims to investigate the mutations of EGFR and KRAS gene in NSCLC, and the relationship between the genotype and clinicopathologic features. METHODS: The hotspot mutations in EGFR and KRAS gene in 754 tissue samples of stage I-IIIa NSCLC from Department of Pathology, Peking Union Medical College Hospital were detected by modified amplification refractory mutation system (ARMS) real-time PCR kit, and analyzed their correlation with clinical variables. RESULTS: The hotspot mutation rates in EGFR and KRAS were 34.5% and 13.1% respectively, and there were EGFR-KRAS double mutations in 3 samples. The mutation rate of EGFR was higher in females than that in males (39.5% vs 29.4%, P=0.076), significantly increased in adenocarcinomas (38.7%) compared to that in the other forms of NSCLC (P<0.01), but still lower than that reported in some Asian studies of advanced adenocarcinoma (-50%). Meanwhile, the mutation rate of KRAS was remarkably higher in males than that in females (16.6% vs 9%, P=0.048), increased in adenocarcinomas compared to that in the other forms of NSCLC, but the difference was not significant (P=0.268). Samples harbored EGFR mutation were younger than those harbored KRAS mutation (P=0.031,5), and had significant difference in gender between the two groups (P<0.01). CONCLUSIONS: The mutation rate of EGFR in stag I-IIIa NSCLC patients was lower than that in advanced NSCLC patients. And the percentage of the NSCLC patients with EGFR-KRAS double mutations is 0.9%.

A sensitive and practical method to detect the T790M mutation in the epidermal growth factor receptor.

The current study aimed to develop a method to rapidly, sensitively and practically screen for the epidermal growth factor receptor (EGFR) T790M mutation. This method combines an allele-specific competitive blocker (ACB) with a TaqMan quantitative polymerase chain reaction (PCR) amplification refractory mutation system (ARMS) in a one-step reaction. Using a mimic of a human genomic DNA panel containing serially diluted mutant alleles, the performance efficacy of this method was assessed. Using this method, the EGFR T790M mutation was detected in tyrosine kinase inhibitor (TKI)-naïve samples obtained from 27 non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations. The association between de novo T790M mutations and the clinical benefit of EGFR-TKI treatment was also analysed. The sensitivity of this method was as low as 0.01%. In the samples from the 27 NSCLC patients, this method identified 6 mutant patients (22.2%), which was higher than the detection rate with scorpion ARMS (0.0%). No clinical variables were associated with the occurrence of a de novo T790M mutation. The median progression-free survival time in the TKI-naïve patients with a T790M mutation was shorter that that of patients without the mutation, but the difference was not significant (3.2 vs. 19.5 months, respectively; P=0.256). The median overall survival time in the groups with or without T790M mutation also did not significantly differ (10 vs. 20 months, respectively; P=0.689). Overall, the ACB-ARMS PCR method could be useful for detecting the EGFR T790M mutation in clinical samples that contain only a small number of mutant alleles. The clinical significance of a de novo T790M mutation should be further investigated.

Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique.

OBJECTIVES: The objective of this study is to develop a novel and sensitive method for KRAS codon 12 mutation testing. DESIGN AND METHODS: We developed a sensitive one-step real-time digestion-and-block TaqMan probe PCR (RTDB-PCR) technique that uses a thermostable endonuclease and a minor groove binder (MGB) blocker to detect KRAS codon 12 mutations. Dilution mimic DNA panels were used to assess the sensitivity of this technique. The RTDB-PCR method was performed and compared with three other methods: PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray. A total of 100 formalin-fixed paraffin-embedded (FFPE) metastatic colorectal cancer (mCRC) specimens were also tested by all four methods. RESULTS: The RTDB-PCR was sensitive to as little as 0.01% mutant DNA, significantly higher than other methods. Among the 100 FFPE mCRC specimens examined, 45 tested positive for KRAS codon 12 mutations according to RTDB-PCR, 44 tested positive according to mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, and only 26 samples tested positive according to PCR sequencing. CONCLUSIONS: Compared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR is more cost effective, saves time, and is easier to use, making it suitable for the detection of low-level KRAS mutations in the clinic.

[Detection of EGFR gene mutations in 100 non-small cell lung cancer clinical samples by a real-time polymerase chain reaction method using amplification refractory mutation system specific primers and Taqman fluorescence probes].

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutation is the most important predictor of the efficiency of EGFR-tyrosine kinase inhibitors in the treatment of non-small cell lung cancer (NSCLC). The detection of EGFR gene mutations can guide individual therapies for NSCLC. Numerous methods are used to detect EGFR gene mutation and each method has different features. This study aims to establish a real-time polymerase chain reaction (PCR) method for the detection of EGFR gene mutations using amplification refractory mutation system (ARMS) specific primers and Taqman fluorescence probes. METHODS: ARMS specific primers for the two EGFR gene mutations (E746_A750 and L858R) and Taqman fluorescence probes for the detection of the target sequence were carefully designed by the Primer Premier 5.0 software. Then, using the recombinants containing E746_A750 and L858R mutations as the study objects, we further analyzed the sensitivity and lower limit of this method, and then determined the cutoff ΔCt value to evaluate specific or non-specific amplification. A total of 100 clinical samples were collected and used to detect the EGFR gene mutations using this method. RESULTS: The lower limit of this method for the detection of EGFR gene mutation was 10 copies if no interference of wild-type EGFR gene or background DNA existed. Regarding the method sensitivity, the detection resolution was as high as 1% and 0.1%-0.5% in the background of 500 and 5,000 copies/µL wild-type EGFR gene, respectively. Regarding the method specificity, non-specific amplifications were found when it was used to detect 21 L858R mutations in leukocyte DNA samples from healthy volunteers. However, the minimal ΔCt value was 14.48. Non-specific amplifications were not found when detecting 19 Del mutations. Among the 100 clinical samples, 39 mutations were detected (19 Del and 21 L858R were 21 and 18, respectively) using this method. The total mutation rate was 39.0%. CONCLUSIONS: The proposed ARMS-TaqMan real-time PCR method for the detection of 19 Del and 21 L858R mutations in EGFR gene was rapid, simple, sensitive, specific, and applicable in the clinical setting.

MGB-based one-step multiplex real-time PCR method for rapid detection of HPV.

AIM: For full-scale analysis of Human Papillomavirus (HPV) status in humans, two minor groove binder (MGB)-based one-step multiplex real-time PCR systems were developed: one to screen 16 high-risk HPV (HR-HPV) types, and one to screen a broader spectrum of HPV types (common HPV or C-HPV). METHODS: Sensitivity and specificity were evaluated using diluted reference plasmids and 20 control human DNA samples. For clinical evaluation, 510 cervical scrape samples were evaluated. RESULTS: The sensitivity assays revealed that the C-HPV detection system could detect 10 ~ 100 plasmid copies/reaction, while the HR-HPV detection system detected 10 ~ 500 copies. The specificity test revealed that the systems did not yield positive signals from the 20 human genomic DNA samples. Performance was tested on 510 usable clinical samples. The HR-HPV results were compared to those from the Hybrid Capture 2 (HC2) test, which assesses 13 HR-HPV types; the concordance level between the two methods was 90.8% with a kappa value of 0.813. CONCLUSIONS: These results showed that our novel MGB-based one-step multiplex real-time PCR method may be used for the diagnosis and mass screening of HPV in clinical and large-scale epidemiological studies.

Simultaneous detection and identification of enteric viruses by PCR-mass assay.

Simultaneous detection of enteric viruses that cause similar symptoms (e.g. hand, foot and mouth disease) is essential to the prevention of outbreaks and control of infections. In this study, a novel PCR-Mass assay combining multiplex polymerase chain reaction (PCR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed and used for simultaneous detection of eight distinct human enteric viruses. Enteric viral isolates and standard viral RNAs were examined to determine the sensitivity and specificity of the PCR-Mass assay. Clinical performance was evaluated with a total of 101 clinical specimens from patients suspected of having hand, foot and mouth disease (HFMD). The results were compared to those of previous analyses using real-time RT-PCR. The identification of specific viruses and clinical specimens shows that the PCR-Mass assay performed as well as or better than standard methods with respect to indicating the presence of multiplex pathogens in a single specimen.

A novel method for detection of mutation in epidermal growth factor receptor.

BACKGROUND: For the rapid and sensitive screening of epidermal growth factor receptor (EGFR) hot-spot mutations, we developed a novel method combining mutant-enriched PCR with amplification refractory mutation system (ARMS) TaqMan real-time PCR in a one-step reaction tube. METHODS AND RESULTS: We designed two pairs of primers to enrich and genotyping each mutation (E746_A750del and L858R): nest primers and ARMS primers. Before the PCR assays were carried out, the restriction enzymes were used to cut wild alleles. The results showed that this method could detect mutant alleles mixed samples containing 0.1% with a cutoff ΔCt value of 12. We used this method in a survey of 73 non-small cell lung cancer (NSCLC) samples, detecting 14 mutant samples of E746_A750del and 12 mutant samples of L858R. The results well agreed with the results of DxS. All unmatched samples were identified by sequencing and the results showed that our method has high specificity. CONCLUSION: The mutant-enriched ARMS TaqMan PCR could be useful in the detection of mutation in clinical samples containing only a small number of mutant alleles.