Jinyin Zhao

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.

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.

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 ĸ 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.

Spectrum of ATP7B mutations and genotype–phenotype correlation in large-scale Chinese patients with Wilson Disease

Wilson disease (WD), an inherited disorder associated with ATP7B gene, has a wide spectrum of genotypes and phenotypes. In this study, we developed a rapid multiplex PCR‐MassArray method for detecting 110 mutant alleles of interest, and used it to examine genomic DNA from 1222 patients and 110 healthy controls. In patients not found to have any mutation in the 110 selected alleles, PCR‐Sanger sequencing was used to examine the ATP7B gene. We identified 88 mutations, including 9 novel mutations. Our analyses revealed p.Arg778Leu, p.Arg919Gly and p.Thr935Met showed some correlations to phenotype. The p.Arg778Leu was related to younger onset age and lower levels of ceruloplasmin (Cp) and serum copper, while p.Arg919Gly and p.Thr935Met both indicated higher Cp levels. Besides, the p.Arg919Gly was related to neurological subtype, and p.Thr935Met showed significant difference in the percentage of combined neurological and visceral subtype. Moreover, for ATP7B mutations, the more severe impact on ATP7B protein was, the younger onset age and lower Cp level presented. The feasibility of presymptomatic DNA diagnosis and predicting clinical manifestation or severity of WD would be facilitated with identified mutations and genotype–phenotype correlation precisely revealed in the study.

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.

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.

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.