Development of Droplet Digital PCR-Based Assays to Quantify HIV Proviral and Integrated DNA in Brain Tissues from Viremic Individuals with Encephalitis and Virally Suppressed Aviremic Individuals

We developed ddPCR assays to quantitatively measure HIV DNA and used this ddPCR assays to detect and quantitatively measure HIV DNA in the archived brain tissues from HIV patients. The tissue viral loads assessed by ddPCR was highly correlative with those assessed by qPCR.

Accuracy and Clinical Relevance of Intra-Tumoral Fusobacterium nucleatum Detection in Formalin-Fixed Paraffin-Embedded (FFPE) Tissue by Droplet Digital PCR (ddPCR) in Colorectal Cancer

The use of droplet digital PCR (ddPCR) to identify and quantify low-abundance targets is a significant advantage for accurately detecting potentially oncogenic bacteria. Fusobacterium nucleatum (Fn) is implicated in colorectal cancer (CRC) tumorigenesis and is becoming an important prognostic biomarker. We evaluated the detection accuracy and clinical relevance of Fn DNA by ddPCR in a molecularly characterized, formalin-fixed, paraffin-embedded (FFPE) CRC cohort previously analyzed by qPCR for Fn levels. Following a ddPCR assay optimization and an analytical evaluation, Fn DNA were measured in 139 CRC FFPE cases. The measures of accuracy for Fn status compared to the prior results generated by qPCR and the association with clinicopathological and molecular patients’ features were also evaluated. The ddPCR-based Fn assay was sensitive and specific to positive controls. Fn DNA were detected in 20.1% of cases and further classified as Fn-high and Fn-low/negative, according to the median amount of Fn DNA that were detected in all cases and associated with the patient’s worst prognosis. There was a low agreement between the Fn status determined by ddPCR and qPCR (Cohen’s Kappa = 0.210). Our findings show that ddPCR can detect and quantify Fn in FFPE tumor tissues and highlights its clinical relevance in Fn detection in a routine CRC setting.

Biomarkers of Castrate Resistance in Prostate Cancer: Androgen Receptor Amplification and T877A Mutation Detection by Multiplex Droplet Digital PCR

Androgen Receptor (AR) alterations (amplification, point mutations, and splice variants) are master players in metastatic castration resistant prostate cancer (CRPC) progression and central therapeutic targets for patient management. Here, we have developed two multiplexed droplet digital PCR (ddPCR) assays to detect AR copy number (CN) and the key point mutation T877A. Overcoming challenges of determining gene amplification from liquid biopsies, these assays cross-validate each other to produce reliable AR amplification and mutation data from plasma cell free DNA (cfDNA) of advanced prostate cancer (PC) patients. Analyzing a mixed PC patient cohort consisting of CRPC and hormone sensitive prostate cancer (HSPC) patients showed that 19% (9/47) patients had AR CN amplification. As expected, only CRPC patients were positive for AR amplification, while interestingly the T877A mutation was identified in two patients still considered HSPC at the time. The ddPCR based analysis of AR alterations in cfDNA is highly economic, feasible, and informative to provide biomarker detection that may help to decide on the best follow-up therapy for CRPC patients.

Development of a Multiplex Droplet Digital PCR Assay for Detection of Enterovirus, Parechovirus, Herpes Simplex Virus 1 and 2 Simultaneously for Diagnosis of Viral CNS Infections

Background: Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification especially in low concentration samples.Methods: Targeting four common viruses of EV, HPeV, HSV1, and HSV2 in cerebrospinal fluid (CSF), we developed a multiplex ddPCR assay using probe ratio-based multiplexing strategy, analyzed the performance, and evaluated it in 97 CSF samples collected from patients with suspected viral CNS infections on a two-channel ddPCR detection system.Results: The four viruses were clearly distinguished by their corresponding fluorescence amplitude. The limits of detection for EV, HPeV, HSV1, and HSV2 were 5, 10, 5, and 10 copies per reaction, respectively. The dynamic range was at least four orders of magnitude spanned from 2000 to 2 copies per reaction. The results of 97 tested clinical CSF specimens were identical to those deduced from qPCR/qRT-PCR assays using commercial kits.Conclusion: The multiplex ddPCR assay was demonstrated to be an accurate and robust method which could detect EV, HPeV, HSV1, and HSV2 simultaneously. It provides a useful tool for clinical diagnosis and disease monitoring of viral CNS infections.

Non-Invasive Prenatal Detecting of Achondroplasia In Earlier Stage of Pregnancy By Droplet-Digital PCR

Background: Achondroplasia (ACH) is generally detected by abnormal prenatal ultrasound findings in the late stage of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained invasively. Most ACH cases appear to be de novo mutations with FGFR3 gene, so it is a challenge to screen ACH fetus out in the early stage of pregnancy.Objective: The aim of this study was to validate the possibility of detect fetus ACH along with non-invasive prenatal screening(NIPS) routinely in the early stage of pregnancy.Methods: 5927 cases of pregnant women undergoing NIPS were enrolled in this study. An additional 5ml of blood was collected together with NIPS blood sampling. Cell free DNA was extracted for the detecting of fetus ACH. Droplet-digital PCR(ddPCR) method based on the amplification of the two possible mutant alleles (c. 1138G>A and c. 1138G>C) of FGFR3 gene was performed to screen fetus ACH. Prenatal ultrasound and amniocentesis were then performed to confirm the positive screening result of ACH cases. The mutation sites of fetus were identified via Sanger sequencing by using amniotic fluid cells. For the screen negative cases of pregnant women, we followed up the results of prenatal diagnosis or the general conditions of the newborns.Results: One pregnant woman with fetus ACH were screened out at 22 weeks by Non-invasive prenatal detecting. Later prenatal ultrasound confirmed fetal skeletal dysplasia. Sanger sequencing confirmed de novo FGFR3 1138G>A mutant of the fetus. No ACH fetus or newborns were found in the rest detected negative cases of enrolled pregnant women.Conclusion: ddPCR technology could effectively identify de novo mutation like ACH of fetus noninvasively. We prospect the clinical application of ddPCR can expand the range of prenatal screen in the future.

OPTIMIZACIÓN DE ddPCR Y DESARROLLO DE MÓDULOS MULTIPLEX PARA EL ANÁLISIS DE EVENTOS DE MODIFICACIÓN GENÉTICA EN MAÍZ

La siembra, comercialización y consumo de maíz transgénico es motivo de debate en México, ya que es centro de origen y diversificación de variedades y razas de maíz nativo. La reacción en cadena de la polimerasa digital por goteo (droplet digital PCR, ddPCR) ha demostrado ser más eficiente que el estándar de oro para el análisis de maíz modificado genéticamente. Por tanto, el objetivo fue optimizar pruebas de ddPCR para el análisis de transgenes en maíz y generar módulos multiplex de detección. Por medio de un análisis bioinformático se evaluó la aplicabilidad de los iniciadores. Después, con gradientes se determinó una temperatura de hibridación y elongación óptima y se evaluó la concentración de iniciadores, la recuperación de gotas y el efecto “lluvia”. La optimización se verificó por medio de pruebas dúplex, y finalmente se desarrollaron módulos multiplex. La temperatura que permitió una separación clara entre clústeres de gotas positivas y negativas para las 11 pruebas símplex fue 58.5 °C, con concentraciones relativamente bajas de iniciadores y sondas. Las gotas recuperadas resultaron adecuadas según el propósito de los análisis y la “lluvia” obtenida resultó irrelevante. En las pruebas dúplex se confirmó el potencial para la cuantificación sin usar curvas de calibración; la mayoría de los ensayos concordaron con los reportes de validación o con los porcentajes teóricos de variabilidad genética. Por último, en los módulos multiplex se realizaron variaciones en la concentración de iniciadores y sondas para diferenciar clústeres de gotas positivas en un canal detector único. Las mejores diferencias se lograron con concentraciones con diferencias de 1:3. Los resultados mostraron que las pruebas optimizadas son adecuadas para la detección y posible cuantificación de 10 transgenes en maíz.

Development of SARS-CoV-2 packaged RNA reference material for nucleic acid testing

Nucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80–8.23% and is stable at 4 °C for 1 week and at −70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.