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. 2021 Sep 25;11(10):1761.
doi: 10.3390/diagnostics11101761.

Design of InnoPrimers-Duplex Real-Time PCR for Detection and Treatment Response Prediction of EBV-Associated Nasopharyngeal Carcinoma Circulating Genetic Biomarker

Mai Abdel Haleem Abusalah  1 , Siti Asma Binti Hassan  1   2 , Norhafiza Mat Lazim  2   3 , Baharudin Abdullah  2   3 , Wan Fatihah Binti Wan Sohaimi  2   4 , Azlan Husin  2   5 , Kueh Yee Cheng  2   6 , Chan Yean Yean  1   2
Affiliations

Design of InnoPrimers-Duplex Real-Time PCR for Detection and Treatment Response Prediction of EBV-Associated Nasopharyngeal Carcinoma Circulating Genetic Biomarker

Mai Abdel Haleem Abusalah et al. Diagnostics (Basel). .

Abstract

Nasopharyngeal carcinoma (NPC) is an epithelial tumor with high prevalence in southern China and Southeast Asia. NPC is well associated with the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) 30 bp deletion by having its vital role in increased tumorigenicity and decreased immune recognition of EBV-related tumors. This study developed an InnoPrimers-duplex qPCR for detection of NPC blood circulating LMP1 30 bp deletion genetic biomarker for early diagnosis and treatment response prediction of NPC patients. The analytical and diagnostic evaluation and treatment response prediction were conducted using NPC patients' whole blood (WB) and tissue samples and non-NPC cancer patients and healthy individuals' WB samples. The assay was able to detect as low as 20 ag DNA per reaction (equivalent to 173 copies) with high specificity against broad reference microorganisms and archive NPC biopsy tissue and FNA samples. The diagnostic sensitivity and specificity were 83.3% and 100%, respectively. The 30 bp deletion genetic biomarker was found to be a good prognostic biomarker associated with overall clinical outcome of NPC WHO type III patients. This sensitive and specific assay can help clinicians in early diagnosis and treatment response prediction of NPC patients, which will enhance treatment outcome and lead to better life-saving.

Keywords: 30 bp deletion NPC genetic biomarker; Epstein-Barr virus; latent membrane protein 1; nasopharyngeal carcinoma; real-time PCR.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Alignment of reference strain (V01555.2) (first line) with 25 sequences of AB and FNA samples from NPC patients. Dot areas (…………………………) represent the location of 30 bp deletion.
Figure 2
Figure 2
Schematic diagram of the InnoPrimers-duplex qPCR (innovative combination of the gap-filling mutant primer and multi-points degenerative reverse primer blocker) EBV LMP1 30 bp deletion assay.
Figure 3
Figure 3
Preliminary functionality of the InnoPrimers-duplex qPCR for detecting of 30 bp deletion NPC genetic biomarker among MT gBLOCK, WT gBLOCK, NPC AB and FNA samples. Abbreviations: AB, archive biopsy sample; FNA, fine needle aspiration.
Figure 4
Figure 4
Standard curve of the InnoPrimers-duplex qPCR showing amplification of 10-fold dilutions of 30 bp deletion synthetic DNA NPC genetic biomarker (MT gBLOCK).
Figure 5
Figure 5
The functionality of the developed InnoPrimers-duplex qPCR. Abbreviations: AB, archive biopsy sample; FNA, fine needle aspiration.
See this image and copyright information in PMC

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