Milford Molecular Diagnostics

2044 Bridgeport Ave, Milford, CT 06460


Lyme Disease DNA Test

"DNA sequencing-based diagnostics are our specialty and routine diagnostic DNA sequencing is the gateway to practicing personalized molecular medicine."

Sin Hang Lee, MD, F.R.C.P.(C), FCAP


Sanger sequencing for definitive detection of SARS-CoV-2

Under the best of circumstances, RT-qPCR assays granted emergency use authorization for presumptive detection of SARS-CoV-2 in clinical specimens generate at least 30% false-positive results and 20% false-negative results [32]. The World Health Organization (WHO) has acknowledged “In some circumstances, the distinction between background noise and actual presence of the target virus is difficult to ascertain” [33]. The WHO further advised that SARS-CoV-2 gene sequencing can be used for improved diagnostics [34]. However, most laboratories require specimens with high viral loads for sequencing [35]. Milford Molecular Diagnostics Laboratory has developed a routine sequencing assay for definitive detection of SARS-CoV-2 by partial sequencing of a 398-bp amplicon of the N gene cDNA and a 437-bp amplicon of the S gene for routine screening of 8 common amino acid mutations of concern. All isolates positive for at least one of these 8 amino acid mutations will be further sequenced for variant differentiation. Every report of positive sample will contain at least two electropherograms showing an N gene for detection (upper electropherogram) and an S gene for possible amino acid mutations (lower electropherogram) as follows.


Electropherogram (below) showing the ACE 2 receptor binding domain of the SARS-CoV-2 S gene with a solitary E484K amino acid mutation

This routine DNA sequence electropherogram covering the ACE 2 receptor binding domain shows 7 vertical black bars from left to right, pointing to 7 key nucleotides identified as A, G, T, G, A, T and A in the S gene of SARS-CoV-2 in a nasopharyngeal swab specimen taken from a patient with acute respiratory infection. The nucleotide A pointed by the 5th bar is a mutated base, indicating an E484K amino acid mutation (G>A nucleotide mutation). All other 6 black bars point to wildtype bases, confirming the lack of K417N, K417T, L452R, S477N, E484Q, S494P and N501Y mutations in this virus isolate. All known newly emerging SARS-CoV-2 variants of concern are associated with at least one of the 8 amino acid mutations identified in this sequence. Specimens harboring any of these 8 amino acid mutations will be further sequenced for A67V, Δ69/70, T95I, Δ144Y, Y145del, H146del, W152C and V1176F mutations for B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617, B.1.168, P.1 and P.2 variant differentiation, said Dr. Sin Hang Lee, Director of Milford Molecular Diagnostics Laboratory