Analytical validation of the droplet digital PCR assay for diagnosis of spinal muscular atrophy

Park, Sunggyun; Lee, Hyeonah; Shin, Saeam; Lee, Seung Tae; Lee, Kyung A.; Choi, Jong Rak

doi:10.1016/j.cca.2020.09.024

Cited by 15 publications

(7 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Except for qPCR, NGS and MLPA, there are other methods that be used for SMN1 copy number detection. The droplet digital PCR is another emerging technique for SMN1 copy number assay 47 – 51 . Although, the improved digital PCR technology has been reported 52 , the most are operational complexity and the related equipment and reagents are expensive.…”

Section: Discussionmentioning

confidence: 99%

Next generation sequencing is a highly reliable method to analyze exon 7 deletion of survival motor neuron 1 (SMN1) gene

Zhao

Wang

Xin

et al. 2022

Sci Rep

View full text Add to dashboard Cite

Spinal muscular atrophy (SMA) is one of the most common and severe genetic diseases. SMA carrier screening is an effective way to identify couples at risk of having affected children. Next-generation sequencing (NGS)-based expanded carrier screening could detect SMN1 gene copy number without extra experiment and with high cost performance. However, its performance has not been fully evaluated. Here we conducted a systematic comparative study to evaluate the performance of three common methods. 478 samples were analyzed with multiplex ligation probe amplification (MLPA), real-time quantitative polymerase chain reaction (qPCR) and NGS, simultaneously. Taking MLPA-based results as the reference, for 0 copy, 1 copy and ≥ 2 copy SMN1 analysis with NGS, the sensitivity, specificity and precision were all 100%. Using qPCR method, the sensitivity was 100%, 97.52% and 94.30%, respectively; 98.63%, 95.48% and 100% for specificity; and 72.72%, 88.72% and 100% for precision. NGS repeatability was higher than that of qPCR. Moreover, among three methods, NGS had the lowest retest rate. Thus, NGS is a relatively more reliable method for SMN1 gene copy number detection. In expanded carrier screening, compared with the combination of multiple methods, NGS method could reduce the test cost and simplify the screening process.

show abstract

Section: Discussionmentioning

confidence: 99%

Next generation sequencing is a highly reliable method to analyze exon 7 deletion of survival motor neuron 1 (SMN1) gene

Zhao

Wang

Xin

et al. 2022

Sci Rep

View full text Add to dashboard Cite

show abstract

“…qPCR, DHPLC, and HRM hold better application potential in screening for SMA, which has been demonstrated to be feasible and cost-effective by pilot screening [ 15 , 16 , 29 ]. dPCR could provide more comprehensive and exact results about copy numbers of SMN1 and SMN2 [ 17 , 18 ]. However, all these methods need experienced technicians to conduct tests on special instruments and to process data with professional software.…”

Section: Discussionmentioning

confidence: 99%

“…Several techniques to detect SMA have been developed, such as multiplex ligation-dependent probe amplification (MLPA), quantitative PCR (qPCR), high-resolution melt profiling (HRM), denature high-performance liquid chromatography (DHPLC), and digital PCR (dPCR) [ 13 , 14 , 15 , 16 , 17 , 18 ]. However, these technical platforms rely on bulky instruments and trained personnel, hindering their application in developing countries or low-resource regions.…”

Section: Introductionmentioning

confidence: 99%

Cas12a and Lateral Flow Strip-Based Test for Rapid and Ultrasensitive Detection of Spinal Muscular Atrophy

Zhang

Chen

et al. 2021

Biosensors

View full text Add to dashboard Cite

Spinal muscular atrophy (SMA) is characterized by severe lethality and irreversible progression. Early diagnosis of SMA is of more practical significance with the emergence of effective therapy. However, existing techniques to identify SMA patients rely on cumbersome instruments, hindering their accessibility and application. An SMA-Cas12a-strip assay was developed with the integration of Cas12a-based nucleic acid detection, isothermal amplification, and lateral flow strip. The analytical performance of the assay was assessed with clinical samples. To explore its extensible utility, various specimens were tested. Validated with 168 clinical samples, the sensitivity and specificity of the SMA-Cas12a-strip assay were both 100%. The minimum detectable concentration of genomic DNA containing the target gene achieved 526 aM. The assay was compatible with specimens from several sources, and the turnaround time could be within 1.5 h. We developed a simple, cost-effective, and highly sensitive and specific assay to detect SMA patients. With little and field-portable equipment, the assay holds great promise in the detection of SMA patients, particularly in low-resource regions.

show abstract

“…Additionally, these techniques cannot easily distinguish unit differences in SMN1 or SMN2 when the copy number is greater than three [ 78 , 85 , 109 ]; however, recent refinements to MLPA assays can accurately measure four or five copies of SMN1 or SMN2 [ 110 ]. Digital PCR (dPCR) can accurately measure SMN1 and SMN2 over a large range of unit copies (0–6) without the need for an external calibration curve [ 70 , 93 , 111 , 112 , 113 , 114 , 115 ]. Next-generation sequencing approaches have recently been shown to be useful for SMA carrier detection [ 116 , 117 , 118 , 119 , 120 ] as well as for SMN2 copy number measurements [ 120 ].…”

Section: Measurement Of Smn1 and Smn2 Cnvmentioning

confidence: 99%

Genomic Variability in the Survival Motor Neuron Genes (SMN1 and SMN2): Implications for Spinal Muscular Atrophy Phenotype and Therapeutics Development

Butchbach

2021

IJMS

View full text Add to dashboard Cite

Spinal muscular atrophy (SMA) is a leading genetic cause of infant death worldwide that is characterized by loss of spinal motor neurons leading to muscle weakness and atrophy. SMA results from the loss of survival motor neuron 1 (SMN1) gene but retention of its paralog SMN2. The copy numbers of SMN1 and SMN2 are variable within the human population with SMN2 copy number inversely correlating with SMA severity. Current therapeutic options for SMA focus on increasing SMN2 expression and alternative splicing so as to increase the amount of SMN protein. Recent work has demonstrated that not all SMN2, or SMN1, genes are equivalent and there is a high degree of genomic heterogeneity with respect to the SMN genes. Because SMA is now an actionable disease with SMN2 being the primary target, it is imperative to have a comprehensive understanding of this genomic heterogeneity with respect to hybrid SMN1–SMN2 genes generated by gene conversion events as well as partial deletions of the SMN genes. This review will describe this genetic heterogeneity in SMA and its impact on disease phenotype as well as therapeutic efficacy.

show abstract

Analytical validation of the droplet digital PCR assay for diagnosis of spinal muscular atrophy

Cited by 15 publications

References 7 publications

Next generation sequencing is a highly reliable method to analyze exon 7 deletion of survival motor neuron 1 (SMN1) gene

Next generation sequencing is a highly reliable method to analyze exon 7 deletion of survival motor neuron 1 (SMN1) gene

Cas12a and Lateral Flow Strip-Based Test for Rapid and Ultrasensitive Detection of Spinal Muscular Atrophy

Genomic Variability in the Survival Motor Neuron Genes (SMN1 and SMN2): Implications for Spinal Muscular Atrophy Phenotype and Therapeutics Development

Contact Info

Product

Resources

About