The homogenous genotyping test combines allele-specific tailed primers with SYBR-Green to facilitate fast and accurate detection of PI*S and PI*Z alleles of SERPINA1.
Credit: PixaBay
Researchers have developed a novel homogeneous genotyping test allowing for the detection of PI*S and PI*Z alleles of the SERPINA1 gene using any typical re
al-time PCR platform, facilitating reliable and quick identification of genetic variants causing alpha-1 antitrypsin deficiency (AATD).1
Developed by combining allele-specific tailed primers with SYBR-Green, the test offers a cost-effective method for genotyping PI*Z and PI*S variants of AAT compared with probe-based techniques based on the principles of allele-specific PCR and amplicon melting analysis with a fluorescent dye.1
“AATD is an underdiagnosed disease, as only about 10–15% of cases have been estimated to be properly diagnosed and increasing rates of AATD screening and diagnosis are currently in demand,” José Antonio Pérez-Pérez, PhD, an associate professor at the University of La Laguna in Spain, and colleagues wrote.1 “Therefore, accurate and cost-effective detection methods for PI*S and PI*Z alleles are necessary, and the present work aims to satisfy this demand.”
According to the Alpha-1 Foundation, AATD can be challenging to diagnose because it often presents with the same symptoms as more common conditions like chronic obstructive pulmonary disease (COPD) and asthma. Given the progressive nature of AATD, prompt diagnosis and treatment are essential for improving outcomes, highlighting the need to address the current underdiagnosis of the disease.2 The PI*Z (rs28929474) and PI*S (rs17580) alleles represent the majority of diagnosed AATD cases, so improving their detection is essential for increasing rates of AATD screening and diagnosis.1
To develop a homogeneous genotyping test for the detection of PI*S and PI*Z alleles based on the principles of allele-specific PCR and amplicon melting analysis with a fluorescent dye, investigators assayed 60 individuals previously genotyped by real-time PCR with fluorescent probes with tailed allele-specific primers and SYBR Green dye in a real-time PCR machine. Among the cohort, individuals had each one of the 6 commonly observed genotypes resulting from the combination of genetic variants at rs17580 and rs28929474 loci: PI*MM, PI*ZZ, PI*SS, PI*MZ, PI*MS, and PI*SZ.1
Investigators extracted genomic DNA in alkaline conditions from dried blood samples, and the rs17580 and rs28929474 variants were detected in separate PCRs. Amplification reactions contained 2–10 ng of DNA (4 µl of the alkaline extract), 1 × FastStart Essential DNA Green Master Mix (Roche), 2 forward allele-specific primers, and 1 reverse conserved primer.1
As described by investigators, PCRs were incubated in a LightCycler 480 instrument. The thermal profile included a preincubation step (98 °C [208.4 °F] for 10 minutes), followed by 35 amplification cycles with denaturing (95 °C [203 °F] for 5 seconds), primer annealing (65 °C [149 °F] for 10 seconds), and extension (72 °C [161.6 °F] for 10 seconds). A single capture of fluorescence was included at the end of each extension step for monitoring amplification in real-time, and a melting-curve program from 60 °C - 95 °C, with 10 fluorescence acquisitions per °C, was used to calculate the melting temperature (Tm) of each amplicon with the software implemented in the thermal cycler.1
Investigators noted the developed genotyping assays allowed for the accurate detection of genetic variants that define the PI*S and PI*Z alleles and the corresponding wild-type variants. Amplicons obtained with the non-S variant showed a mean Tm (79.77 ± 0.16 °C; n = 50; 91 bp) 2.0 °C greater than amplicons obtained with the S variant (77.76 ± 0.21 °C; n = 30; 99 bp). Investigators pointed out the resolution of melting peaks was improved (2.9 °C) for amplicons derived from non-Z (86.22 ± 0.07 °C; n = 50; 132 bp) and Z (83.29 ± 0.14 °C; n = 30; 128 bp) variants.1
For DNA samples from heterozygous individuals, the proposed genotyping assays produced melting peaks with balanced heights within an annealing temperature range of 58–66 °C for non-S/S and 63–66 °C for non-Z/Z. Thus, both assays can work in the same PCR run using an annealing temperature of 63–66 °C, although investigators recommend using 65 °C for optimal results.1
Based on these results, investigators acknowledged the accuracy and cost-effectiveness of their optimized genotyping method. Because both amplification and genotype determination occur in a single tube, the test overcomes the drawbacks of non-homogeneous approaches such as allele-specific PCR, RFLP, or Sanger sequencing. They also pointed out the present genotyping assay takes advantage of the cheap, stable, and universal dye SYBR Green, avoiding expensive fluorescent DNA probes with limited durability used in other assays.1
“Our novel PI*S and PI*Z genotyping method can be easily implemented on any typical real-time PCR platform, provides reliable and quick results, and notably reduces genotyping costs when compared with probe-based techniques,” investigators concluded.1 “Therefore, this test could be used in the future for large screenings of AATD.”
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