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In recent years, the insertion/deletion (InDel) polymorphism has become a preferred genetic marker in forensic genetics due to its low mutation rates and small amplicon sizes. In this study, a 36-InDelplex identification panel, consisting of autosomal 34 InDel loci, 1 Y InDel locus, and amelogenin, was developed, and gene frequencies in the Turkish population were determined. The loci of the InDel panel with global minimum allele frequencies (MAF) ≥ 0.4 were selected from the 1000 Genomes Project Phase 3 data. The amplicon sizes of the loci were designed in the range of 69-252 bp. In the validation study of the developed panel, analysis threshold, dynamic range, sensitivity, stochastic threshold, inhibitor tolerance, and reproducibility parameters were studied by following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The sensitivity studies indicated that complete and reliable InDel profiles could be obtained with 0.25 ng of DNA. A population study was evaluated using 250 samples from Turkey. The mean observed heterozygosity ratio (Ho) of all loci was 0.48. The combined discrimination power (CPD) is 0.999999999990867 and the combined exclusion probability (CPE) was 0.9930. The population comparison was also made using Turkish and the five major populations from the 1000 Genomes Phase 3 populations' data (Africa, Europe, East Asia, South Asia, and America). In conclusion, the results showed that the 36-InDelplex panel is a reliable, sensitive, and accurate system that is suitable for human identification and population genetics purposes., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In order to promote the widespread application of single-nucleotide polymorphism (SNP)-based genotyping, a new method was developed and called target SNP-seq which combined the advantages of multiplex polymerase chain reaction (PCR) amplification and high throughput sequencing on Illumina X Ten platform. Compared with kompetitive allele-specific PCR (KASP), microchips, and genotyping by sequencing (GBS), target SNP-seq uses perfect SNPs based on the analysis of variome (whole-genome sequence data of different accessions) and is flexible, cost-effective, and highly accurate for genotyping middle-scale SNPs. It could genotype hundreds of SNPs in massive DNA samples within 3 days at the cost of $7 for each DNA sample. The high efficiency and low cost of target SNP-seq make it more competitive than current SNP genotyping methods, and it has excellent potential for application in genetic research, as well as in promoting plant-breeding processes in the near future., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5'- or 3'-ends. In this work, we explain in detail examples of rational design of primers in three different applications, including the use of specific determinants at the 3'-end, to (i) improve PCR efficiency with related sequences for members of a protein family by complete degeneration at a core box of conserved genetic information at the 3'-end with the reduction of degeneration at the 5'-end, (ii) optimize specificity of allelic discrimination of closely related DNA sequences of orthologous by 5'-end fully degenerate primers, and (iii) increase the PCR efficiency of primers by targeting DNA sequences belonging to specific phylogenetic groups, within a large and diverse gene family, allowing the use of multiplex/degenerate PCR., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The Investigator ® 24Plex kits are multiplex PCR kits utilized by forensic laboratories to simultaneously amplify 22 of the most commonly utilized STR markers for human identity testing, including the 20 core CODIS loci, along with the sex marker Amelogenin and 2 novel quality sensors. These quality sensors are unique internal PCR controls that provide useful insight to the analyst regarding possible inhibition or degradation within the sample. This chapter describes the use of the QS version of the kit designed for use with extracted DNA from casework samples, as well as the use of the GO! version of the kit designed for direct amplification of reference samples., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Diagnosis of fruit tree viruses has been challenging for a long time as viral titer is often low and unevenly distributed among different tissues and branches of fruit trees. It is necessary to develop effective and reliable detection systems to identify viral pathogens in fruit trees. In this chapter, I describe RT-PCR and its derivatives tube capture-based reverse-transcription PCR (TC-RT-PCR) and multiplex RT-PCR assays for detection and identification of latent viruses in apple and pear trees. Classical RT-PCR is composed of two steps including transcription of viral RNA using extracted total RNA and PCR amplification of viral cDNA. TC-RT-PCR includes a TC step to capture particles and nucleic acid mixtures from crude plant tissue extracts as template directly for the first single-strand DNA (cDNA) synthesis, followed by PCR to amplify the viral cDNA fragment for viral identification. The cDNA derived from total RNAs can also be used for a one-step multiplex PCR to simultaneously detect several viruses in a given sample. As perennial fruit trees are usually coinfected by several viruses in orchards, multiplex RT-PCR can save time and lower labor and material costs for viral detection. These nucleic acid-based methods are sensitive and may be adapted for detection and identification of diverse viruses from different tissue materials of fruit trees., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

COVID-19 disease caused by the novel SARS-CoV-2 virus represents a new challenge for healthcare systems. The molecular confirmation of infection is crucial to guide public health decision-making. This task could be made more difficult during the next influenza season. Thus, a rapid and user-friendly diagnostic test to discriminate SARS-CoV-2 from influenza viruses is urgently needed. Here, we present a multiplex quantitative polymerase chain reaction (qPCR) assay capable of distinguishing SARS-CoV-2 from influenza A and B cases. This assay benefits from the use of an inhibitor tolerant PCR mix which obviates the need for the rate-limiting extraction step, allowing for a more rapid and accurate analysis., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This chapter presents a genotyping assay that uses DNA isolated from axenic cultures of Cryphonectria parasitica, which discriminates the six known diallelic vic loci and the two mating idiomorphs (MAT gene) based on (i) modified primer, labeled with a fluorescent dye, (ii) multiplex polymerase chain reaction (multiplex-PCR), and (iii) capillary electrophoresis system. Alternatively, we show that the same primer set is suitable for conventional PCR of each vic locus and MAT gene using nonmodified primer and agarose gel electrophoresis., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

An accurate T cell quantification is prognostically and therapeutically relevant in various clinical applications, including oncology care and research. In this chapter, we describe how T cell quantifications can be obtained from bulk DNA samples with a multiplex digital PCR experiment. The experimental setup includes the concurrent quantification of three different DNA targets within one reaction: a unique T cell DNA marker, a regional corrector, and a reference DNA marker. The T cell marker is biallelically absent in T cells due to VDJ rearrangements, while the reference is diploid in all cells. The so-called regional corrector allows to correct for possible copy number alterations at the T cell marker locus in cancer cells. By mathematically integrating the measurements of all three markers, T cells can be accurately quantified in both copy number stable and unstable DNA samples., (© 2022. The Author(s).)

While conventional PCR applications typically focus on a single PCR assay per reaction, multiplex PCR applications are a convenient and scalable solution becoming more routine. Multiplex methods can be applied to virtually any DNA template source (e.g., plant or human DNA, FFPE DNA isolated from clinical samples, bisulfite-converted DNA for DNA methylation analysis), and offers a cheap, convenient, and scalable solution for experiments that require characterization and analysis of multiple genomic regions.This method will detail the procedures to successfully design, screen, and prepare multiplex amplicon libraries; as well as supporting instructions on how to prepare these libraries for sequencing on Illumina, Ion Torrent, and Oxford Nanopore platforms. The flexibility of assay design allows means that custom multiplex panels can range in size from two assays up to a few hundred amplicons or more. Notably, the method described here is also amenable to whatever PCR buffer system the user prefers to use, making the system globally adaptable to the needs and preferences of the end user., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Multiplex quantitative polymerase chain reaction (multiplex qPCR) enables the amplification of more than one target in a single reaction using different reporter dyes with distinct fluorescent spectra. The number of reporter fluorophores is typically restricted to three or four, depending upon the capability of the real-time PCR platform and software used. Each target is amplified by a different set of primers and a uniquely labeled probe that distinguishes each PCR amplicon. Thus, the levels of several targets of interest can be quantified in real time. By combining several reactions in a single tube, multiplex qPCR reduces the quantity, and cost of reagents needed to screen a sample for multiple targets. Specificity and efficiency are not affected by the inclusion of the three assays in a multiplex reaction., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Amplification of different nucleic acid targets in the same reaction (multiplex polymerase chain reaction) is challenging but an extremely useful tool especially for viroid diagnosis. In the amplification mixtures, several pairs of primers work together in the same conditions to detect different targets. Here, we describe the development and use of a multiplex reverse transcription polymerase chain reaction protocol highlighting the most crucial factors that can significantly affect the quality of the method. First, particular attention must be paid to primer design. Then, the amplification mixture and temperature conditions must be calibrated precisely to avoid cross reactivity or loss in sensitivity. Finally, the detection system of the amplification results must allow a specific identification of the amplified target(s)., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Molecular testing of pathognomonic gene fusions is mandatory for small round cell tumor diagnosis, including Ewing sarcoma which is indeed defined by a variety of chimeric genes. Reference laboratories are increasingly implementing NGS-based techniques to overcome several limitations of conventional singleplex determinations. We have been early adopters of a targeted-RNA sequencing method based on Anchored multiplex PCR, which allows assessing several fusion transcripts simultaneously with previous knowledge of only one partner gene. Here we describe in detail our protocol and tips for nucleic acid extraction, library preparation, sequencing, and reporting of gene fusions.

The profiling of EGFR mutations, the most common genetic alterations in non-small cell lung cancer (NSCLC) predictive of targeted therapy efficacy, is crucial to anticipate the patient response to EGFR tyrosine kinase inhibitors. Here, we introduce the naica ® system for 6-color Crystal Digital PCR TM and describe in detail a standardized workflow for the multiplexed, single-assay detection of the 19 most prevalent sensitizing and resistance EGFR mutations in both tumor and circulating tumor DNA (ctDNA) samples. Two major advantages of the 6-color multiplexing system over current digital PCR systems are the rapid time to results, and the large quantity of mutational information obtained per patient sample, rendering the 6-color system highly cost effective. The 6-color Crystal Digital PCR TM technology enables highly sensitive and efficient therapeutic monitoring through liquid biopsy, resulting in the early detection of treatment resistance. While the assay presented here specifically addresses EGFR mutation status monitoring in NSCLC patients, 6-color Crystal Digital PCR TM assays are flexible and evolutive in design. As such, 6-color detection assays can be optimized to monitor mutations associated with a range of cancers and other genetic diseases, as well as to detect genetic changes beyond the oncology and human health domains.

In the past two decades, genetic studies of familial forms of Parkinson's disease (PD) have shown evidence that PD has a significant genetic component. Indeed, 12 genes are strongly involved in PD causality, three of them having dominant inheritance and 9 causing early-onset autosomal recessive forms, including 3 with a typical PD and 6 with an atypical parkinsonism. The aim of this study was to determine the genetic basis of familial PD in Moroccan patients. We selected 18 Moroccan index case with familial forms of PD. Patients were first screened for exon-rearrangements by MLPA kit. They were then analyzed by gene panel next-generation sequencing (NGS). Functional variants with minor allele frequencies < 0.5% in public databases were considered potential candidate variants to PD. In the 18 PD patients with a positive family history that were analyzed, MLPA assays identified PRKN deletions in two patients: a homozygous exon 3-5 deletion and a heterozygous exon 4 deletion. Sixteen rare SNV were identified by NGS, four of them were novel. Seven mutations were categorized as pathogenic, five as likely pathogenic, two to be of uncertain significance, and 3 were predicted to be likely benign but may give a weaker pathogenic effect and could contribute to PD since they were found in late-onset PD patients. Rare or novel mutations that could be related to the disease were identified in 72% of these patients (13/18), including nine with bi-allelic pathogenic/likely pathogenic variants in genes causing recessive PD, particularly PRKN and PINK1. Mutations in genes with dominant inheritance were found in 4/18 patients (22%).

Potato bacterial wilt is caused by the devastating bacterial pathogen Ralstonia solanacearum. Quantitative resistance to this disease has been and is currently introgressed from a number of wild relatives into cultivated varieties through laborious breeding programs. Here, we present two methods that we have developed to facilitate the screening for resistance to bacterial wilt in potato. The first one uses R. solanacearum reporter strains constitutively expressing the luxCDABE operon or the green fluorescent protein (gfp) to follow pathogen colonization in potato germplasm. Luminescent strains are used for nondestructive live imaging, while fluorescent ones enable precise pathogen visualization inside the plant tissues through confocal microscopy. The second method is a BIO-multiplex-PCR assay that is useful for sensitive and specific detection of viable R. solanacearum (IIB-1) cells in latently infected potato plants. This BIO-multiplex-PCR assay can specifically detect IIB-1 sequevar strains as well as strains belonging to all four R. solanacearum phylotypes and is sensitive enough to detect without DNA extraction ten bacterial cells per mL in complex samples.The described methods allow the detection of latent infections in roots and stems of asymptomatic plants and were shown to be efficient tools to assist potato breeding programs., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Reverse transcription (RT) based quantitative PCR (qPCR) for quantifying microRNAs (miRNAs) in in the circulation presents specific challenges. Here, we describe an optimized research protocol to assess serum sample quality and quantify levels of a panel of four test miRNAs (miR-371a-3p, miR-372-3p, miR-373-3p, and miR-367-3p) that enables highly sensitive and specific malignant germ cell tumor (GCT) diagnosis and monitoring. This protocol utilizes a multiplex RT step using Taqman miRNA stem-loop primers. A multiplexed preamplification stage is then employed to increase the sensitivity of the final quantification step, which is performed using standard singleplex Taqman qPCR methodology.

CRISPR-Cas-based genome editing has enabled efficient genetic engineering of a range of organisms and sparked revolutions in many fields of biology. After Streptococcus pyogenes Cas9 was first demonstrated for mammalian genome editing, many CRISPR-associated (Cas) protein variants have been isolated from different species and adopted for genome editing. Furthermore, various effector domains have been fused to these Cas proteins to expand their genome-editing abilities. Although the number of genome-editing tools has been rapidly increasing, the throughput of cell-based characterization of new genome-editing tools remains limited. Here we describe a highly multiplexed genome editing and sequencing library preparation protocol that allows high-resolution analysis of mutation outcomes and frequencies induced by hundreds to thousands of different genome-editing reagents in mammalian cells. We have successful experiences of developing several key genome-editing tools using this protocol. The protocol also is designed to be compatible with robotic liquid handling systems for further scalability.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by deletion or subtle variant of survival motor neuron 1 (SMN1) gene. By multiplex ligation-dependent probe amplification, genomic sequencing, and T-A cloning on cDNA level, we identified one novel SMN1 subtle variant c.835G>C (p.Gly279Arg) in a non-homozygous patient with type 1 SMA. Full-length SMN1 (fl-SMN1) transcripts in the peripheral bloods of the patient were significantly decreased compared with those in healthy individuals and the carries (p < 0.05). And two fragments of SMN1 transcripts including fl-SMN1 and △7-SMN1 were observed by RT-PCR, which indicated Exon 7 skipping of SMN1 gene. To further evaluate its splicing effects on Exon 7, we performed ex vivo splicing analysis, which showed that the mutant mini gene with c.835G>C reduced Exon 7 inclusion to 54%. In addition, self-oligomerization between mutant SMN protein with the c.835G>C (p.Gly279Arg) and wild SMN was decreased in self-interaction assays. Our study clearly demonstrates that the c.835G>C (p.Gly279Arg) variant can lead to a decrease in fl-SMN1 transcripts by interrupting correct splicing of SMN1. What is more, the variant also affects SMN self-oligomerization via amino acid substitution from Gly to Arg at amino acid position of 279. This work presents the first evidence that it does exit double-hit events for the novel variant, which is crucial to understanding a severe SMA phenotype (type 1).

Death due to respiratory infection is commonly encountered at autopsy. With only one opportunity to obtain samples for identification of a causative agent, it is important to ensure that sampling regimes are optimized to provide the greatest detection, without the expense and redundancy that can arise from over-sampling. This study was performed retrospectively using data from Coronial autopsies over the period 2012-2019 from which swabs from the nasopharyngeal region, trachea and lung parenchyma, in addition to samples of lung tissue, had been submitted for multiplex PCR detection of respiratory pathogens. From 97 cases with all four samples, there were 24 with at least one positive result for viral infection. Some cases had multiple positive results and a total of 27 respiratory tract viruses were identified, of which rhinovirus, influenza A virus and respiratory syncytial virus were the most common. Seventeen of the 27 viral infections (63%) were identified in all four samples. However, in nearly all cases (96%) the nasopharyngeal swab detected the infective agent when the multiplex PCR panel had detected infection in any of the four sample types. A nasopharyngeal swab is considered to be an optimal sample for detection of respiratory tract viral infection. As the samples analyzed were acquired before the appearance of the COVID-19 virus, the applicability of this finding for COVID-19 screening is not established.

In terms of diagnosing and treating diseases, our adaptive immune system is the "best doctor." It carries out these tasks with unmatched precision, with the help of both T and B cell receptors, our most diverse set of genes, distinguishing one individual from another. It does this by generating autologous extraordinary diversity in the receptors, ranging from 10 15 to 10 25 for each chain of the rearranged receptors. By combining multiplex PCR and next-generation sequencing (NGS), we have developed high throughput methods to study adaptive immunity. The adaptome is the sum-total of expressed T and B cell receptor genes in a sample, composed of seven chains, including the alpha/beta and gamma/delta chains for T cells, and heavy/lambda or kappa chains for B cells. Immune repertoire is the sum-total of the individual clonotypes within one chain, including individual complementarity-determining regions (CDR) 3 sequences. In order to reflect the breadth and depth of the true adaptome, the following criteria assessing any method needs to be ascertained: (1) Methods need to be inclusive and quantitative; (2) Analysis should consider what questions need to be addressed and whether bulk or single cell sequencing provide the best tools for assessing the underlying biology and addressing important questions; (3) Measures of clonal diversity are key to understand the underlying structure and providence of the repertoire; and (4) Convergent evolution may allow a surprising degree of homologous or identical CDR3s associated with individual disease entities, creating hope for novel diagnostics and/or disease burden assessments. Integrating studies of the peripheral blood, lymph nodes, and tumor allows dynamic interrogation of the alterations occurring with age, treatment, and response to emergent and established therapies.

The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and the products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.

Methicillin-susceptible S. aureus changes to methicillin-resistant S. aureus (MRSA) upon the acquisition of staphylococcal cassette chromosome mec (SCCmec), a genomic island that encodes methicillin resistance. SCCmec elements in S. aureus are classified into different types based on the combination of mec gene complexes and ccr gene complexes, which share variations, five classes in mec and eight in ccr. To date, at least 13 types of SCCmec elements have been identified and each SCCmec type has individual characteristics. It is known that hospital-associated MRSA strains carry SCCmec elements of types, I, II, and III, and the majority of community-acquired MRSA strains carry characteristic SCCmec elements, type IV SCCmec or type V SCCmec. We herein describe multiplex PCR methods to type SCCmec elements by identifying the mec gene complex class and ccr gene complex type.

Multiplex Ligation-dependent Probe Amplification (MLPA) is a method to determine the copy number of up to 60 genomic DNA sequences in a single multiplex PCR based reaction.MLPA probes consist of two oligonucleotides that can hybridize next to each other on a certain DNA sequence of interest, where they are ligated. All ligated probes are subsequently amplified by PCR using a single set of primers. Each amplified MLPA probe has a unique length and can be visualized and quantified by capillary electrophoresis. As the primers are almost 100% consumed in the PCR reaction, the quantity of each PCR amplicon is proportional to the number of copies of each probe target sequence in the DNA sample. A trisomy 21 can therefore be detected by an approximately 50% increased signal of each chromosome 21 specific probe relative to reference samples.MLPA with the P095 Aneuploidy probemix for chromosomes 13, 18, 21, X and Y has been used as a rapid detection method on large numbers of samples from uncultured amniotic fluid or from chorionic villi. As compared to FISH and karyotyping, MLPA is more rapid, has a higher throughput, and is less expensive. MLPA however cannot detect low grade mosaicism, female triploidies, and copy number neutral chromosome abnormalities such as inversions and translocations.

Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.

A fully automated rapid DNA analysis system requires integrating several operational elements performing multiple steps onto one single microfluidic platform. The functions to include on the microfluidic platform consist of substrate lysis, lysate DNA extraction, single or multiplexed PCR amplification, amplicon separation, and product readout. Here we describe a fully automated integrated system for forensic short tandem repeat (STR) analysis of reference samples, achieving buccal swab-in and DNA profile-out.

Citrus can host a number of important vector- and graft-transmissible pathogens which cause severe diseases. Citrus disease management and clean stock programs require pathogen detection systems which must be economical and sensitive to maintain a healthy citrus industry. Rapid diagnostic tests for simultaneous detection of major graft-transmissible disease agents enable reduction of cost and time. The genetic and biological features of viruses and viroids can vary according to the strains/variants, with severe and mild strains described within the same species. The use of diagnostic tests that can allow to selectively discriminate severe strain(s) is a powerful tool to intercept the most harmful strains and to reduce the need for biological indexing. Moreover a combination of these detection methods will facilitate the studies on the interactions between CTV and viroids, a research topic only partially explored so far.