PCR-based Procedure Research Articles

High-Performance PCR for Alleles Discrimination of Chromo-Helicase-DNA Binding Protein (CHD1) Gene in Bird Sexing.

Genetic analyses aiming at assessing the presence of specific sequences or alleles are often carried out by PCR. Sexing of most birds is nowadays based on PCR with "universal" primers and relies on the assessment of the presence of the sex-linked CHD1-Z and -W alleles. The entire workflow is relatively time-consuming, especially for batch analyses, whereas methods that allow carrying out the entire procedure in a short time are highly desirable. The only method for outdoor analyses reported so far relies on LAMP; however; it fails to work properly in Procellariiformes. Besides improving the LAMP test; we have developed a PCR-based DNA amplification procedure (named high-performance PCR); whose unique features allow it to outperform standard PCR; making possible the direct, in-tube visual reading of results. We tested it with specifically designed Procellariiformes-targeted primer sets for rapid sexing of the birds using fluorimetric detection. The protocol, combined with rapid DNA extraction, allows for fast reading of results without electrophoresis within less than 1 h from sampling. The technique could be extended to other species, as well as to many other applications.

Genetic Characterization of Brucella spp.: Whole Genome Sequencing-Based Approach for the Determination of Multiple Locus Variable Number Tandem Repeat Profiles.

Brucellosis is an important zoonosis that is emerging in some regions of the world, gaining increased relevance with the inclusion of the causing agent Brucella spp. in the class B bioterrorism group. Until now, multi-locus VNTR Analysis (MLVA) based on 16 loci has been considered as the gold standard for Brucella typing. However, this methodology is laborious, and, with the rampant release of Brucella genomes, the transition from the traditional MLVA to whole genome sequencing (WGS)-based typing is on course. Nevertheless, in order to avoid a disruptive transition with the loss of massive genetic data obtained throughout the last decade and considering that the transition timings will vary considerably among different countries, it is important to determine WGS-based MLVA alleles of the nowadays sequenced genomes. On this regard, we aimed to evaluate the performance of a Python script that had been previously developed for the rapid in silico extraction of the MLVA alleles, by comparing it to the PCR-based MLVA procedure over 83 strains from different Brucella species. The WGS-based MLVA approach detected 95.3% of all possible 1,328 hits (83 strains×16 loci) and showed an agreement rate with the PCR-based MLVA procedure of 96.4% for MLVA-16. According to our dataset, we suggest the use of a minimal depth of coverage of ~50x and a maximum number of ~200 contigs as guiding “boundaries” for the future application of the script. In conclusion, the evaluated script seems to be a very useful and robust tool for the in silico determination of MLVA profiles of Brucella strains, allowing retrospective and prospective molecular epidemiological studies, which are important for maintaining an active epidemiological surveillance of brucellosis.

Abstract 2080: Tumor microsatellite instability detection method using paired tumor-normal sequence data

Abstract Background: Microsatellite instability (MSI) is an important indicator of larger genome instability and has been linked to many genetic diseases. MMR gene mutations usually lead to the loss of MMR proteins, resulting in high instability of tumor DNA microsatellites (MSI-H), and leading to a high tumor mutation burden (TMB-H). In current clinical practice, MSI detection is performed by experimental methods such as MSI-PCR and MMR-IHC. However, the PCR-based detection procedure is laborious and time-consuming, the accuracy of the results depends on the naked eye judgment of the analysts. With the development of next-generation sequencing (NGS) technology, a large number of MSI detection software based on high-throughput sequencing data have been developed. In this study, we compared the two methods provide a computational method that quantifies MSI status based on genome sequencing data. Methods: 2523 tested samples, including 80 MSI-H samples and 2443 MSS samples, detected by MSI-PCR and NGS-Seq simultaneously, were enrolled in this study. In the MSI-PCR method, MSI status was classified as Microsatellite stable (MSS) and MSI-high (MSI-H, over 2 markers unstable). For the NGS-Seq method, the status of 54 microsatellite site was evaluated by the MSI sensor (https://github.com/ding-lab/msisensor) and the proportion of unstable microsatellite site (RatioUMS) was calculated, and MSI status was classified as MSS (RatioUMS<35), MSS-To Be Determined (MSI-TBD, 35≤RatioUMS≤55), and MSI-H (RatioUMS>55). For MSI-TBD, either TMB is greater than 30 (TMB>30) or TMB is greater than 10 and had a non-silent mutation in an MMR gene (TMB>10&MMR+), MSI-H is considered, otherwise, MSS is considered. Results: In our test, NGS results were inconsistent with MSI-PCR method in 8 samples, among which 5 samples were categorized incorrectly by MSI-PCR after reverification. According to the above results, the consistency of two methods was 99.9%. We also tested the classifier using 114 microsatellite sites in 425 samples and 309 microsatellite sites in 2098 samples. Compared with MSI-PCR, the consistency was 100% and 99.6%, respectively. For the 25 MSI-TBD samples, adding two parameters, MMR gene mutation and TMB, greatly improved the accuracy of the judgment. Conclusion: The results show NGS method are highly consistent with the gold standard MSI-PCR, and the judgmental errors caused in MSI-PCR by human factors can be avoided. The different number of microsatellite sites used in the NGS method has little effect on the final results. NGS approach offers the advantages to simultaneous detection of predictive markers, including MSI, TMB and clinically relevant genomic alterations, will save valuable material, time and costs, which can provide more comprehensive and reasonable recommendations for medication and treatment. Citation Format: Hongling Yuan, Danhua Wang, Honglin Zhu. Tumor microsatellite instability detection method using paired tumor-normal sequence data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2080.

Pool Testing as a Strategy for Prevention of SARS-CoV-2 Outbreaks in Schools: Protocol for a Feasibility Study.

BackgroundSchool closures are a widely implemented strategy for limiting infection spread in the current COVID-19 pandemic. The negative impact of school closures on children and young people is increasingly apparent, however.ObjectiveWe aim to evaluate the feasibility of an infection monitoring program in schools to enable targeted quarantining to replace school closures. The program is currently being implemented in two model schools in Magdeburg, Germany, within the framework of the Study of Coronavirus Outbreak Prevention in Magdeburg Schools (Studie zur Ausbruchsvermeidung von Corona an Magdeburger Schulen [STACAMA]).MethodsFive pupils per class are pseudorandomly selected twice a week and asked to provide a gargle sample over a 16-week evaluation period. RNA is extracted from each sample individually in a laboratory and pooled according to school class for real-time reverse transcription polymerase chain reaction (rRT-PCR) analysis. Immediate individual sample testing will be carried out in the case of a positive pool test. Individual RNA extraction prior to pooling and application of rRT-PCR result in high test sensitivity. Testing will be performed in strict adherence to data protection standards. All participating pupils will receive a 16-digit study code, which they will be able to use to access their testResultsWhen the study commenced on December 2, 2020, 520 (52%) pupils and their families or guardians had consented to study participation. The study was suspended after four test rounds due to renewed school closures resulting from rising regional infection incidence. Testing resumed when schools reopened on March 8, 2021, at which time consent to participation was provided for 54% of pupils. We will quantitatively and qualitatively evaluate the logistics and acceptability of the program.ConclusionsThe findings from this study should inform the design of infection surveillance programs in schools based on gargle samples and a PCR-based pool testing procedure, enabling the identification of aspects that may require adaptation before large-scale implementation. Our focus on each step of the logistics and on the experiences of families should enable a robust assessment of the feasibility of such an approach.International Registered Report Identifier (IRRID)DERR1-10.2196/28673

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Detection and Serogrouping of Dichelobacter nodosus Infection by Use of Direct PCR from Lesion Swabs To Support Outbreak-Specific Vaccination for Virulent Footrot in Sheep.

Virulent footrot is an economically significant disease in most sheep-rearing countries. The disease can be controlled with vaccine targeting the fimbriae of virulent strains of the essential causative agent, Dichelobacter nodosus However, the bacterium is immunologically heterogeneous, and 10 distinct fimbrial serogroups have been identified. Ideally, in each outbreak the infecting strains would be cultured and serogrouped so that the appropriate serogroup-specific mono- or bivalent vaccine could be administered, because multivalent vaccines lack efficacy due to antigenic competition. If clinical disease expression is suspected to be incomplete, culture-based virulence tests are required to confirm the diagnosis, because control of benign footrot is economically unjustifiable. Both diagnosis and vaccination are conducted at the flock level. The aims of this study were to develop a PCR-based procedure for detecting and serogrouping D. nodosus directly from foot swabs and to determine whether this could be done accurately from the same cultured swab. A total of 269 swabs from the active margins of foot lesions of 261 sheep in 12 Merino sheep flocks in southeastern Australia were evaluated. DNA extracts taken from putative pure cultures of D. nodosus and directly from the swabs were evaluated in PCR assays for the 16S rRNA and fimA genes of D. nodosus Pure cultures were tested also by the slide agglutination test. Direct PCR using extracts from swabs was more sensitive than culture for detecting and serogrouping D. nodosus strains. Using the most sensitive sample collection method of the use of swabs in lysis buffer, D. nodosus was more likely to be detected by PCR in active than in inactive lesions, and in lesions with low levels of fecal contamination, but lesion score was not a significant factor. PCR conducted on extracts from swabs in modified Stuart's transport medium that had already been used to inoculate culture plates had lower sensitivity. Therefore, if culture is required to enable virulence tests to be conducted, it is recommended that duplicate swabs be collected from each foot lesion, one in transport medium for culture and the other in lysis buffer for PCR.

Site-directed mutagenesis in Brucella abortus S19 by overlap extension PCR-based procedure

Introduction of a site-directed mutation can be effective method to evaluate properties of various genes. Brucellosis is one of the most common zoonotic infectious diseases, which causes great economic losses. Thus, determination of pathogenicity factors in the genus Brucella can lead to the control of this health problem. Due to the importance of site-directed mutations in identification of genomic structure, overlap extension polymerase chain reaction (PCR) has been introduced as an improved technique for the removal and replacement of gene targets. In this study, three DNA fragments were amplified and combined using a two-step PCR with specific primers. The resulting fusion PCR product, obtained without any change in the nucleotide sequence, was cloned in a specific position in the pBluescript II SK (-) plasmid using restriction enzymes. Finally, the construct was transferred into cells of Brucella abortus S19 by electroporation and replaced the target gene (wbkA) in the genome of the bacterium. PCR analysis was performed on kanamycin-resistant colonies to provide genetic evidence that the B. abortus wbkA gene was interrupted by the kanamycin cassette. The results of this study show that the optimized modified technique, splicing by overlap extension PCR, is effective in creating mutations in the bacterial genome and can easily be used in Brucella spp.

Development of Simple Multiplex Real-Time PCR Assays for Foodborne Pathogens Detection and Identification On Lightcycler

Abstract Most acute intestinal diseases are caused by food-borne pathogens. A fast and simple real-time PCR-based procedure for simultaneous detection of food contamination by any of the five food-borne pathogens: Campylobacter jejuni, Mycobacterium bovis, Enterobacter sakazaki, Shigella boydii, Clostridium perfrigens using multiplex EvaGreen real-time PCR for LightCycler was developed and evaluated. Real-time qPCR showed excellent sensitivity. Tm calling and Melting Curve Genotyping (MCG) were used for analysis of PCR product melting curves. The Melting Curve Genotyping option showed good performance for discrimination of positive samples containing DNA of single pathogen or pathogen mixtures from negative samples.

A Rapid and Sensitive Method for Simultaneous Screening of Nine Foodborne Pathogens Using High-Performance Liquid Chromatography Assay

We developed a multiplex PCR-based procedure followed by high-performance liquid chromatography (mPCR-HPLC) assay for high-throughput screening foodborne pathogens, including Salmonella spp., Listeria monocytogenes, Enterobacter sakazakii, Staphylococcus aureus, Shigella spp., Escherichia coli O157:H7, Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus. Vibrio species-specific primers were designed targeting dnaJ gene, and pathogen-specific primers for L. monocytogenes, Salmonella spp., E. sakazakii, E. coli O157:H7, Shigella spp., and S. aureus were designed targeting iap, fimY, 16S ribosomal RNA (rRNA), rfbE, ipaH, and 442 genes, respectively. PCR products were analyzed using WAVE-4500 DNA system equipped with PS-DVB-C18 particles DNASep column and each specific amplicon generated a characteristic chromatographic profile. Detection limit of mPCR-HPLC assay was ca. 101 CFU/mL in pure cultures and less than 102 CFU/g in contaminated matrixes. A total of 395 bacterial strains were used for specificity testing of the mPCR-HPLC assay, and specific HPLC profiles were only produced in strains belonging to the target, showing a high specificity. Applying the assay to 2677 samples collected from clinical, food, and environmental sources revealed that 917 samples were positive, in accordance with bacterial isolation. The high sensitivity and specificity of mPCR-HPLC assay indicate its great potential to be a powerful tool for high-throughput screening foodborne pathogens.

Inability of 'Whole Genome Amplification' to Improve Success Rates for the Biomolecular Detection of Tuberculosis in Archaeological Samples.

We assessed the ability of whole genome amplification (WGA) to improve the efficiency of downstream polymerase chain reactions (PCRs) directed at ancient DNA (aDNA) of members of the Mycobacterium tuberculosis complex (MTBC). Using extracts from a variety of bones and a tooth from human skeletons with or without lesions indicative of tuberculosis, from multiple time periods, we obtained inconsistent results. We conclude that WGA does not provide any advantage in studies of MTBC aDNA. The sporadic nature of our results are probably due to the fact that WGA is itself a PCR-based procedure which, although designed to deal with fragmented DNA, might be inefficient with the low concentration of templates in an aDNA extract. As such, WGA is subject to similar, if not the same, restrictions as PCR when applied to aDNA.

Detection of Mycoplasma Contamination Directly from Culture Supernatant Using Polymerase Chain Reaction

Ensuring mycoplasma-free cell culture is of prime importance as they severely affect cellular characteristics leading to experimental artefacts and spurious results. Various methods persist for mycoplasma detection; out of the whole array of methods polymerase chain reaction (PCR) is the most favoured one because it is highly sensitive, specific and quick. The PCR-based detection procedure involves three steps: cell culture supernatant collection, DNA isolation, and PCR. We have modified this procedure so that cell culture supernatant can directly be used for PCR without the need for DNA extraction. This modification makes the procedure quicker and more sensitive because loss of mycoplasma DNA is prevented and this loss becomes more significant when the level of mycoplasma contamination is very low.

MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform.

Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities.

Quantitative PCR for the diagnosis of cutaneous leishmaniasis from formalin-fixed and paraffin-embedded skin sections

The present report describes a real-time PCR-based procedure to reliably determine the quantity of Leishmania amastigotes in relation to the amount of host tissue in histological skin sections from canine and equine cases of cutaneous leishmaniasis. The novel diagnostic Leishmania-PCR has a detection limit of <0.02 amastigotes per μg tissue, which corresponds well to the detection limit of immunohistochemistry and is far beyond that of conventional histology. Our results emphasise the importance of PCR to complement routine histology of cutaneous leishmaniasis cases, particularly in laboratories in which no immunohistochemical assay is available.

Detecting the H3F3A mutant allele found in high-grade pediatric glioma by real-time PCR.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brain tumor with a median survival of 1 year after diagnosis. It has been reported recently that about 80% of DIPG cases and 70% of midline glioblastomas contain a mutation at one allele of the H3F3A gene (encoding histone H3 variant H3.3), replacing the lysine 27 with methionine (K27M). In order to facilitate diagnosis of DIPG patients, a quick and reliable method to identify the H3F3A K27M mutation is needed. Here, we describe a real-time PCR-based procedure involving a mutant-specific primer, a blocker oligonucleotide, and a reverse primer that can differentiate samples with H3F3A K27M mutation from those that do not. We first tested four different mutant-specific primers for their ability to selectively amplify H3F3A K27M-mutant allele and found that one primer amplified the mutant allele more efficiently than the rest. We then determined the optimal concentration of blocker oligo that significantly improved amplification of the H3F3A K27M-mutant allele. Using this optimized real-time PCR assay, we analyzed eleven samples, two of which containing H3F3A K27M mutation, and found that these two samples were differentially amplified from the nine others. In addition, we were able to discern the H3F3A K27M mutation in a newly obtained pediatric brainstem glioblastoma sample whose H3.3 status was not known previously, and in three other DIPG samples as well as paraffin embedded samples. These results demonstrate that we have developed a new reliable procedure for detecting the H3F3A K27M mutation in pediatric glioblastoma patient samples.

AB141. Multiplex PCR-based procedure establishment for simultaneous detection of two mutations occurring most frequently in FMS-like tyrosine kinase-3

Acute myeloid leukemia (AML) is a hematological disorder strongly correlated to several gene mutations which have been used in diagnosis and prognosis of disease. FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase, involving in cell differentiation and proliferation. In AML, abnormal haematopoietic cells carry most commonly two FLT3 mutations, namely internal tandem duplication (ITD) and tyrosine kinase domain (TKD) found in approximately 25% and 5% among patients, respectively. Previously, two separate PCR based procedures was widely used to detect two mutations. In this study, we established a detection procedure for both mutations consisted of two major steps: multiplex PCR for simultaneous amplification of a 329 base-pair amplicon for ITD containing region and a 146 bp amplicon for TKD prone region, and digestion of the PCR products with EcoRV followed by analysis on 8% polyarylamide gel. The procedure was performed with previously confirmed ITDwt/TKDwt, ITDmt/TKDwt, and ITDwt/TKDmt samples and showed consistency with earlier result obtained by using separate procedures for each mutations. Moreover, the procedure is rapid, consuming 6 hours compared to 10 hours and more effective, using less reagents for PCR and polyacrylamide electrophoresis in detection of two mutants. In conclusion, we successfully established more productive multiplex PCR-based method for detection of two common AML-related mutations in FLT3 gene.

In-house Validation of PCR Based Procedure for Specific Detection of Clostridium Botulinum Types C and D

Abstract A PCR-based procedure for detection of C. botulinum C and D in corn silage samples was validated. During the validation, method specificity, sensitivity, and accuracy were determined according to PN - EN ISO 16140:2004. Additionally, the specificity of the validated methods was proved by sequence analysis of PCR products obtained from examination of samples connected with botulism cases in cattle and mallard ducks. Limit of detection was estimated according to the Spearman - Kärber formula and expressed as LOD50. The obtained results showed that a 100% specificity was achieved. The sequencing of PCR products revealed 99% identity with sequences of bont/C and bont/D genes deposited in the GenBank. The sensitivity value ranged from 63.3% for C. botulinum type C to 75% for type D. The accuracy value varied from 72% for type C to 81.3% for type D. LOD50 was estimated at the levels of 0.272 (0. D 188-0395) spore/g for type C and 0.17 (0.1-0.289) spore/g for type D. The described PCR-based procedure enabled detection of C. botulinum C and D at the stage of liquid culture. This makes examination of feed samples possible without isolation process. The presented procedure could support the diagnosis of botulism by faster and specific laboratory examination process.

RNA splicing regulates the temporal order of TNF-induced gene expression

When cells are induced to express inflammatory genes by treatment with TNF, the mRNAs for the induced genes appear in three distinct waves, defining gene groups I, II, and III, or early, intermediate, and late genes. To examine the basis for these different kinetic classes, we have developed a PCR-based procedure to distinguish pre-mRNAs from mRNAs. It shows that the three groups initiate transcription virtually simultaneously but that delays in splicing characterize groups II and III. We also examined the elongation times, concluding that pre-mRNA synthesis is coordinate but splicing differences directly regulate the timing of mRNA production.

Development of a new multiplex quantitative real‐time PCR assay for the detection of the mtDNA4977 deletion in coronary artery disease patients: A link with telomere shortening

Mitochondrial DNA (mtDNA) and telomere shortening have been proposed as important contributors to vascular disease and atherogenesis. The role of mitochondrial and telomere alterations has been examined frequently, but usually separately. Recently, an integrated model in which DNA damage and metabolic pathways intersect in age-associated cardiovascular disease has been proposed. In this study we developed a fast and reliable real-time PCR-based procedure to investigate relative quantification of the 4,977 bp mitochondrial DNA deletion (also indicated as "mtDNA(4977) deletion"), employing TaqMan probes with a multiplex approach. As a validation of the assay, a nested PCR coamplification was performed. Telomere shortening was evaluated by a real-time monochrome multiplex PCR technique employing a SybrGreen-based analysis. The study of mtDNA(4977) deletion and telomere shortening was carried out in atrial biopsies from 11 patients undergoing coronary artery (n = 5) and valve surgery (n = 6). The relative quantifications showed that the amount of mtDNA(4977) deletion was greater in tissue of patients with coronary artery disease (CAD) (P = 0.01) and that telomere length (expressed as telomere length relative to a single copy reference gene) was significantly shorter in tissue of CAD patients, compared to patients without CAD (P = 0.03). Moreover, most conventional risk factors were significantly more frequent in CAD patients, smoking and dyslipidemia having the strongest association with the degree of mtDNA(4977) deletion and a significant correlation with telomere attrition (P = 0.02 and P = 0.006, respectively). In conclusion, the present study suggests that mtDNA(4977) deletion and telomere shortening may represent additional and synergic major risk factors for the pathogenesis of CAD and its complications.

Development of a polymerase chain reaction for the detection of abalone herpesvirus infection based on the DNA polymerase gene

A 5781-base pair (bp) fragment of genomic DNA from the Taiwanese abalone herpesvirus was obtained and showed 99% (5767/5779) homology in the nucleotide sequence and 99% (1923/1926) in the amino acid sequence with the DNA polymerase gene of the abalone herpesvirus strain Victoria/AUS/2007. Homology of the amino acid sequence with the DNA polymerase of ostreid herpesvirus 1 was 30% (563/1856). In this study, a PCR-based procedure for detecting herpesvirus infection of abalone, Haliotis diversicolor supertexta, in Taiwan was developed. The method employed primer sets targeting the viral DNA polymerase gene, and was able to amplify DNA fragments of the expected size from infected samples. Primer sets of 40f and 146r were designed for amplification of an expected PCR product of 606 bp. Combining the new PCR protocol with histopathology, this assay can serve as a reliable diagnostic for herpesvirus infections in abalone.

Ksp1 Kinase Regulates Autophagy via the Target of Rapamycin Complex 1 (TORC1) Pathway

Macroautophagy (hereafter autophagy) is a bulk degradation system conserved in all eukaryotes, which engulfs cytoplasmic components within double-membrane vesicles to allow their delivery to, and subsequent degradation within, the vacuole/lysosome. Autophagy activity is tightly regulated in response to the nutritional state of the cell and also to maintain organelle homeostasis. In nutrient-rich conditions, Tor kinase complex 1 (TORC1) is activated to inhibit autophagy, whereas inactivation of this complex in response to stress leads to autophagy induction; however, it is unclear how the activity of TORC1 is controlled to allow precise adjustments in autophagy activity. In this study, we performed genetic analyses in Saccharomyces cerevisiae to identify factors that regulate TORC1 activity. We determined that the Ksp1 kinase functions in part as a negative regulator of autophagy; deletion of KSP1 facilitated dephosphorylation of Atg13, a TORC1 substrate, which correlates with enhanced autophagy. These results suggest that Ksp1 down-regulates autophagy activity via the TORC1 pathway. The suppressive function of Ksp1 is partially activated by the Ras/cAMP-dependent protein kinase A (PKA), which is another negative regulator of autophagy. Our study therefore identifies Ksp1 as a new component that functions as part of the PKA and TORC1 signaling network to control the magnitude of autophagy.