A review of digital PET-CT technology: Comparing performance parameters in SiPM integrated digital PET-CT systems.

OBJECTIVE: The objective of this study was to perform a narrative review of digital Positron emission tomography-computed tomography (PET-CT) scanners, focussing on the current development in the technology of optimized crystal size and design, the time of flight (ToF) resolution, sensitivity, and axial field of view (AFOV). KEY FINDINGS: It was observed that significant developments were carried out on the optimization of scintillation crystal size which results in the improvement of spatial resolution. such developments include the upgrade in the AFOV after the integration of SiPM technology, which results in dynamic parametric imaging acquisition in PET and sensitivity boost. The improvement in ToF resolution and the better ToF resolution values, which result in a boost in adequate sensitivity and signal-to-noise ratio (SNR). Other upgrades include the use of the smallest crystal size of 2.76 × 2.76 mm, and the use of the lowest ToF resolution of 214 ps. The use of the largest AFOV of 194 cm with the highest observed NEMA sensitivity of 225 cps/kBq for the total body PET-CT system. CONCLUSION: Digital PET-CT systems offer various advantages such as a reduction in radiation dose from injected radiopharmaceuticals doses and the overall PET acquisition time with an improved diagnostic certainty. This is because of the better performance of the SiPM detector. Digital PET-CT also has added benefits of the dynamic acquisition and Patlak modeling capabilities into routine clinical practice with the advancement in higher AFOV PET systems. IMPLICATION: This will help the users choose the best system during the evaluation of the PET-CT for purchase in clinical and research applications. This review will further help in teaching the latest technology and developments in PET-CT systems.

First observation of Afromorgus chinensis (boheman, 1858) (Coleoptera: Trogidae) on a rabbit (Oryctolagus cuniculus L., 1758) carcass and its implications in forensic entomology.

Beetles (Coleoptera) are known to constitute forensic evidence in medico-legal investigations as their presence can be used to date human remains in almost all decomposition stages. Many forensic studies focus on the successional colonization pattern of flies (Diptera); however, beetles have not so far been studied extensively for this aspect. A beetle of the genus Afromorgus Scholtz, 1986, A. chinensis (Boheman, 1858) (Scarabaeoidea: Trogidae), was found beneath a late decaying rabbit carcass at Paya Indah Wetland, Dengkil, Malaysia, for the first time. Both genus and species are already known to occur in Malaysia from literature.

Post-mortem pulmonary tuberculosis: comparison of available diagnostic methods.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a serious public health threat with the World Health Organisation (WHO) reporting 5.8 million cases and 1.3 million deaths in the year 2020 due to TB. TB can be diagnosed by imaging, histopathological and bacteriological methods with culture remaining the gold standard. This study was performed to look at the sensitivity and specificity of post-mortem computed tomography (PMCT) imaging when compared to culture in diagnosing pulmonary tuberculosis. This was a retrospective comparative study looking at post mortem cases where lung tissue samples sent for TB culture at Hospital Kuala Lumpur were compared against PMCT imaging. Exclusion criteria included contaminated samples, decomposed cases, immunocompromised subjects and those below 18 years of age. Subjects included 80 medico-legal autopsy cases at the National Institute of Forensic Medicine, Hospital Kuala Lumpur, Malaysia who had whole body PMCT done in accordance with the Institute's protocol and tissue samples sent for bacteriology culture for tuberculosis. PMCT findings were positively associated with acid-fast organisms in 23.5 out of 33 cases (71.2%). Our study also showed that PMCT had a sensitivity of 71.3% and specificity of 54.3% (95% CI: 39.5-68.4) in diagnosing TB based on the protocol set in this study. This study showed that there was relatively good agreement between radiological PMCT findings and bacterial culture, suggesting that radiological examination is a relatively reliable tool for preliminary screening and possible diagnosis of TB prior to a postmortem examination which would be beneficial in reducing the risk of transmission of TB to health workers during autopsy.

Technical and clinical assessment of latest technology SiPM integrated digital PETCT scanner.

OBJECTIVE: The aim of this study was to conduct a technical and clinical evaluation of a Silicon Photomultiplier (SiPM) integrated digital Positron Emission Tomography - Computed Tomography (PETCT) Scanner using National Electrical Manufacturers Association (NEMA) NU 2- 2018 standards. METHODS: System sensitivity was measured by using a NEMA sensitivity phantom. Scatter fraction, count-rate performance, accuracy of count loss, and timing resolution were all computed. Clinical images were acquired and image quality was assessed and compared with published studies. RESULTS: At 1 cm, tangential, radial, and axial spatial resolutions were 3.02 mm, 3.02 mm, and 2.73 mm at full width half maximum (FWHM), respectively. Sensitivity at centre and 10 cm was 10.359 cps/kBq and 9.741 cps/kBq, respectively. The timing resolution was measured at 372 ps. CONCLUSION: The digital PETCT exhibits a high-spatial resolution and a superior timing resolution, which advances the diagnostic ability to detect small lesions and boosts the diagnostic confidence. IMPLICATIONS FOR PRACTICE: Increases clinical relevance by improving the ability to detect and differentiate tiny or low-contrast lesions without compromising radiopharmaceutical dose or overall scan time.

Post-mortem computed tomography differentiation of putrefactive gas and air embolism: A case of traumatic carotid-jugular arteriovenous fistula.

INTRODUCTION: PMCT is superior to autopsy for identification of intravascular or extravascular gas pockets and their distribution. However, differentiation between air embolism and putrefactive gas can prove challenging due to overlapping imaging findings. CASE REPORT: We report a case of a healthy young man who was involved in a fight, sustained a slash wound to the right side of his head by a kitchen knife and died at the scene. Pre-autopsy PMCT demonstrated complex fractures of the right mastoid bone extending to the right petrous apex and jugular bulb, exposing the right sigmoid sinus. There was also asymmetric intravascular air distribution suspicious of air embolism with ancillary findings of traumatic carotid-jugular pseudoaneurysm and arteriovenous fistulous formation. Post-mortem examination revealed a slash wound measuring 12x2 cm at the right side of the head, cutting through the scalp, right temporal bone, right temporal meninges, right sigmoid venous sinus and part of the right occipital lobe. No intracranial haemorrhage was found on both PMCT and autopsy. DISCUSSION: PMCT findings of air embolism versus putrefactive air on PMCT are discussed in this case. Detailed history on mechanism, circumstances, time of death and careful analysis of intravascular and extravascular air distribution patterns on PMCT are essential in guiding differentiation of true fatal air embolism and "normal" post-mortem putrefactive air. Needless to say, it is recommended that PMCT be performed as early as possible after death to reduce the chances and presence of artifactual decomposition changes.

IGF-1 stabilizes goat sperm mitochondrial transmembrane potential and reduces dna fragmentation.

BACKGROUND: Antioxidant present in sperm cells protects them from oxidative damage. However, sperm are more susceptible to peroxidative damages due to the loss of these enzymes during cryopreservation and their survival and fertility may be compromised. Insulin like growth factor-1 (IGF-1) has an antioxidant effect and could maintain sperm motility. OBJECTIVE: To improve seminal parameters, mitochondrial membrane potential (MMP), oxidative status and DNA integrity of buck semen after freeze-thawing by fortification of goat semen diluent with various concentrations of IGF-1. MATERIALS AND METHODS: Fifty ejaculates were collected and were extended with tris- citric acid- fructose diluent with 10% egg yolk and 6% glycerol with sperm concentrations of 1×108 mL-1. Post-cryopreserved sperm were assessed for motility and a range of other functional parameters. RESULTS: In post-thaw semen sperm motility, live sperm count, acrosome integrity, hypo-osmotic swelling positive spermatozoa, malondialdehyde (MDA), protein carbonyl content (PCC), TUNEL positive sperm differed significantly (P<0.05) with the various concentrations of IGF-1 used. Sperm functional parameters post-thawing were significantly (P<0.05) better in 250 ng/mL IGF-1. IGF-1 protects against lipid peroxidation by lowering MDA and PCC production, thus reducing the harmful effect of reactive oxygen species. The kidding percentage using the artificial insemination technique was significantly higher ( i.e., 40%) in the group supplemented with 250 ng/mL of IGF-1 than in the non-supplemented group (i.e., 30%). CONCLUSION: IGF-1 may be used to improve post-thaw semen quality and fertility as measured by actual kidding rate. Doi.org/10.54680/fr23610110312.

Exotic Dark Matter Search with the CDEX-10 Experiment at China's Jinping Underground Laboratory.

A search for exotic dark matter (DM) in the sub-GeV mass range has been conducted using 205 kg day data taken from a p-type point contact germanium detector of the CDEX-10 experiment at China's Jinping underground laboratory. New low-mass dark matter searching channels, neutral current fermionic DM absorption (χ+A→ν+A) and DM-nucleus 3→2 scattering (χ+χ+A→ϕ+A), have been analyzed with an energy threshold of 160 eVee. No significant signal was found; thus new limits on the DM-nucleon interaction cross section are set for both models at the sub-GeV DM mass region. A cross section limit for the fermionic DM absorption is set to be 2.5×10^{-46} cm^{2} (90% C.L.) at DM mass of 10 MeV/c^{2}. For the DM-nucleus 3→2 scattering scenario, limits are extended to DM mass of 5 and 14 MeV/c^{2} for the massless dark photon and bound DM final state, respectively.

Genetic and population diversity of Toxocara cati (Schrank, 1788) Brumpt, 1927, on the basis of the internal transcribed spacer (ITS) region.

The present investigation was aimed to study the sequence, phylogenetic and haplotype analyses of Toxocara cati based on the ITS region, along with the genetic diversity, demographic history and population-genetic structure. The maximum likelihood tree based on Kimura 2-parameter model was constructed using the complete ITS region of all the nucleotide sequences (n = 57) of Toxocara spp. and other related ascarid worms available in the GenBank™. It placed all the sequences of T. cati into four major clades designated as T. cati genotypes 1-4 (TcG1-G4). A total of 66 signature nucleotides were identified in the ITS region between genotypes. The median-joining haplotype network displayed a total of 24 haplotypes, with China exhibiting the highest number of haplotypes (h = 20) followed by India (h = 4), and Japan and Russia (h = 1). It indicated a clear distinction between all the four genotypes. The pairwise FST values between all the genotypes indicated huge genetic differentiation (> 0.25) between different T. cati genotypes. Moreover, the gene flow (Nm) between T. cati genotypes was very low. Results of AMOVA revealed higher genetic variation between genotypes (92.82%) as compared to the variation within genotypes (7.18%). The neutrality indices and mismatch distributions for the G1-G4 genotypes, Indian isolates and the overall dataset of T. cati indicated either a constant population size or a slight population increase. The geographical distribution of all the genotypes of T. cati is also reported. This is the first report of genotyping of T. cati on the basis of the ITS region.

Association between mental health and executive dysfunction and the moderating effect of urban-rural subpopulation in general adolescents from Shangrao, China: a population-based cross-sectional study.

OBJECTIVES: To examine the association between mental health and executive dysfunction in general adolescents, and to identify whether home residence and school location would moderate that association. DESIGN: A population-based cross-sectional study. SETTING: A subsample of the Shanghai Children's Health, Education, and Lifestyle Evaluation-Adolescents project. 16 sampled schools in Shangrao city located in downstream Yangtze River in southeast China (December 2018). PARTICIPANTS: 1895 adolescents (48.8% male) which were divided into three subpopulations: (A) adolescents who have urban hukou (ie, household registration in China) and attend urban schools (UU, n=292); (B) adolescents who have rural hukou and attend urban schools (RU, n=819) and (C) adolescents who have rural hukou and attend rural schools (RR, n=784). MEASURES: The Depression Anxiety and Stress Scale-21 was used to assess adolescent mental health symptoms, and the Behaviour Rating Inventory of Executive Function (parent form) was applied to measure adolescent executive dysfunction in nature setting. RESULTS: Mental health symptoms were common (depression: 25.2%, anxiety: 53.0%, stress: 19.7%) in our sample, and the prevalence rates were lower among UU adolescents than those among the RR and RU, with intersubgroup differences in screen exposure time explaining most of the variance. We found the three types of symptoms were strongly associated with executive dysfunction in general adolescents. We also observed a marginal moderating effect of urban-rural subgroup on the associations: UU adolescents with depression (OR 6.74, 95% CI 3.75 to 12.12) and anxiety (OR 5.56, 95% CI 1.86 to 16.66) had a higher executive dysfunction risk when compared with RR youths with depression (OR 1.93, 95% CI 0.91 to 4.12) and anxiety (OR 1.80, 95% CI 1.39 to 2.33), respectively. CONCLUSIONS: Rural adolescents experienced more mental health symptoms, whereas urban individuals with mental health problems had a higher executive dysfunction risk.

Establishment of effective and safe recipient preparation for germ-cell transplantation with intra-testicular busulfan treatment in pre-pubertal Barbari goats.

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3-5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P > 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P < 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and ß-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats.

Immunophenotypic Characterization and Ploidy Analysis of Neoplastic Plasma Cells by Multiparametric Flow Cytometry.

Flow cytometric (FCM) immunophenotyping is an important tool for generating diagnostic and prognostic information in plasma cell dyscrasias. This study aimed to evaluate the immunophenotype and ploidy status of plasma cells (PCs) in patients of myeloma and its correlation with other laboratory parameters. Bone marrow of 70 newly diagnosed cases of myeloma were subjected to FCM using a panel of antibodies; CD138, CD38, CD19, CD45, CD28, CD81, CD56, CD200, and CD229. FxCycle Violet (FCV) dye was used for the ploidy analysis of clonal PCs. Median age was 60 years with M:F ratio of 3.2:1. A positive correlation was noted between the morphological and FCM-based PC enumeration (r = 0.4, p = 0.001). Aberrant expression of CD56, CD200, CD28, CD117, CD81 and CD19 and was observed in 88.5%, 77%, 29%, 37%, 23% and 17% cases respectively. Two aberrant antigens were noted in all cases. CD81 + cases had a relatively higher quantity of monoclonal-protein (> 1 g/dl, p < 0.05) and renal insufficiency (Cr > 2 mg/dl, p < 0.05) as compared to the CD81- cases. CD229 was expressed in all the cases, with a median MFI in PCs significantly higher than other hematopoietic elements. Hyperdiploid PCs (median DI-1.59, range, 1.16-2.6) were noted in 80% cases (n = 48), diploid/ near-hyperdiploid PCs in 8% (n = 5) cases and hypodiploidy in 3% (n = 1) cases. Bright CD56/CD200 and CD45- can identify abnormal PC in the majority of the cases. CD81 appears to correlate with disease burden and might be useful as a prognostic marker. CD229 is a reliable gating marker for plasma cells. Ploidy analysis may be incorporated in routine workup to guide in the identification of patients with poor prognosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-021-01477-y.

Identification of Health Systems Science in a Problem-Based Learning Clinical Reasoning Exercise.

The creation of developmentally appropriate and meaningfully complex clinical reasoning exercises in the pre-clerkship curriculum is a common challenge for many medical schools. We provide an overview of one component of the pre-clerkship clinical reasoning curriculum at Case Western Reserve University School of Medicine, and present evidence that inclusion of Health Systems Science in this exercise facilitates integrated thinking in a Problem-Based Learning curriculum.

Prevalence of incidental and total human immunodeficiency virus, hepatitis B and hepatitis C seropositivity among patients posted for cataract surgery at a tertiary care center in India.

PURPOSE: To study the prevalence of human immunodeficiency virus (HIV), hepatitis B (HBV), and hepatitis C (HCV) viral seropositive among the patients posted for cataract surgery at a tertiary care center in north India. METHODS: It was a cross-sectional study done for 30 months duration. All the patients posted for cataract surgery underwent comprehensive ophthalmic evaluation followed by routine hematological workup, including viral markers for HIV, Hepatitis B surface antigen (HBsAg), and anti-HCV. Data were analyzed by the Statistical Package for Social Science (SPSS Version 20). RESULTS: A total of 7,316 individuals underwent cataract surgery from Jan 2016 to August 2018, 4,073/7,316 (55.7%) were males. The prevalence for HIV was 58/7,316 (0.8%), HBsAg was 151/7,316 (2.1%), and HCV was 11/7,316 (0.1%); 28/58 (48.3%) HIV positives were unaware of their seropositivity till testing, as were 37/151 (24.5%) of HBsAg positives, and 4/11 (36.4%) HCV positives. There was a significant relationship between the mean age in the patients with HIV (P = 0.002) and anti-HCV (P = 0.045). A majority of the seropositive patients were found to be illiterate (45.6%), followed by educated up to high school level (29.1%), and graduate (25.0%). CONCLUSION: Viral seropositivity was significant among the patients posted for cataract surgery. The eye care providers could refer these patients for counseling and further management for the patient's and their caretaker's benefit.

Association of TYRP1 with hypoxia and its correlation with patient outcome in uveal melanoma

Purpose Molecular mechanisms of uveal melanoma development in association with high pigmentation are unclear. Tyrosinase Related Protein (TYRP1) is not only one of the important melanogenesis marker that contributes to melanin synthesis, but can also prevents the melanocyte death. The induction of melanogenesis leads to induction of HIF-1α which can affect the behavior of melanoma cells and its surrounding environment. The aim of our study was to determine the expression of TYRP1 and HIF-1α at the protein and RNA level and determine its prognostic significance. Methods In the present study, the expression of TYRP1 and HIF-1α was investigated on 61 formalin-fixed paraffin-embedded choroidal melanoma samples by immunohistochemistry. Fresh 50 samples were validated by real-time PCR. Results were correlated with clinicopathological parameters and Kaplan–Meier was performed to determine the prognostic significance. Results High immunoexpression of TYRP1 and HIF-1α was present in 61 and 54% of patients, respectively. Both TYRP1 and HIF-1α correlated well with high pigmentation and BAP1 (BRCA1 Associated Protein-1) loss (p < 0.05) at IHC level as well as transcriptional level. There was reduced metastatic free survival in patients with necrosis and this was statistically significant (p = 0.010). Conclusion Our findings indicate that TYRP1 can be used as a potential biomarker in the development of targeted therapy in UM. Further studies on melanogenesis markers associated with TYRP1 could provide us a better understanding in this field (AU)

Differential gene expression in liver of colored broiler chicken divergently selected for residual feed intake.

Feed constitutes about 60-70% of the total cost of poultry production. So maximizing the feed efficiency will reduce production cost. The rapid growth in the juvenile period is essential to achieve higher body weight. Therefore, identifying the genes and pathways involved in rapid growth at an early age with a lesser requirement of feed is of utmost importance to further economize the broiler production. The efficiency of feed utilization was measured using RFI (residual feed intake). The present study aimed to estimate the RFI (0-5 week) in a population of indigenously developed colored broiler sire line chicken as well as identifying the differentially expressed genes influencing RFI in high and low RFI groups. The liver samples of high and low RFI broiler chicken aged 35 days were used for microarray analysis. A total of 2798 differentially expressed genes (DEGs) were identified, out of which 913 genes were downregulated and 1885 were upregulated. The fold change varied from - 475.17 to 552.94. A subset of genes was confirmed by qRT-PCR, and outcomes were matched well with microarray data. In the functional annotation study of DEGs, the highest significant GO (Gene Ontology) terms in the biological process included protein transport, protein localization, regulation of apoptosis, and mitochondrial transport. Gene network analysis of these DEGs plays an important role to understand the interaction among genes. Study of the important genes which were differentially expressed and the related molecular pathways in this population may hold the potential for future breeding strategies for augmenting feed efficiency.

Expression of the developmental important candidate genes in oocytes, embryos, embryonic stem cells, cumulus cells, and fibroblast cells of buffalo (Bubalus bubalis).

The present study was undertaken to study the expression of the developmental important gene transcripts in immature oocytes, mature oocytes, different stages of IVF produced embryos, embryonic stem (ES), cumulus (BCC), fetal fibroblast (BFF), newborn fibroblast (NBF) and adult fibroblast (BAF) cells of buffalo by semi-quantitative RT-PCR. The expression of GLUT1, HSP70.1, POL A Polymerase, GDF9, BMP15, and SURVIVIN transcripts was found in immature oocytes, mature oocytes, 2-cell, 4-cell, 8-16 cell, morula, and the blastocyst. Interestingly, the CX43 expression was found in oocytes, embryos, and other cell types, but it was not detected in the blastocyst. However, the IFNT expression was found in the blastocyst only, but not in other cells. The buffalo ES cells showed the expression of intracellular and cell surface markers (NANOG, OCT4, SOX2, FOXD3, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) and alkaline phosphatase activity. Two ES cell lines (S-line and M-line-II) were continued to survive up to 98th passages (~630 days) and 97th passages (~624 days), respectively. It was interesting to note that GLUT1, CX43, HSP70.1, POL A Polymerase, GDF9, BMP15, and SURVIVIN transcripts (except the IFNT) were expressed in buffalo ES, BCC, BFF, NBF and BAF cells. This is the first preliminary report that the buffalo ES, BCC, BFF, NBF, and BAF cells expressed the several developmental important candidate genes. It is concluded that the expression of the major developmental important genes was not only expressed in the oocytes and embryos but also expressed in the ES, BCC, BFF, NBF, and BAF cells of buffalo.

Status of Beta Defensin-1 and its Effect on Post-thaw Semen Fertility Gene Expression in Indian Goat Breed.

BACKGROUND: Defensins are antimicrobial peptides and uniformly spans the entire sperm surface and is not exclusive to a specific domain. Goat ß-defensin-1 helps in initiation of motility and capacitation of sperm. OBJECTIVE: To know the status of ß-defensin-1 in blood, semen and its effect on post thaw fertility gene expression in Indian goat breeds. MATERIALS AND METHODS: Semen was extended and divided for estimation of ß-defensin-1 and cryopreserved having different concentrations of ß-defensin-1. RESULTS: Bet defensin-1 concentration (pg/mL) in neat semen, sperm pellet and seminal plasma was significantly higher (P< 0.05) in goat breed Barbari followed by Jamunapari and Jakhrana. ß-defensing-1 was also high in Jakhrana blood followed by Barbari and Jamunapari. The post thaw motility, live sperm, acrosome intactness and hypo osmotic swelled sperms were significantly higher (P< 0.05) with 10 ng/mL ß-defensin in the semen dilutor. CONCLUSION: Beta defensin (10 ng/mL) in semen dilutor may be used as immuno-modulator to get better post thaw quality suitable for artificial insemination.

Improvement in molecular detection of phytoplasma associated with rose by selection of suitable primers and development of a multiplex PCR assay.

Sensitive and effective phytoplasma DNA amplification in symptomatic rose cultivars is a long unresolved problem. In the present study, improvement in standardization for PCR assay for phytoplasma detection was established with rose samples by selection of various combinations of nested primer pairs of 16S ribosomal gene and secA gene. CTAB DNA extraction method was slightly modified by adding 2% polyvinyl pyrrolidone and increased the isopropanol volume which yielded better quality DNA. Best amplification results were achieved in nested PCR assay employing P1/P7, R16mF2/R16mR2 and R16F2n/R16R2, P1/P7 and R16mF2/R16mR2, and R16mF2/R16mR2 and fU5/rU3 primer pairs. Besides, a multiplex PCR assay was also developed and optimized for consistent identification of phytoplasma in rose samples by employing primer pairs of 16S rRNA and secA genes together in a single PCR reaction by optimizing annealing temperature at 55 °C.